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					Reproduction & Development
These notes were made by Hadley Wickham, hadley@technologist.com and are licensed under the
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Table of Contents
Table of Contents ........................................................................................................................................ 2

Hormones - Hypothalamus......................................................................................................................... 4
   Hypothalamic/Pituitary/Gonadal Axis.......................................................................................4

Hormones - Pituitary ................................................................................................................................... 8
   Somatomammotrophic Polypeptides .........................................................................................8
   Glycoprotein Hormones .............................................................................................................8
   Gonadotrophins..........................................................................................................................9

Hormones - Secondary ............................................................................................................................. 11
   Steroids ....................................................................................................................................11
   Sex Steroids .............................................................................................................................11
   Steroid Hormone Action ..........................................................................................................13
   Inborn Errors of Steroid Metabolism .......................................................................................14
   Prostanoids ............................................................................................................................... 16

Reproductive Organs ................................................................................................................................ 19
   Ovary .......................................................................................................................................19
   Menstrual Cycle – Uterus and Female Tract ...........................................................................21
   Testes & Male Tract ................................................................................................................22

Fertilisation, Pregnancy, Birth and Development ................................................................................... 28
    Fertilisation .............................................................................................................................. 28
    Placenta ....................................................................................................................................30
    Endocrinology of Pregnancy ...................................................................................................34
    Maternal Adaptation to Pregnancy ..........................................................................................37
    Fetal Growth and Nutrition ......................................................................................................38
    Fetal Imaging ...........................................................................................................................39
    Partuition..................................................................................................................................40
    Pre-term Labour .......................................................................................................................41
    Post Natal Growth....................................................................................................................43
    Lactation ..................................................................................................................................44
    Milk..........................................................................................................................................46
    Hormones and Contraception ..................................................................................................48
    Puberty .....................................................................................................................................49
    Infertility ..................................................................................................................................51
    Menopause ............................................................................................................................... 52
    Ageing......................................................................................................................................55

Metabolic Endocrinology ......................................................................................................................... 57
   Adrenal Cortex.........................................................................................................................57
   Adrenal Medulla ......................................................................................................................59
   Calcium Metabolism ................................................................................................................60
   Calcium Clinical ......................................................................................................................63
   Glucose Homeostasis ...............................................................................................................66
   Diabetes Mellitus .....................................................................................................................68
   Hypoglycaemia ........................................................................................................................72

Development of Reproductive System .................................................................................................... 77

Hadley Wickham                                                                    2                                                                            16/06/11
   Development of Gonads ..........................................................................................................77
   Development of Genital Ducts ................................................................................................ 79
   Development of External Genitalia .........................................................................................80
   Disorders of Sexual Differentiation .........................................................................................81
   Thyroid ....................................................................................................................................73
   Histology of Endocrine Glands ............................................................................................... 84




Hadley Wickham                                                             3                                                                        16/06/11
Hormones - Hypothalamus
Hypothalamic/Pituitary/Gonadal Axis
  reproductive processes are mediated by hierarchal arrangement of endocrine glands,
     ultimately under control of CNS
  complex regulatory mechanisms operate between different levels of hierarchy
      External Cues
           few influences of human sexual cycle (cf. oestrus cycles in mammals)
           few behaviour changes in humans
           number of external cues can influence reproduction:
             Light: red deer mate in autumn (controlled by day length), biological clock takes
                 about 18 months to completely resynchronise, nocturnal rats ovulate so that egg
                 is optimum position at night-time
             Smell: women living together tend to have synchronised cycles, mediated by
                 pheromones?
             Touch: suckling release oxytocin from posterior pituitary, stimulating milk release
             Sound: milk release can become conditioned to release when baby cries
             Psychosocial: sperm counts decrease with increasing stress, severe stress in
                 females can lead to amenorrhoea, dominant male monkey produce testosterone,
                 non-dominant don’t, non-dominant female monkeys don’t produce LH surge
                 (mediated by  PRL)
             Nutritional Status: protein/fat ration critical in proper functioning of menstrual
                 cycle, critical body weight necessary for menstruation to occur

Hypothalamus
  very complicated structure with many interconnections with rest of brain (especially
     autonomic areas, reticular core of brainstem, limbic forebrain and suprachiasmatic nuclei)
      Location
           relatively small area of brain, ~4g
           base beneath pituitary stalk, between diverging optic tract
           derived from diencephalon ( most posterior part of forebrain)
           funnel shaped
      Boundaries
           sup: hypothalamic sulcus (groove)
           ant: laminar terminalis (edge)
           post: mamillary bodies (swelling)
           surrounds wall of 3rd ventricle at its entry
           Funnel Metaphor
             hypothalamus = thick walled funnel
             pituitary stalk = solid tube at base of funnel
Hadley Wickham                                    4                                                16/06/11
                3rd ventricle = contents
                optic chiasm = clamp at front of funnel
                mamillary bodies = two swellings on back of funnel
                laminar terminalis = anterior side of funnel
                tuber cinerium = lowest part of hypothalamus
                median eminence = bottom part of funnel, highly vascular
      Relationship to Pituitary
           pituitary stalk connects hypothalamus to pituitary
           Posterior Pituitary
             axons of magnocellular nuclei project directly to post lobe via
                 hypothalamohypophoseal tract
             hormones are synthesised in cell bodies, packaged with binding protein
                 (neurophysin) and transported along axons by axoplasmic flow
             stored in post lobe of pituitary, where they are released into bloodstream
           Anterior Pituitary
             hypothalamus connects to anterior pituitary by an indirect vascular route
             parvicellular axons terminate in precapillary space of 10 portal plexus of vessels in
                 median eminence
      Composition
           hypothalamic neurons (cell bodies + axons)
           non-hypothalamic neurons (axons + terminals)
           axons passing through hypothalamus (fornix)
           support cells
           blood vessels
      Nuclei
           nucleus = cells bodies collected together in clusters, not always anatomically discrete
      Hormones
                                               Nuclei                 Hormones
               Magnocellular (big)     Supraoptic x2            oxytocin
                                       Paraventricular          ADH
               Parvicellular (small)   Arcuate (infundibular)   TRH, GnRH, GHRH, CRH,
                                       Ventromedial             PRH
                                       Dorsomedial              GH-RIH (somatostatin),
                                                                PIH (dopamine)
                                       Preoptic
                                       Tuberalis
                                       Posterior
                                       Paraventricular




Hadley Wickham                                                    5                             16/06/11
                                                               Location                                      Comments
                                 Somatotroph    post/lat PD                                usually rounded                                      50%

                    Acidophils
                                 Lactotroph     diffuse, most prominent in lat wings       large and numerous during pregnancy, shape          <20%
                                                                                           variable
     Chromophils




                                 Corticotroph   ant-med, some post-med, often              variable in size and shape                         15-20%
                                                associated with somatotrophs
                    Basophils




                                 Thyrothroph    ant-med basophilic wedge, scattered in     usually large and angular with irregular nuclei,      5%
                                                among somatotrophs                         granules concentrated peripherally
                                 Gonadotroph    scatted mainly post-lat                    always round of oval, usually small, lightly       5-10%
                                                                                           granulated
                                                tend to be in dorsal part of lobe          often form acini, degranulated
     Chromophobes




                                some specialisation of hormone production, but generally widespread:
                                  arcuate nucleus: generally GnRH; dorsomedial, PIH; ventromedial, GH-RIH;
                                     ventrolateral, GHRH
                                  paraventricular nucleus: parvicellular component produces TRH and CRF
                                GnRH secreted by large group of neurons caudal medial septal nucleus,
                                 periventricular medial preoptic nucleus and adjacent hypothalamic areas
         Projections of Axons
                                to posterior pituitary
                                to capillaries of median eminence
                                to capillaries of lower infundibular stalk
                                to lining of 3rd ventricle ( capillaries of median eminence via tanycytes)

Pituitary
         Relationship to Hypothalamus
                                lies in hypophoseal fossa of sphenoid bone
                                almost encased in bone and very well protected
                                overlapped by circular fold of dura, called diaphragma sellae, which has small circular
                                 opening through which pituitary stalk phases
                                can get to through lacrimal bone  nasal sinus  pituitary fossa
         Origin
                                anterior lobe derived from Rathke’s pouch, small diverticulum pinched off from dorsal
                                 pharynx
                                  pars distalis arises from one side of pouch
                                  pars intermedia arises from other side of pouch (collapsed in humans)
                                closely applied to smaller posterior lobe, derived from neuroectoderm, origin reflect
                                 by connection to hypothalamus by stalk of nervous tissue, the infundibulum
                                inside of pouch often filled with darkly staining glycoprotein
         Blood Supply
                                all comes from internal carotid a. at circle of Willis
                                superior hypophoseal a. (SHA) supplies median eminence, and via trabecular a.
                                 (which passes through pars distalis, but doesn’t supply it) lower infundibular stalk


Hadley Wickham                                                                         6                                                               16/06/11
           veins from SHA don’t drain into systemic circulation, instead they form portal system
             blood from SHA forms venules drain into long portal vessels and supplies pars
                 distalis
             blood from trabecular a. does same, via short portal vessels
           inferior hypophoseal a. (IHA) supplies pars nervosa
           veins from IHA drain via short vessels into dural sinuses
           magnocellular axons secrete hormones into IHA capillary bed  systemic circulation
           parvicellular axons secrete hormones into SHA capillary bed  pars distalis




Hadley Wickham                                  7                                             16/06/11
Hormones - Pituitary
Somatomammotrophic Polypeptides
  three members: prolactin (PRL), placental lactogen (PL; aka placental somatomammotrophin) and
     growth hormone (GH, aka somatotrophin)
                                               PRL                                            PL                                     GH
  Secreted by              ant. pituitary lactotrophs,                        cytotrophoblast then                ant. pituitary somatotrophs (GH-N)
                           placental decidua                                  syncytiotrophoblast cells of        syncytiotrophoblast cells (GH-V)
                                                                              placenta
  Acts upon                Leydig cells                                       Maternal intermediary metabolism    Breast development
                           Seminal vesicle & prostate                         Mammary gland                       Many other tissues
                           Ovarian follicles                                  Fetal growth
                           Corpus luteum
                           Mammary gland
                           Amnion
  Molecular weight         ~23,000                                            ~22,000                             ~22,000
  Composition              199 aa polypeptide, post-translation               191 aa polypeptide                  191 aa polypetide, 1 CHO
                           modification gives variety of forms
  Homology                                                67%                                                             96%
  Receptors                PRL-R                                              PRL-R (same affinity),              PRL-R (GH-V binds best)
                                                                              GH-R (1/2000 affinity)              GH-R (GH-N binds best)
  Actions                                                Widespread supportive role (mediated through IGF-1 and related factors)
                           facilitate action of LH                            unknown (perhaps general            protein: +ve N balance, anabolic,
                           milk proteins  (must be primed)                   metabolic changes associated with   increase aa uptake
                                                                              pregnancy)                          carbohydrate: induces insulin
                                                                                                                  resistance, blood glucose  (utilisation
                                                                                                                  , production )
                                                                                                                  fat: FA release , oxidation 
                                                                                                                  mineral: increased uptake of Ca2+,
                                                                                                                  Mg2+, PO34-, bone growth 

      Disorders
               IFG-1 deficiency: Laron type dwarfism, normal levels of IGF-2 indicate relative importance
               GH deficiency: dwarfism, treated with recombinant hGH
               GH overproduction in adults: acromegaly, growth of short bones
               GH overproduction in childhood: gigantism, growth of all bones

Glycoprotein Hormones
  family contains four important hormones:
      thyroid stimulating hormone (TSH)
          short- and long-term effects on thyroid, leading to stimulation of most thyroid functions
          responsible for overall control of metabolic processes (eg. too little = lethargic, too much =
                     hyperactive)
                 very tightly controlled by negative feedback
               follicle stimulating hormone (FSH) & lutenising hormone (LH)
                 regulate gonadal function by promotion of sex steroid production and gametogenesis
               chorionic gonadotropic (CG)
                 increases in blood shortly after conception and is used as basis of pregnancy tests


Hadley Wickham                                                            8                                                                           16/06/11
Gonadotrophins
                                          LH                                        FSH                                       CG
  Secreted by        Ant. pituitary gonadotrophs                   Ant pituitary gonadotrophs             cytotrophoblast then syncytiotrophoblast
                         (LH and FSH usally secreted by different cells, although some secrete both)      cells of placenta

  Acts upon          Leydig cells                                  Sertoli cells                          Luteal cells
                     Thecal cells (antral follicles)               Granulosa cells (follicles)
                     Granulosa cells (pre-ovulatory follicles)
                     Luteal cells (corpus luteum)
                     Interstitial glands of ovary
  Molecular          ~28,000                                       ~28,000                                ~37,000
  weight
  Composition        α-chain (92 aa, 2 CHO)                        α-chain (as LH)                        α-chain (as LH)
                     β-chain (121 aa, 2 CHO)                       β-chain (111 aa, 2 CHO)                β-chain (145 aa, 6 CHO)
  Receptor           85-95 kDa glycoprotein,                       146 kDa multimeric glycoprotein,       as LH
                     G protein coupled                             G protein coupled
  Actions            1. testosterone  (lipoprotein receptors ,   1. general cell activity , androgen   steroid production (hydroxylation of
                     cholesterol ester hydrolase )                receptors                             bound cholesterol, uptake from LDL)
                     2. androgens                                 2. aromatase 
                     3. maturation 
                     4. maintains corpus luteum

  long-term trophic effect unusual for peptide hormones, indicating that gene transcription changes
     must be involved

Structure
  heterodimers (α & β sub-units strongly associated by non-covalent interactions)
      α sub-unit (92 aa) common (except for some differences in CHO) to all four hormones,
                essentially interchangeable
               β sub-unit confers hormonal specificity but are homologous
                 β units roughly same size, except hCG has an extra 31 aa chain at C terminus
                 15% homology between α and β sub-units
      Carbohydrate
               both α and β sub-units have ASP linked CHO, accounting for 15-30% of dry weight
               considerable size heterogeneity for each hormone, reflecting differential patterns of
                glycosylation (may give subtle physiological differences)
               contain many negatively charged sugars
               necessary for biological activity and stability in bloodstream
                 deglycosylated FSH has antihormonal effect (probably through CHO interactions with cell
                   membrane lectins)
                 deglycosylated FSH has much shorter half life than normal FSH (probably through
                   decreased glomerular filtration and/or decreased breakdown by enzymes)

Mechanism of Action
  generally G protein coupled (activation of adenylate cyclase with short-term increase (40-100x) in
     cAMP production)
  likely to involve cAMP response elements and cAMP response element binding protein (CREB)
     nuclear receptors in long-term actions




Hadley Wickham                                                          9                                                                            16/06/11
Actions
                       Follicular Development                                   Ovulation                                    Luteal Function
 FSH         elevated concentrations of LH and FSH                                                             low concentrations of LH and FSH prevent
             promote development of follicle beyond                                                            development of follicles
             primary stage
             binds to granulosa cells, induces aromatising
             activity
             accumulation of FSH and E2 promotes: fluid
             formation, granulosa  , maturation of P4
             synthesis capacity, LH receptors 
 LH          binds to thecal cells, stimulates androgen and   LH surge: follicular androgen and E2 ,          low plasma conc. maintains P4 production
             oestrogen production                             granulosa , initiates function and structural
                                                              lutenisation, PG , proteolytic enzymes
 Other       prolactin involved in developing capacity to      P4 accompanied by reduced aromatising          in pregnancy, CG prevents luteal regression
             produce P4 in response to LH                     activity


Secretion
  secreted in pulsatile mechanism in response to GnRH concentration
      LH and GnRH peaks coincide and approximately every 90 minutes (circhoral)
      crucially important for gonadotropin section – ensures that cells don‟t become desensitised to
               circulating levels associated with receptor down-regulation
         




Hadley Wickham                                                           10                                                                          16/06/11
Hormones - Secondary
Steroids
   four main families of steroids: progestogen, androgens, oestrogens, and corticosteroids
       all structurally similar and may be regarded as being part of family (progestogens parental and
                   oestrogens filial to androgens)
                  interconversion occurs in multienzyme where substrate is passed from one enzyme to next

Sex Steroids
                                      Progestogens                                       Oestrogens                                       Androgens
                      Progesterone (P) (100%)                           Oestradiol (E2) (100%)                           5α-hydrotestosterone (DHT) (100%)
                      17α-hydroxyprogesterone (40-70%)                  Oestriol (E3) (10%) (menopausal)                 Testosterone (T) (50%)
                      20α-hydroxyprogesterone (5%)                      Oestrone (E1) (1%) (pregnancy)                   Androstenedione (8%)
                                                                                                                         Dehydroepiandrostenedione (4%)
 Properties           1. prepare uterus to receive conceptus            1. stimulate 20 sex characteristics (female)     1. induce and maintain differentiation of
                      2. maintain uterus during pregnancy               2. prepare uterus for sperm                         male somatic tissues
                      3. stimulate growth of mammary glands,            3. vascular permeability , tissue oedema       2. stimulate 20 sex characteristics (male),
                         suppress secretion of milk                                                                         body hair (female)
                                                                        4. growth and activity of mammary gland
                                                                                                                         3. induce and maintain some 20 sex
                      4. mild effect of Na+ loss                           and endometrium 
                                                                                                                            characteristics of males
                      5. general mild catabolic effect                  5. prepare endometrium for progestogen
                                                                                                                         4. support spermatogenesis
                      6. regulate secretion of gonadotrophins              activity
                                                                                                                         5. influence sexual and aggressive
                                                                        6. mildly anabolic, calcification
                                                                                                                            behaviour
                                                                        7. active during pregnancy
                                                                                                                         6. promote protein anabolism, somatic
                                                                        8. regulates secretion of gonadotrophins            growth and ossification
                                                                                                                         7. regulate secretion of gonadotropins


                      preparations and maintenance of pregnancy         development and maintenance of feminine          development and maintenance of masculine
                                                                        characteristics and fertility                    characteristics and fertility
 Structure            21C, C4-C5 double bond,                           18C, aromatised ring, C3 hydroxyl, C17 β-        19C, C19 β-hydroxyl, C3 ketone structure
                      C17 β-acetyl, C13 β-methyl                        hydroxyl


                 Progesterone (P4 )                                        Oestradiol (E2)                                        Testosterone (T)
preovulation:          <4 nM                             male:      <0.16 nM                                     male:       11 – 40 nM
during LH surge:       4-10 nM                           normal female:         0.2-1.1 nM
postovulation:         25-120 nM                         postmenopausal:        <0.1 nM                          female:     2.5 – 0.5 nM
endometrium: stroma and glandular epithelium ,          endometrium: stroma and glandular epithelium ,         facilitates libido and sexual potency in males
tubular gland coiling , water content ,                endometrium thickness , tubular gland length ,        facilitates aggressiveness
vascularity of stroma , thick glandular secretions      P4 receptors , serous glandular secretions
myometrium: activity                                    myometrium: activity 
endocervical canal: mucus secretion , sperm             endocervical canal: mucus secretion , viscosity
transport and survival                                  , elasticity , cervical os softens and opens
vagina: epithelial cells                                vagina: epithelial cells 
acts through CNS to increate basal body
temperature by 0.3 – 0.50C
FSH stimulates initial secretion of oestrogens from growing follicles                 LH stimulates Leydig cells to produce testosterone (PRL and inhibin facilitate
LH stimulates further development (and further oestrogen secretion),                  action of LH)
ovulation, formation of corpus luteum, and production of hormones by corpus           FSH stimulates production of androgen receptors on Sertoli cells (androgens
luteum                                                                                stimulate production of FSH receptors)
                                                                                      T has inhibitory effect at both pituitary and hypothalamus (believed to be via
                                                                                      conversion to oestradiol)
small amount produced in non glandular tissues           testes secrete small amount of oestradiol (12% of       also produced in extraglandular tissues from
increased in polycystic ovary disease (relatively        total)                                                  circulating androstenedione (minor in men,
common, 22% have echographic appearance)                 secondary site of oestrogen production (important       significant in women)
                                                         in male and postmenopausal women) is adipose
                                                         tissue, which converts androstenedione in to E2

   all steroids synthesised from cholesterol

Hadley Wickham                                                                  11                                                                              16/06/11
           conversion of cholesterol to pregnenolone is first and common step in formation of all major
            sterioids (rate limiting, occurs on inner mitochondrial membrane, further steps occur in sER)
           rate limiting step is conversion of hydroxymethyl glutaryl CoA to mevalonic acid (catalysed
            by HMG CoA reductase)
           major site of synthesis is liver
           transported out from liver in variety of packages, most important being LDL
           taken up by cells in periphery and utilised
  close relationship between steroids means that defect in biosynthesis of one may have far reaching
     effects
           eg. absence of enzyme to make ACTH leads to increased androgens
  main steroid producing organs are gonads, adrenal cortex, and placenta
  steroids are lipid soluble and pass easily through cell membranes but do not dissolve well in plasma
       to increase solubility they are bound to plasma binding proteins
                         Binding Protein               Progestogens        Androgens   Oestrogens   Cortisol
               Albumin                                     48                 32          63          20
               Cortisol binding globulin                   50                  1                      70
               Sex steroid binding globulin                                   66          36
               Free steroid                                 2                  1           1          10

           can also be conjugated (with glucoronic acid or sulphate), although this reduces biological
            activity and deconjugation is necessary to regain full activity
           SHBG
             glycoprotein, Mr = 95,000
             synthesised in liver, production stimulated by E2
             high affinity for testosterone

Biosynthesis of Steroids
  see page 28 of Essential Reproduction

Male Sex Hormones
                           Time                  Amount (mg/ml)
                 13-15 weeks (pre)         2
                 5-6 months                0.8
                 2 months (post)           2-3
                 3-4 months                0.5
                 prepuberty                0.1
                 post puberty              2-3
                 adult                     5-9

      5α Dihydrotestosterone
           principle intracellular androgen in number of important tissues (eg. male reproductive tract,
            skin)
           most arises from local production from testosterone and androstenedione (also some produced
            by testes)
             catalysed by 5α reductase (two forms, share only 50% homology)
             75% comes from testosterone in males, 20% in females (remainder comes from
                 androstenedione)



Hadley Wickham                                                        12                                       16/06/11
                                             Type 1                                                       Type 2
                         5p                                                 2p
                         dominant form in adult scalp                       dominant form in genital tissue, including prostate
                         expressed in skin tissue throughout body           defective in men with 5α reductase deficiency
                                                                            more readily inhibited by finasteride (5α inhibitor used to treat
                                                                            BPH)



Steroid Hormone Action
  steroid-like hormones are all hydrophobic and include:
                 Group                       Members                           Example
       steroid hormones                glucocorticoids         cortisol
                                       mineralocorticoids      aldosterone
                                       progestogens            progesterone
                                       oestrogens              oestradiol
                                       androgens               testosterone
       antirachitics                   vitamin D               1,25 dihydroxycholecalciferol
       insect hormones                 ecdysteroids            20-OH ecdysone
       thyroid hormones                                        tri-idothyroninee
       derivatives of vitamin          retinoids               retinoic acid

  act on specific target cells by changing transcription rate of specific genes in highly coordinated
     fashion
  mostly stimulate transcription, however, corticosteroids tend to inhibit some enzymes and
     stimulate others
  early studies indicating that steroid hormones alter rate of transcription came from studies of
     ecdysone on larval salivary cells
             larval salivary cells contain extremely large chromosomes visible with LM, replication has
              occurred without normal mitotic chromosomal separation and millions of copies of DNA are
              aligned side-by-side
             active areas of chromosome are visible as “puffs” in which DNA is less condensed and
              appears swollen
             treatment of such cells with 20-OH ecdysone causes sequential rapid, coordinated, transient
              puffing and then depuffing of bands, corresponding to different genes
  2D electrophoresis of radiolabelled proteins have shown that mammalian steroids also induce or
     repress synthesis of certain proteins
           Steroid Type                      Protein                 Action                                 Tissue
       glucocorticoids           tyrosine amino transferase                          liver
                                 pro-opiomelanocortin                                pituitary
       progestogens              ovalbumin                                           oviduct of birds
       oestrogens                vitellogenins                                       liver of birds (major egg yolk proteins)
       thyroid hormones          fatty acid synthetase                               liver
       vitamin D                 calcium binding protein                             intestinal ep


Mechanism
  receptors for steroid hormone, when unoccupied by steroids, are either located in
      nucleus, bound to DNA
      cytoplasm, bound to HSP 90 (heat-shock protein)
  binding of hormone to receptor causes change (transformation) which induces release of original
     ligand, dimerisation with other bound receptors and movement into nucleus
  transition changes binding of receptor to DNA to tighter and more specific form (specific
     palindromic nucleotide sequence)

Hadley Wickham                                                                13                                                                16/06/11
      Receptor Structure
           steroid receptors are present in very small amounts
           affinity chromatography using immobilised steroid and very enriched tissue sources of
            receptors has enabled receptors to be purified and characterised
             cDNA probes have been subsequently discovered allowing screening for other steroid
                 receptors using low stringency hybridisation
           typical steroid receptor (in its monomeric form) has between 400 and 1,000 aa
           three domains within receptor can be identified:
             variable domain: found at N-terminal, specific to each receptor, variable length and
                 sequence
             DNA binding region: ~67 aa., residues, highly homologous(40-90%) between different
                 steroid receptors, containing pattern of 8 cysteine residues which coordination two zinc
                 atoms
                  8 cysteine residues + 2 Zn = 2 zinc fingers, bind either side of major groove of DNA
                  each receptor binds to partially palindromic defined DNA sequence, usually 15 b.p.
                   long called steroid response element
             hormone binding domain: ~240 aa., C-terminal, moderate homology (15-60%)

Inborn Errors of Steroid Metabolism
Congenital Adrenal Hyperplasia (CAH) and Related Disorders
  genetic disorder which involves deficiency in one of enzymes required in steroid hormone synthesis
  in enzyme defects associated with cortisol deficiency, hyperplasia of adrenal gland may occur due to
     excessive stimulation by ACTH
  when enzyme deficiency leads to virilisation of affected females (masculinisation of ext genitalia)
     disorder sometimes referred to as adreno-genital syndrome
           wolffian duct development consistently absent in virilised females, who, therefore, have
            normal internal sex organs
           why development is not stimulated by excess androgen is not understood, but high local
            concentrations created by testes may be required
      21-Hydroxylase deficiency
           commonest cause of CAH, accounting for ~90% of recognised cases
           severe (classical) form occurs in about 1/14,000 live births
           presents clinically in two forms:
             simple virilisation, compensated (production of aldosterone largely unaffected)
             salt-wasting form (75% of cases, production of aldosterone compromised)
           aldosterone biosynthesis affected by defect with  aldosterone leading to Na+  and K+ 
           life-threatening crisis may occur within first two weeks unless disorder recognised and treated
           leads to high concentrations of 17-hydroxyprogesterone and high urinary pregnanetriol
            (diagnostic)
           treated with glucocorticoids and mineralocorticoids (in salt-wasting form)
           less severe, non-classical, form doesn‟t usually present until puberty with hirsutism and
            amenorrhoea

Hadley Wickham                                       14                                                     16/06/11
      11-Hydroxylase deficiency
           results in hypertensive form of CAH
           decrease in cortisol synthesis, with resultant overproduction of cortisol precursors and
            androgens
           hypertension though to be result of excess production 11-desoxy-corticosterone (DOC), an
            aldosterone precursor, itself possessing mineralocorticoid activity
           external genitalia of female fetus masculinised
      3-Hydroxysteroid Deficiency
           rare enzyme deficiency results in impaired synthesis of cortisol, aldosterone and
            androstenedione, and elevated production of dehydroepiandrostenedione
           disorder may be associated with pseudohermaphroditism and salt wasting

Disorders of Androgen Function
  while T is major circulating androgen, in most of target cells it is converted to 5-DHT by 5α-
     reductase
  disorders of androgen action may involve 5α-reductase or androgen receptor
      5α-reductase Deficiency
           autosomal recessive enzyme defect impairing conversion of T to 5-DHT
           internal male genital tract develops normally but external genitalia are predominantly female
           variable virilisation of external genitalia at puberty
      Disorder of Androgen Receptors (Androgen Resistance Syndrome)
           lest four variants of disorder have been described (arise from mutations in androgen receptor
            gene):
                complete testicular feminisation
                incomplete testicular feminisation
                Reifenstein syndrome
                infertile male syndrome

Aromatase Deficiency
    ambiguous genitalia at birth
    at puberty present with hypergonadotrophic hypogonadism with multicystic ovaries
    no breast development, enlarged clitoris, external genitalia otherwise immature
    stature is short with delayed bone age

Steroid Sulfatase Deficiency
  during fetal life disorder reveals itself as placental sulfatase deficiency
      causes marked decrease in placental oestrogen production, particularity that of E3 (through
            failure of hydrolysis of oestrogen precursors DEAHS)
           pregnancies otherwise normal but may be associated with failure to progress into labour at
            term,
  in postnatal life, affected individuals develop a dry scaly skin (ichthyosis) usually within three
     months of birth, and sometimes corneal opacities


Hadley Wickham                                        15                                                 16/06/11
  X-linked recessive

Prostanoids
  hormone-like substances, found in almost all animals cells
  play an important role in regulating cellular function (sites of action include: gonads, reproductive
     systems, GI tract, cardiovascular system, renal and CNS)
  half-life of 3-10 minutes, act locally as paracrine messengers

Structure
  all based on prostanoic acid, 20-C carboxylic acid
      Prostaglandins and thromboxanes
           formed from oxygenation of arachidonic acid, catalysed by cyclooxygenase
           named according to when they were found (eg. PGA, PGB, PGC etc.)
           endoperoxide intermediates (PGG and PGH) are not biologically active, merely precursors of
            PGE and PGF
             can also be converted into thromboxanes (platelet aggregation) and prostacyclin (platelet
                 aggregation)
           TXs contain an oxygen in ring structure
      Leukotrienes
           formed from conjugation of arachidonic acid, catalysed by 5-lipoxygenase
           analogous compounds also formed from eicosotrienic acid
           LTC4, LTD4 and LTE4 considered to be responsible for biological activity of SRA-S (slow
            releasing substance of anaphylaxis)

Biosynthesis
  except for semen, most body fluids and tissues contain few free prostanoids
  prostanoids are not accumulated in stores, instead they are rapidly activated (and inactivated)
     according to need
  arachidonic acid in central intermediate derived from EFAs for conversion to prostanoids
      arises from two sources:
          endogenous synthesis from linoleic acid
          release from cell membrane phospholipid (catalysed by phospholipase A2)

Functions
      Prostaglandins
                        Pathological          Physiological
                 fever                 temperature control
                                       bronchial tone
                 peptic ulcers         stomach mucosa
                 pain                  intestinal motility
                 dysmenorrhoea         myometrial contractility
                 bone degeneration     semen viability
                 inflammation          vasodilatation

           PGJ thought to have anti-tumour and anti-viral effects


Hadley Wickham                                                    16                                  16/06/11
           PGG & PGH (produced by same enzyme) are precursors to all other PGs
           TX and prostacyclin are antagonists

Reproductive Function
      Menstruation
           Dysmenorrhoea (painful menstruation)
             two types: primary (idiopathic, unknown cause), secondary (acquired, known cause)
             PGF2 and PGF2α are greatly raised in dysmenorraghic women (especially in luteal phase)
             administration of PG inhibitor relieves symptoms
      Implantation
           PGE2 and prostacyclin increased at implantation site (PG , implantation )
           human decidual tissue from 1st-trimester abortions has extremely low concentrations of PG
           related to recognition of “foreign” embryo
      Parturition
           PGF increases with cervical dilation in spontaneous labour
             originally though to be causal effect
             some think is related to fetal head trauma, but some still seen even if sampled
                 transabdominally
           PGF metabolites in blood increased during and decreased after labour
           oxytocin, produced by gestational membrane, brings on labour, but is very painful
      Cervical Ripening
           PGs increased during pregnancy (because of head trauma etc.)
           help activate collagenases which break down collagen in cervix, relaxing it and allow baby to
            get out
      Fetus
           Patency of umbilical vessels
             prostacyclin helps to keep vessels open
             PG inhibitors can‟t be given to prevent pre-term labour because of this
             high prostacyclin concentration in umbilical vessels
           Patency of ductus arteriosus
                PGE2 helps to keep ductus patent
                after birth PGE2 decreases and ductus closes
                neonates with patent ductus usually have high levels of PGs
                PGE2 is clinically useful in cyanotic congenital heart disease (allows pulmonary flow),
                 coarctation of aorta (allows systemic flow) and transposition of great arteries (improves
                 mixing and flow)




Hadley Wickham                                       17                                                  16/06/11
Reproductive Organs
Ovary
 differentiates later than testes and endocrine activity not necessary during fetal life, but must occur
   postnatally for full sexual maturity at puberty

Oogenesis and Hormonal Control
    Production of Follicles
          Primordial
           located around edges of ovaries
           flat follicular cells located around large oocyte, surrounded by stromal cells
          Primary (Preantral)
           flat follicular cells  cuboidal granulosa cells
           increase in size of follicle (20-400 μm) and oocyte (to final size of 60-120 μm)
           zona pellucida (jelly-like shell) secreted by oocyte, separates granulosa from oocyte by
             thin avascular layer of glycoproteins, but contact still maintained by cytoplasmic processes
           stromal cells start to organise into two layers known as theca interna (inner, glandular,
             vascular layer) and theca externa (outer fibrous layer)
           single layer of granulosa cells proliferate to up to 12 layers
             increasing numbers of gap junctions form between neighbouring cells
             small substance passed through to oocyte (only supply of nutrition as granulosa is
                avascular)
              as granulosa cells continue to proliferate viscous fluid, known as follicular fluid,
                appears between them, composed partly of mucopolysaccharides and partly of serum
                transudate
              drops of fluid coalesce to form an antrum, marking beginning of antral phase of
                development
          Secondary (Antral)
           formed on first day of menstrual cycle
           entire structure becomes larger (~2 mm, visible with naked eye) over next two weeks
             increase size depends mainly on increase in size of follicular antrum
           as follicular antrum grows oocyte, surrounded by dense mass of granulosa cells called
             cumulus oophorus becomes suspended in fluid
          Tertiary/Mature/Graafian
           very large (2 cm in diameter)
           extends outward on ovary (visible as bulges on surface)
           granulosa cells closest to oocyte form corona radiata, which supplies oocyte with nutrients
    Selection of Dominant Follicle
         follicular growth is continuous and independent of gonadotropins up to antral stage (ie.
          development occurs independently of an extraovarian controls)
           at beginning of menstrual cycle 15 – 20 follicles are recruited to develop further
             without FSH/LH follicles become atretic when they reach diameter of ~2 mm
             recruitment due to chance (based on stage of development), keyed by P4, LH/FSH
             FSH is crucial hormone for follicular recruitment (ie. FSH recruited follicles, FSH
                 recruited follicles)
           selection of (usually) one dominant follicle based essentially on size and stage of development
             FSH (due to inhibin  and E2 )
             less well developed follicles become atretic due to lack of FSH (androgens )
             dominant follicle has sufficient FSH receptors to survive, in addition FSH triggers
                 production of LH receptors linked to same pathway
           emergence of only one dominant follicle can be overcome by administration of extra
            gonadotropins
      Rupture
           after rupture follicle forms large corpus luteum (yellow body)
             blood flows back into antrum and clots within it
             stimulates granulosa and theca interna cells to proliferate hugely and start producing E2
                 and P4
             cytoplasm stains less darkly because of sER (necessary for steroid production)
             granulosa cells expand to fill cavity as clot resolves
           prepares uterus to receive fertilised egg
             corpus luteum degenerates after 12-14 days if implantation doesn‟t occur
             if implantation does occur, corpus luteum lasts about 3-5 months until placenta takes over
           Mechanism
             not due to pressure release
             due to weakening of follicle wall stimulated by FSH/LH via cAMP and P4, LK3 and PG‟s
                 activating latent collegenase

Luteal Phase
  E2 and P4 both high, combined high level giving negative feedback on FSH and LH
  FSH/LH levels recover in late luteal phase as E2 and P4 
      uterus also detects falling levels and menstruation begins
      LH/FSH Pulse Frequency and Amplitude
           day 2: pulses of LH every 90 minutes, but none during sleep
           day 7: LH , but pulse continues through night and frequency 
           day 14: baseline , at moment of LH surge baseline , frequency , not much happens to
            FSH
           LH surge + 1: still lots of LH but decreasing baseline and frequency (peak amplitude )
           luteal phase: baseline = 0, pulses far apart, peaks reasonably high, triggers P4 peak, -ve
            feedback on LH
           Hormonal Actions
             P4: pulse freq 

Hadley Wickham                                      20                                                    16/06/11
             E2: pulse amplitude 

Control of Pituitary (LH & FSH)
      Effect of Testosterone
           same as P4

Menstrual Cycle – Uterus and Female Tract
Concept of Cycles
      Historical
           concept of “normal” menstrual cycle relatively recent phenomenon
           early ancestors (eg. 10th century) had only 30-40 cycles in lifetime
             puberty was later (~17 years, declining for past hundred years) and 1st 1-2 years are
                 anovulatory
             each pregnancy lasts 9 months and is followed by 2-3 years of lactational amenorrhoea
             average of five children
             short life span
           present day have about 400 cycles
             average two pregnancies and early abandonment of breast feeding
      Phase Lengths
           more cycles occur with lengths of 27 or 28 days
           normal considered to be between 21 and 36 days
           considerable within individual variation as well as population variation
           follicular phase (17 ± 4 days) more variable than luteal phase (12 ± 1 days)
           highest percentage of normal cycles occur between 26 and 35 years of age
             women with reproductive problems experience rapid drop off after this age
           cycle lengths  with age
           basal body temperature increases by about 0.40C over course of cycle (more population than
            an individual phenomenon)

Phases of Menstrual Cycle
  changes in E2 and P4 levels cause cyclic changes in structure of female reproductive tract, esp
     endometrium
  although menstrual cycle divided into 3 phases for descriptive purposes menstrual cycle is
     continuous process
      Menstrual Phase
           first day of menstruation defined as beginning of cycle
           usually lasts 4 – 5 days
           functional layer of uterine wall is sloughed off and discarded with menstrual flow
           menses (menstrual flow) is discharged through vagina and consists of varying amounts of
            blood combined with small pieces of endometrium


Hadley Wickham                                      21                                                16/06/11
      Proliferative Phase
           lasts about 9 days
           coincides with growth of ovarian follicles and is controlled by E2 secreted by follicles
            (follicular phase)
           two- to threefold increase in thickness of endometrium and in its water content
           early during phase surface ep reforms and covers endometrium
           glands increase in number and length, spiral aa. elongate
      Secretory Phase
           lasts about 13 days
           coincides with formation, functioning and growth of corpus luteum (luteal phase)
           P4 produced by corpus luteum stimulates glandular ep to secrete glycogen-rich material
           glands become wide, tortuous and saccular
           endometrium thickens because of P4 and E2 from corpus luteum and increase in fluid in ct
           as spiral aa. grow into superf compact layer they become increasing coiled
           venous network becomes complex and shows large lacunae (a-v shunts prominent feature)
           hormonal withdrawal results in stoppage of glandular secretion, loss of interstitial fluid and
            marked shrinking of endometrium
           toward end of ischaemic phase spiral aa. become constricted for longer periods, results in
            venous stasis and patchy ischaemic necrosis in superf tissues (result of P4 )
           eventually rupture of vessel walls follows and blood seeps into surrounding ct  bleeding
            into uterine lumen and vagina

Testes & Male Tract
  postnatal growth of is slow and outputs of androgens low (albeit higher than females)
  at puberty rapid growth and maturation of testes is accompanied by increased androgen output

Structure
  both exocrine (sperm) and endocrine (hormones, principally testosterone) gland
      occurs in two distinct compartments: sperm within tubules, androgens between tubules
      compartments structurally and physiologically distinct (separated by tight junctions at level of
            Sertoli cells, called blood-testes barrier)
           prevents sperm leaking into systemic circulation (causing an immune reaction against their
            „foreign‟ antigens) and allows control over intratubular fluid (markedly different from blood)
      Descent of testes
           begins at ~7 months, attached to gubernaculum
           develop in lumbar region, normally pass through inguinal canal into scrotum just before birth
           site of inguinal canal first indicated in embryo by gubernaculum, ligament that extends from
            testes through ant. abdominal wall and inserts into internal surface of scrotum
           later finger-like process of peritoneum, processus vaginalis, follows gubernaculum and
            evaginates ant. abdominal wall to form inguinal canal




Hadley Wickham                                       22                                                  16/06/11
           maldescent (cryptorchidism) of testis is common event (3% of full-term, 30% of pre-term),
            most commonly bilateral, can be partially or completely undescended, usually descends in
            first few weeks after birth, results in infertility if untreated
      Changes at Puberty
           cords become tubules (ie. gain lumens)
           marked increase in proliferation of spermatogonia
           beginning of sperm production
      General Structure of Adult Testes
           divided into segments by fascial partitions, each segment containing many blind ending
            tubules
           these run in to rete testes (network of tubules) and then in to efferent ductules (15-20),
            epididymis, and ductus deferens
           fibrous capsule (tunica albuginea) surrounds tubules
           rich blood supply cooled by pampiniform plexus
           Leydig cells form cords of cells outside tubules
      Intratubular Fluid
           proteins , PGs 
           Na+ , K+ , inositol , testosterone 

Spermatogenesis
      Early Development
           dependent on testosterone
           Leydig cells start secreting testosterone at 8-10 weeks after conception
      Mitotic Proliferation (Spermatocytogenesis) (1st Stage)
           interphase prospermatogonial cells of immature testis are reactivated at puberty to enter
            mitosis, and are now called spermatagonial stem cells
           at intervals group with distinct morphology arises from this reservoir, these are A1
            spermatogonia, and mark beginning of spermatogenesis
           these spermatogonia divide about 5 times ( number characteristic of species) before becoming
            terminally differentiated B spermatogonia
           all type B spermatogonia divide to form resting primary spermatocytes
           an remarkable feature of mitotic phase is that while nuclear fission is completed successfully,
            cytoplasmic division is incomplete, so all primary spermatocytes from one A1
            spermatogonium are connected by thin cytoplasmic bridges
           all of proliferative phase takes place in basal intratubular compartment of testes
      Meiosis (2nd Stage)
           resting primary spermatocytes double their DNA content then push through into adluminal
            tubular compartment by transient disruption of zonula junctions between Sertoli cells
           then enter first prolonged meiotic prophase, during which sister chromatids come together and
            recombine, shuffling genetic material before they pull apart
           spermatocytes very sensitive to damage during this time



Hadley Wickham                                         23                                                16/06/11
           first meiotic division ends with separation of homologous chromosomes and subsequent
            cytokinesis yielding two secondary spermatocytes, each containing single set of chromosomes
           chromatids then separate and move to opposite ends of meiotic spindle, cell divides and forms
            two haploid early spermatids
           only genes from autosome expressed, sex chromosomes deactivated
      Packaging (Spermiogenesis) (3rd Stage)
           major visible changes during spermatogenesis occur during considerable cytoplasmic
            remodelling
                tail is generated for forward propulsion
                midpiece, containing mitochondria, forms
                equatorial and post-acrosomal cap region forms (important in sperm-oocyte fusion)
                acrosome develops to act like enzymic knife
                nucleus contains compact packaged haploid chromosomes
                residual body removes left over cytoplasmic components and is phagocytosed by Sertoli
                 cell
           composed of four phases: golgi, cap, acrosomal and maturation
           Golgi Phase
             quite round cells with large nuclei and small tail (from centrioles)
             golgi body forms on opposite side of nucleus to tail, starts to form proacrosomal vesicles
           Cap Phase
             golgi vesicles coalesce forming cap over head of nucleus
             centrioles migrate to nucleus
           Acrosomal Phase
             cap begins to cover entire nucleus
             body and nucleus start to streamline
             mitochondria start to move towards central strand of tail
           Maturation Phase
                streamlining continues
                tail gains fibrous sheath (important for movement)
                mitochondria wrap around sheath
                cytoplasm starts to get pinched off and taken up by Sertoli cells
                RNA production ceases and DNA becomes highly compacted (heterochromic)
      Sperm Maturation
           composition of intratubular fluid changes as sperm move into rete testes
           takes place as sperm leave seminiferous tubules
           passage through efferent ductules takes between 6-12 days, and changes sperm behaviour
            profoundly
             sperm entering ductules are incapable of movement
             when enter cauda epididymis they are fully capable (although little movement shown until
                 they are activated and leave reproductive tract)


Hadley Wickham                                       24                                               16/06/11
             changes in functional capacity accompanied by changes in biochemistry and morphology,
                 androgens critically important in these changes
                                                                                             Changes
                   Concentration                   100-fold increase (from 5 x 106/ml to 5 x 108/ml)
                   Completion of sperm             nuclear condensation and acrosomal modelling complete
                   modelling                       cytoplasmic drop squeezed down tail and shed
                   Metabolism                      cholesterol and phospholipids selectively metabolised
                                                   dependence on external fructose 
                                                   pH 
                   Mobility                        disulphide cross-linking in tail, stiffness, beat strength
                                                   cAMP content of tail 
                                                   acquires capacity for forward motion
                   Membrane                        coated with glycoproteins
                                                   rise in surface charge and change in profile of surface proteins
                                                   membrane fluidity and lipid content changes

           passage from epididymis to ductus deferens is no longer result of fluid movement, but of
            muscular contractions
      Organisation
           takes ~64 days to complete journey from basement membrane to lumen, but because several
            stages are in production at one time, sperm are released every 16 days
           as spermatogenic cycle takes approximately ¼ of time required for maturation there must be
            four stages occurring at one time
                takes 16 days for six meiotic divisions to occur
                takes 32 days for spermatocyte to complete meiosis and early modelling
                takes 16 days for modelling to be completed
                entry into meiosis will always occur at same time as entry into mitosis, beginning of
                 elongation and release of sperms
           rate of sperm release and entry into spermiogenesis remarkably constant – independent of
            hormones and other externally applied substances
           if all spermatogonia entered cycle at same time, sperm release would occur at same time,
            leading to cyclical male fertility, however sections of tubule enter sequentially so at any one
            time sperm will be in all stages of development
           150 million produced per day (300-600/s/gram of tissue)
           200-300 million in each ejaculate

Organisation of Duct and Accessory Structures
      Seminal Fluid
           seminal fluid derived largely from major accessory sex glands, with only small contribution
            from epididymis
                   Gland            Percentage                              Components
             Seminal vesicles      13-33%        fructose, PGs, pH 7.3
             Prostate              46-80%        acid phosphatase, citric acid, spermin, fibrinolysin, pH 6.5
             Ampulla/ductus        ~10%

           not essential for sperm (as sperm taken directly from epididymis can still fertilise eggs, but
            provides transport medium with protective and nutritive functions
           particulate matter also found because of apocrine secretion




Hadley Wickham                                                  25                                                    16/06/11
                        Constituent       Conc (mM)                 Major Source                              Function
             Spermatozoa                 50-150          testis
             Fructose                    8-40            seminal vesicles & ampulla   anaerobic fructolysis
             Inositol                    1-3             testes and epididymis        preserves osmolarity?
             Citric acid                 5-70            prostate                     Ca2+ chelator
             Glycerylphosphorylcholine   2-3             epididymis                   see below
             Acid phosphatase            2500 units/ml   prostate                     cleaves choline from glycerylphosphorylcholine for
                                                                                      use in phospholipid metabolism

      Fractionation
           initially few drops of fluid from urethral and bulbourethral glands (not part of ejaculation,
            helps to lubricate urethra)
           acid phosphatase (prostate) rich fraction                                                                    (pre sperm)
           some fluid from seminal vesicles + sperm                                                          (sperm rich fraction)
           fructose rich fraction (viscous fluid from seminal vesicles)                                                 (post sperm)
      Erection
           elicited by psychogenic stimuli (eg. visual cues and erotic imagery), integrated by brain
            (probably limbic system) which then influences somatic and autonomic efferents to genitalia
             pelvic nerve (PNS, promotes erection)
             hypogastric nerve (SNS, depresses erection)
             pudendal nerve (somatic, promotes erection)
           same efferents can be activated reflexively by tactile stimulation of penis and perineum
           erection achieved entirely by haemodynamic changes in man
             PNS stimulation counters SNS (maintaining tone within smooth muscle), thereby reducing
                 muscle tone and increasing arteriolar dilation
             increased blood flow into cavernosa, decreased intracavernous resistance, increased
                 volume
             A-V shunts which normally bypass sinuses now direct blood into them
             venous outflow from corpora cavernosa decreased (probably by compression of veins)
           mediated by NO (GMP as secondary messenger), Ach and VIP
           GMP broken down by phosphodiesterase (inhibitors, eg. Viagra, increase erectile response)
      Ejaculation
           two phases: emission and expulsion (time separated)
             emission (SNS): release of sperm from duct by contraction of smooth muscle
             expulsion (PNS): expulsion of semen by contraction of somatic muscle (bulbo- and
                 ischiocavernosus muscles)
           retrograde ejaculation into bladder is prevented by contraction of vesicular urethral sphincter
      Erectile Dysfunction
           52% of men suffer from some degree of impotence in life
      Male Infertility
           affected by
             testosterone
             frequency of emission
             temperature (-2.20C)
Hadley Wickham                                               26                                                                    16/06/11
      Analysis of Semen
           liquefaction                        (should occur within 15 minutes)
           volume               (problem: <1 ml)
           concentration        (problem: <20 x 106/ml)
           motility             (problem: <40% motile)
           grade of motility (3 good forward, 2 moderate forward, 1 poor, 0 twitching)
           morphology           (considerable range, can have up to 70% abnormal)
           debris               (eg. WBCs, not a good sign)

Hormone Production
  most important hormones produced by testis are androgens, but oestrogen, inhibin, activin, oxytocin
     and Müllerian inhibiting hormone (MIH) are also produced
      Steroids
           principal testicular androgen is testosterone (synthesised from acetate and cholesterol by
            Leydig cells)
           4-11 mg secreted daily by males, enters both blood (larger quantity because of greater flow)
            and lymph (important for transmission to accessory sex organs)
           some also passes through cell membranes into seminiferous ducts where it is converted into
            more active DHT
      Growth factors and Peptides
           inhibin and activin (growth factors) are produced by Sertoli cells
           about 25% leaves via lymph, and most of remainder passes into seminiferous tubules
           eventually reach systemic circulation and bind to receptors on pituitary
           oxytocin (small peptide) is produced by Leydig cells, shown to increase seminiferous motility
            via action of myoid cells

Hormonal Control
  hypophysectomy  testes weight  (ie. pituitary always required for testicular function)
  castration  FSH & LH                       (ie. testes regulates pituitary)
  testosterone injection  FSH & LH  (ie. testosterone regulates pituitary)
      Function
           necessary for meiosis (especially 1st prophase)
           necessary for spermatid maturation (at higher levels than required for meiosis)
           acts via Sertoli cells to increase production of enzymes related to spermatogenesis, inhibin
            and ANP
      Role of Oestrogen in Males
                       Male           Female
                      (ng/ml)         (ng/ml)
             T    5             0.3
             E2   0.05          0.3

           negative feedback on pituitary
           enhances actions of testosterone (eg. fibromuscular growth of accessary sex organs)
            

Hadley Wickham                                              27                                             16/06/11
Fertilisation, Pregnancy, Birth and Development
Fertilisation
Gamete Transport
      Oocyte
           E2 , cilia , secretion 
           P4  (against E2 background) cilia , secretion 
           muscle activity not required for transport, passively carried by fluid and cilia
           takes about 6 days to get to uterus
      Spermatozoa
           maturation occurs in epididymis
           overall sperm have survival time of about 48 hours in female reproductive tract (vagina 2
            hours, cervix 48, uterus 24, oviduct 48)
             nourished by mucoid secretions in deep cervical crypts
             mucus only permits further sperm travel in absence of P4
           takes 2-7 hours (minimum) to get to ampulla of oviduct
             vaginal, cervical and uterine movements (during pre- and orgasmic phases) are not
                 required but may assist it
             probably move through under own propulsion and fluid movements set up by cilia
             tend to linger and become immotile in isthmus of oviduct, only during ovulation do they
                 regain movement and pass through ampulla-isthmus junction to site of potential
                 fertilisation
           only about 50,000 reach ampulla

Prerequisites
      Capacitation
           glycoproteins (laid down in male tract) must be stripped from head of sperm
             experimentally reversible
           process of capacitation has two elements:
             change in movement characteristic to hyperactivated motility pattern with undulating
                 wave-like motion replaced by whip-lashing beats
             change in surface membrane that renders sperm responsive to further signals
           associated by acquisition of four properties
                stability of surface membrane 
                Ca2+ permeability 
                adenyl cyclase 
                tyrosine kinase  (required for subsequent acrosomal reaction)



Hadley Wickham                                      28                                                  16/06/11
      Acrosome Reaction
           Ca2+  (perhaps in response to P4 in cumulus?)
           acrosome swells, membrane fuses with overlying plasma membrane, vesiculated appearance
            is created and both content of acrosomal vesicle and inner acrosomal membrane become
            exocytosed
           ZP3 binds sperm and helps to induce acrosome reaction
           ZP2 holds sperm and ZP in contact, essential for passage of sperm through ZP towards oocyte

Activation
      Gamete Fusion
           acrosomal reaction exposed proteases which digest path through ZP, along which sperm
            passes aided by whiplash motion of hyperactivated tail (takes between 5 and 20 minutes)
           sperm eventually comes to lie tangential to oocyte surface between ZP and oocyte membrane
            in perivitelline space
           surface membrane overlying middle and post half of sperm head is site of fusion with oocyte
           only capacitated sperm that have undergone acrosomal reaction are capable of fusion
           once fusion has occurred, sperm dramatically ceases to move and its nucleus (together with
            some of mid piece and tail) passes into ooplasm
           better fertilisation occurs if cumulus is present, and egg doesn‟t travel down oviduct as well
            without it (may just be that bigger object is easier to move than smaller)
      Establishment of diploidy
           newly fertilised oocyte must prevent further sperm from entering (androgenetic polyploidy)
            and must complete meiosis (gynogenetic triploidy)
           immediately after fusion, there is dramatic increase in Ca2+ (mostly from intracellular stores),
            that sweeps like wave across oocyte from point of entry
           first rise followed by series of Ca2+ spikes, each lasting 1-2 minutes, and growing more
            synchronised
           associated with change in pattern of phosphorylation in oocyte
           rise in Ca2+ has two important actions:
             fusion of cortical granules with oolemma, releasing contents into perivitelline space
                 (cleave ZP3, digest ZP2 and cross-link ZP preventing further sperm penetration)
             stimulates completion of meiosis
      Initiation of Development
           cytoplasmic contents of sperm cells membrane pass into ooplasm
           sperm nuclear membrane breaks down and highly condensed chromatin starts to swell,
            releasing filamentous strands of chromatin into cytoplasm
           decondensation actively induced by factor in ooplasm that develops in terminal phases of
            intrafollicular maturation
           between 4-7 hours after fusion, two sets of chromosomes each become surround by distinct
            membranes and are now know as pronuclei (male is usually larger)




Hadley Wickham                                        29                                                 16/06/11
Development Prior to Implantation
      Division
             Cells        Time (hours)
        2            30
        16           72
        60           96
        100+         4 ½ days

             cells get progressively smaller because ZP restricts growth
             eventually ZP splits and egg „hatches‟, lot of debris gets left behind in ZP
      Maintenance
             of endometrium by hCG
             of embryo by substrates supplied by oviduct (as it grows enzyme systems become more
              mature and can digest more substrates)

Placenta
  placenta is primary site of nutrient and gas exchange between mother and fetus
  placenta and umbilical cord function as transport system for substances passing between mother and
     fetus
             nutrients and O2 pass from maternal blood through placenta to fetal blood
             waste material and CO2pass from fetal blood through placenta to maternal blood
  placenta and fetal membranes are responsible for:
      protection
      nutrition
      respiration
      excretion
      hormone production

Development of Placenta
  by end of 3rd week anatomical arrangements necessary for physiological exchanges between mother
     and embryo are established
  complex vascular network is established in placenta by end of 4th week, facilitating maternal-
     embryonic exchange of gases, nutrients and metabolic waster products
  chorionic villi cover entire chorionic sac until beginning of 8th week
      as sac grows villi associated with DC are compressed, reducing blood supply and soon
               degenerate, producing a relatively avascular bare area, smooth chorion
             as villi disappear those associated with DB grow rapidly, forming bushy portion of chorionic
              sac known as villous chorion
  growth in size and thickness of placenta continues rapidly until about 20 weeks
  fully developed placenta covers 15-30% of decidua and weighs about 1/6 that of the fetus
  placenta has two parts
      fetal component of placenta formed by villous chorion
      maternal component of placenta formed by decidua basalis, almost completely replaced by
               fetal component by end of 4th month

Hadley Wickham                                        30                                               16/06/11
      Fetomaternal Junction
           fetal placenta attached to maternal placenta by cytotrophoblastic shell (CS) (external layer of
            trophoblastic cells of maternal placenta)
           stem chorionic villi are attached firmly to DB through CS and anchor chorionic sac
           endometrial aa. and vv. pass freely through gaps in CS and open into IVS
           shape of placenta determined by shape of persistent area of chorionic villi
             usually is a circular area, giving placenta discoid shape
           as chorionic villi invade decidua basalis during placental formation decidual tissue is eroded
            to enlarge the intervillous space (IVS)
           erosion process produces several wedge-shaped areas of decidua, placental septa, that project
            towards chorionic plate
             divide fetal placenta into irregular convex areas called cotyledons
             each cotyledon consists of two or more stem villi and their many branch villi
             by end of 4th month DB almost entirely replaced by cotyledons
           DC forms capsule over external surface of sac
                as conceptus enlarges, DC bulges into uterine cavity and becomes greatly attenuated
                eventually contracts and fuses with DP, obliterating uterine cavity
                by 22-24 weeks reduced blood supply causes degeneration and collapse
                smooth chorion fuses with DP
      Intervillous Space
           contain maternal blood from spiral endometrial a.
           drained by endometrial vv. found over entire surface of DB
           results from coalescence and enlargement of syncitioblastic lacunae
           divided into compartments by placenta septa, but free communication occurs between them
      Amniochorionic Membrane
           amniotic sac enlarges faster than chorionic, resulting in fusion to form amniochorionic
            membrane
           soon fuses with DC and then the DB
           ruptures during labour (premature rupture results in premature labour)

Placental Circulation
  many branch chorionic villi provide large surface for transport across very thin placental membrane
     (separating maternal and fetal circulations)
      Fetal Placental Circulation
           low O2 blood leaves fetus and passes through umbilical aa. to placenta
           at attachment to placenta these divide into many chorionic aa., branching freely over
            chorionic plate
           form extensive aterio-capillary-venous system within chorionic villi, bringing maternal and
            fetal blood very close together
           provides large surface area for gas transport



Hadley Wickham                                       31                                                 16/06/11
           normally, no intermingling of maternal and fetal blood occurs, but very small amounts of fetal
            blood enter maternal circulation through minute defects
      Maternal Placental Circulation
           blood in IVS temporarily outside maternal circulation
           contains 150ml of blood, replenished 3-4 times per minute
           enter through 80-100 spiral endometrial aa., discharged into IVS by gaps in cytotrophoblastic
            shell
           blood flow from aa. pulsatile and propelled in jet-like spurts towards chorionic plate
           pressure decreases and blood flows slowly around branch villi, allowing exchange to take
            place
      Placental Membrane
           composite membrane of extrafetal tissue separating maternal and fetal blood
           until 20th week contains four layer (syncytiotrophoblast, cytotrophoblast, ct of villus and
            endothelium of capillaries)
           after 20th week histological changes take place:
             syncytiotrophoblast thins and disappears
             capillaries move closer to cytotrophoblast (some fuse to form vasculosyncytial placental
                 membrane)
           during 3rd trimester multinucleate aggregations of syncytiotrophoblast form syncytial knots
             continually break off and travel into maternal circulation where they are rapidly broken
                 down
             towards end of pregnancy, fibrinoid material forms on surface of villi as a result of aging

Functions of Placenta
  placenta has three main functions
      metabolism
      transport of gases and nutrients
      endocrine secretion
      Placental Metabolism
           placenta synthesises glycogen cholesterol and fatty acids, as energy source for fetus
           many of metabolic activities critical for other two functions
      Placental Transport
           transport of substances facilitated by very large surface area
           almost all substances are transported by either simple diffusion, facilitated diffusion, active
            transport or pinocytosis
                      Substance          Transport Rate           Mechanism                     Examples
                 essential for life   mg/sec              diffusion, active transport   CO2, O2, urea, Na+, K+
                 nutritional          mg/min              active transport, diffusion   glucose, vitamins, etc.
                 growth modifying     mg/hour             slow diffusion                hormones
                 immunological        mg/day              pinocytosis?                  plasma proteins

           Gas Transport
             O2, CO2 and CO cross placental membrane by simple diffusion
             flow limited rather than diffusion limited
Hadley Wickham                                               32                                                   16/06/11
                                     Uterine            Umbilical
                                 A             V    V           A
                     PO2        95       33        15        28
                     PCO2       33       46        55        40

               although PO2 lesser in fetus, oxygen affinity and [Hb] greater (12 vs 16 mg/100g), so
                   oxygen carrying capacity is greater (15 vs 20-25 ml/100ml)
              Nutritional Substance
                  constitute bulk of substances transferred
                  water and electrolytes rapidly and freely exchange
                  glucose is transported rapidly by facilitated diffusion
                  little cholesterol, FFA, TG or PL are transported
              Maternal Antibodies
               fetus produces only small amounts of antibodies
               some passive immunity conferred by maternal antibodies, especially IgG

Amniotic Fluid
    play major role in fetal growth and development
    initially, most ultrafiltrate of maternal plasma
    before keratinisation of skin, fluid equilibrates across skin
    from 4-20 weeks, from foetus and surrounding tissues
    after 20 weeks from urine, placenta and umbilical cord
    beginning 11th week fetus contributes to amniotic fluid by expelling urine into amniotic cavity
             by late pregnancy, about 0.5 L added daily
            Week          Volume (ml)
       10            30
       20            350
       37            700-1000

      Circulation
             water content changes every 3 hours
               large amounts of water pass into maternal tissue and uterine capillaries
               exchange with fetal blood occurs through umbilical cord
               amniotic fluid is swallowed by the foetus and absorbed by respiratory and digestive tracts
             during final stages of pregnancy fetus swallows up to 400ml of amniotic fluid per day
      Composition
             water (99%)
             desquamated epithelial cells
             equal proportions of organic and inorganic salts
             changes as pregnancy advances as fetal excreta are added
      Function
             permits symmetrical growth of embryo
             acts as barrier to infection
             permits normal fetal lung development

Hadley Wickham                                                      33                                  16/06/11
           cushion embryo against injuries
           helps control embryo‟s body temperature
           enables fetus to move freely, aiding muscular development
           involved in fluid and electrolyte homeostasis

Endocrinology of Pregnancy
  in nine months of existence this organ effects marked changes in homeostasis of mother, important
     for the maintenance and success of the pregnancy
  hormone production by placenta independent of maternal endocrine system
  factors important to placental hormone production
      mass of trophoblast
      maternal-placental and feto-placental blood flow (governs rates of precursor and nutrients
            supply, and hormone and other substance removal)
  cyto- and syncytiotrophoblast produces almost every hormone (except insulin and PTH)
  most ends up in fetus (95%)
  physiological significance of many of these in unclear though some may act locally in regulation of
     other hormone production

Human Chorionic Gonadotrophin
  plays vital roles during early gestation to ensure continuance of pregnancy
      immediately following implantation of blastocyst, primitive trophoblasts begin secreting hCG
      maintains corpus luteum and stimulates further development to corpus luteum of pregnancy
      secretion of P4 and E2 (necessary for maintenance of pregnancy)
      P4 particularly important as it inhibits myometrial contractility
      by 6-8 weeks ovary has been replaced by placenta as main source of these hormones
  detectable in mother‟s blood between 7 and 12 days post-conception
      begins to appear in urine about 15 days after conception
      by 6th week, urinary levels are usually in excess of 1000 IU/L
      placental production increase exponentially and peak at 8-10 weeks (40-200,000 IU/L)
      production then decreases to much lower rate (8-20,000 IU/L)
      urinary excretion closely matches serum levels
      most common test for pregnancy, elevated with trophoblastic disease (tumour marker),
            depressed with ectopic pregnancy
  also plays important role in sexual differentiation of male (10-18 weeks)

Human Placental Lactogen
  detectable in blood from 5th week
      concentration rises to reach maximum value of ~5-9 mg/L at ~36 weeks and plateaus
      production directly related to placenta size
      ~1 g produced per day in late pregnancy


Hadley Wickham                                     34                                               16/06/11
  wide range of activity demonstrated and probably many major metabolic changes can be attributed
     to HPL
  elevates maternal blood levels of free fatty acids, glucose and insulin

Progesterone
    fetus lacks 17α-hydroxylase so can‟t metabolise P4 any further
    synthesised in placenta mainly from maternal LDL-cholesterol, largely independently of fetus
    secreted to mother and to fetus where it undergoes further metabolism to other steroids
    production increases during pregnancy until a few weeks before term (300 mg/day)

Oestrogens
  production of oestrogens intimately involves fetus, and depends on
      live fetus
      functioning fetal adrenal glands
      intact feto-placental circulation
      functioning placenta
  believed that oestrogen synthesis in human pregnancy involves following mechanism:
      fetal adrenal produced and secretes DHEAS at very high levels
      DHEAS passes to placenta where it is converted to E1 and E2
      shunted backed to fetus and some converted to E3
          major pathway to E3 involves precursor 16-OH DHEAS (produced mainly by fetal liver
                 from DHEAS, and fetal adrenal from de novo synthesis)
             placenta in turn converts 16-OH DHEAS to E3 through sequence of enzyme reactions (first
                 step is removal of sulphate)
  DHEAS secreted by maternal adrenal can also be utilised by placenta
      in last 10 weeks, 50% of E1 and E2 come from mother, but only 10% of E3
  E2 production rises almost 1000x during pregnancy

Hormonal Actions of P4 and E2 in Pregnancy
  in later weeks of pregnancy production of P4 estimated to be 250 mg/day and E 80-160 mg/day
       reason for such large quantities is unknown
       fetus protects itself from the high levels through highly active hydroxylation and conjugation
            systems
  initial action of E and P4 in pregnancy is production of uterine environment implantation of fetus
     and nourishment of embryo
  P4 necessary throughout for maintenance of pregnancy by keeping uterus in quiescent state
  E and P4 working synergistically stimulate growth of uterus
      also growth response to mechanical tension from growing conceptus
      uterine growth continues relatively normally with low levels of E
  E promote growth and softening of tissues of birth canal to allow passage of fetus without trauma
      high E levels stimulate maternal liver to produce CBG, SBG and TBG

Hadley Wickham                                    35                                                    16/06/11
  in breast E cause proliferation of duct, while P4 and E stimulates development of gland tissue
       also act directly on breast to inhibit milk secretion
       E not essential for preparing breast for pregnancy
  principal role of E in human pregnancy may be concerned with mechanism of parturition
       induction of labour may be difficult and labour prolonged in pregnancies with low E

Pregnancy and Maternal Endocrine System
      Hypothalamus
           little is know about changes, GnRH probably fully suppresses
      Anterior Pituitary
           size  (E2 mediated?)
           gonadotropic hormones suppressed, slight increase in ACTH and TSH
           PRL secretion increases progressively (8-20x normal levels at term)
             concentration remain high in following 7 days even in absence of suckling
             in non-breast feeding mothers, PRL declines and reaches normal levels 15-20 days after
                 birth
             in fully breast feeding mothers, PRL levels remain high during lactation, although there is
                 some decline
           pituitary secretion of GHs increase progressively to 3x normal
      Posterior Pituitary
           oxytocin and ADH released in increasing amounts from early pregnancy
           levels rise slowly with onset of labour and reach peak values in 2nd stage
           levels of degrading enzymes also raised
      Thyroid Function
           thyroid becomes moderately enlarged
           by 7th week of pregnancy, plasma thyroxine levels rise and eventually reach about 2x normal
           free thyroxine level stays the same
           changes mainly due to E2 induced increase in TBG and increased thyroxine secretion
           BMR increases 10-30%, mainly result of metabolic activity of conceptus
      Adrenal
           levels of CBG and total cortisol rise significantly, half-life of cortisol increased
           unbound cortisol levels show a slight rise
           aldosterone secretion increased considerably and responds markedly to changes in dietary Na+
           plasma levels of renin and AgII are also raised
      Pancreas
           glucose tolerance tends to be impaired, due to
             increased insulin resistance
             increased cortisol secretion
             diabetogenic properties of HPL

Hadley Wickham                                       36                                               16/06/11
             increased body weight
      Ovary
           corpus luteum fails to regress if implantation occurs, due to support by placental hCG
           synthesis of P4 and E stimulated by hCG
           by 6-8 weeks placenta has become main source of sex steroids

Maternal Adaptation to Pregnancy
Fetal-maternal Communication System
  maintenance of pregnancy dependent on and controlled by conceptus
  biomolecular/endocrine communication system between conceptus and mother established before
     implantation and functions throughout pregnancy even though parturition
  feto-maternal communication system has two major components
       placental part, responsible for endocrine and nutritive functions
       paracrine part, responsible for acceptance of semiallogeneic fetal graft, pregnancy
            maintenance, and physical protection

Maternal Adaptation to Pregnancy
  profound changes occur in anatomy, physiology and biochemistry of women during pregnancy
  equally remarkable is short time (few weeks) it takes to reverse changes after pregnancy
  maternal organs and physiological systems affected by pregnancy include: ovaries, uterus, cervix,
     vagina, abdominal wall, metabolic changes, endocrine, haematological, CV and urinary
      Ovary
           corpus luteum sustained in its function at least up to six weeks
      Uterus
           increases rapidly in size from (70 g  1000 g)
           cavity volume increases (~10 ml  ~5 L)
           uteroplacental blood flow increases to ~500 ml/min in late pregnancy
           contractility increases
      Cervix
           pronounced softening and cyanosis occurs from early pregnancy (one of early clinical signs)
           collagen rich ct undergoes structural rearrangement in preparation for parturition
      Vagina
           vascularity increases
           walls undergo structural changes in ct and smooth muscle in preparation for distension
      Metabolic Changes
           weight gain




Hadley Wickham                                      37                                               16/06/11
      Haematological Changes
                                             Pregnant             Non-pregnant
              Blood volume (L)         6                    4
              Plasma volume (L)        4.2                  2.6
              Hct (%)                  30-40                35-47
              Hb (g/L)                 100-140              115-165

      Cardiovascular
              CO  very early in pregnancy
                generalised enlargement of heart, especially LV
                10% increase in SV
              fetus pushes diaphragm up  ant-lat displacement of heart
              resting pulse rate  (10-15%)
              smooth muscle relaxes
                dilation of peripheral resistance vessels and veins
                peripheral resistance 
                refractoriness of AgII (ie. effect )
      Urinary System
              kidneys increase in size slightly
              GFR and RPF increase early in pregnancy
      Breasts
              in early weeks of pregnancy often tender or tingling
              after 2nd month breasts increase in size, nipple becomes larger and areolae broader

Fetal Growth and Nutrition
                  Postnatal Growth                                      Fetal Growth
    growth normally to genetic potential                growth normally constrained by maternal
                                                        environment
    provide minimum requirements met growth             provided minimum endocrine requirements met
    regulate by endocrine potential                     growth regulated by substrate supply

  difficult to distinguish between the environment and genetic contributions by the mother
      mother has a greater influence than the father (eg. Shetland ponies and Clydesdales)
      DZ twins have same birth weight as MZ twins
      cross-breeding in animals shows size of mother more important than father
           flawed if imprinting occurs, but occurs even when embryos are swapped
  natural constraint is the ability of the mother to supply nutrients
      normal phenomenon – ie. fetus can‟t grow as big as it wants
      way of matching fetal growth to maternal capacity
      very easy to show in sheep but not in humans because of the complex chain of events linking
               maternal to placental nutrition
              nutrition  maternal circulation  uterine blood flow  placental transport  umbilical
               blood flow  fetal circulation  tissue uptake
              makes easy to categorise causes of low birth weight



Hadley Wickham                                                           38                              16/06/11
Hormones
      Insulin
           quite important growth regulator shown by classical ablation experiment
           pancreatomy  insulin , growth , insulin replacement  growth 
           but increasing insulin above normal doesn‟t increase growth, so insulin is merely permissive
      GH
           no fetal GH-R present, so no feedback and fetal GH very high
           congenital absence of GH leads to baby with birth weight ½ sd. below mean
           large doses don‟t increase birth weight
      IGF
           probably fetal growth hormone, if there is one
           plasma IGF-1, birth weight , if IGF-1 given not much change in birth weight (although
            organ weight )
           IGF-2 important in the first half of pregnancy, IFG-1 important in the second half
           fetal nutrition regulates IGF-1
      Thyroid hormones
           congenital absence leads to slightly smaller babies (like GH)
      Sex Steroids
           boys are bigger at birth, but it is a small effect

Significance
  common, causes lots of problems for neonates, children and adults
  increased risk of adult diseases if birth weight is low
      David Barker hypothesis
           wildly varying rates of CHD in the UK, but similar risk factors
           strongly correlated with infant mortality (a reflection of average birth weight) 50 years
            previous
           strong inverse relationship between birth weight and risk of a number of disease (eg. diabetes,
            hypertension, CHD, hyperlipidaemia)
           may be related to high levels of maternal glucocorticoids (cortisol causes: poor vascular
            development, insulin resistance, β-cells , liver growth)
           placenta usually protects fetus from maternal cortisol but if mother stressed or placenta small,
            some may get through

Fetal Imaging
  embryology books talks about dates from fertilisations but obstetricians talk about dates from last
     menstrual period
  early embryological development runs like clockwork
      very few differences between babies
      can absolutely predict size embryo should be at any time
      only from 20 weeks do you start to see differences

Hadley Wickham                                        39                                                16/06/11
  all important stuff (organogenesis etc.) happens in 1st 12 weeks, everything else is just growth and
     maturation
      Time table
               Week                                                              Event
           3              blastocyst invades endometrium (eccentric)
           4              double decidual sac sign (functional layer of endometrium)
                          gestation sac = little bubble with double ring of tissue
           5              by end of 4th week can see little sac inside bubble = 20 yolk sac
                          should be 5.6 mm in normal pregnancy (10% don‟t develop past gestation sac = afetal sac)
                          (>8 mm visible TV, >27 mm visible TA)
           6-7            sac + yolk sac + FH
           6-10           primitive gut develops, basically doubles each week
                              6 weeks – 4 mm
                              7 weeks – 8 mm
                              8 weeks – 15 mm
                              9 weeks – 22 mm
           12+            fetal period

              must see FH is CRL >9 mm (TA) or >5 mm (TV)
      Abnormal Sac
                      Abnormality                  Size (mm)
           lack of yolk sac                     >20
           lack of double decidual sign         >10
           lack of embryo                       >25
           amnion, but no yolk sac

      Abnormal Implantation
              ectopic (90% in oviduct), 1/80 – 1/250 pregnancies (increased with PID)
                remember pregnancy test (β-hCG)
                hCG should normally double every 3 days, but will plateau in ectopic pregnancy
                do 2 blood tests, 3 days apart

Partuition
Sheep (endocrine)
    cortisol , P4 , E2 
    induces 17α-hydroxylase + other enzymes responsible for converting P4 to E2
    PGE2 and PGF2α 
    cervical dilation

Humans (paracrine)
  humans lack 17α-hydroxylase so it can‟t be induced by cortisol
  no remarkable changes in P4 or E2 before labour
  CRH and activin may be important in humans
      CRH dramatically increases (from 0) in last 5 weeks of pregnancy, produced by placenta
      CRH-BP also decrease during last few weeks, leading to a large increase in active hormone
      seems to be lower for post-term and higher for pre-term babies
      placental clock theory proposes that placenta is programmed for 40 weeks of life, alarm signal
               is CRH

Hadley Wickham                                                            40                                         16/06/11
  activin levels also increase in last 5 weeks
      pre-term babies have raised levels of activin
      NO inhibits CRH release (NO synthetase decreases during pregnancy)
      CRH stimulates PG synthesis, PG stimulates CRH synthesis
      activin A stimulated by cytokines
      activin A, CRH and PGF2α stimulate oxytocin release
      CRH-R adenyl cyclase reduced during end of pregnancy  increase gap junctions and
              organisation of contraction
             CRH potent vasodilator
      Placenta
        cytokines 
                          CRH 
        NO synthetase 




                 PGF2α + PGE2 + oxytocin 




           cytokines 
                          activin A

      Myometrium
             increasing CRH
               PGE2 + PGF2α 
               myometrial contractile response to oxytocin and PGE2 increase
             CRH-R
               adenyl cyclase activity  cAMP  myometrial contractility 
               onset of labour and delivery
             passive role for E2 (?) – giving E2 in humans doesn‟t induce labour
             cortisol stimulates CRH (?) – but inactivated by placenta and giving cortisol doesn‟t induce
              labour

Pre-term Labour
  pre-term labour = regular uterine contractions leading to cervical effacement and dilation, or
     persistent uterine contractions (>8/hour) if cervix already 2cm dilated or 50% effaced, at less than 37
     weeks (technically, but 32 practically)
  incidence hasn‟t changed in past 40 years
  many women present with preterm labour but few go on to deliver early
  5-8% of deliveries, 75% of perinatal deaths and 85% of deaths between 22 and 37 weeks
      40x more likely to die neonatally
      neurodevelopmental handicaps 22x more common
      <1500g, 3.2 admission in 1st cost
  significant cost to the community (>$100,000/baby)

Hadley Wickham                                       41                                                  16/06/11
  risk factors include
       maternal factors
           low SE status
           non white
           less than 18 or greater than 40 years old
           smoker
           low weight
           substance abuse
           peridontal disease
       maternal history
           previous preterm labour
           previous 2nd trimester abortion
       uterine factors
           anomalies
           trauma
       infection
  no good clinical markers have yet been identified (assessment of cervical length seems to be best)
  biochemical markers may be more successful, but no highly reliable ones currently identified
                            PPV (%)        NPV (%)
        Fibronectin    13-23          81-99
        IL-6           100            79
        E3             27             77


Treatment
  many things have been tried to prevent preterm labour but has been little success
  aims are to prevent preterm labour (either indefinitely or briefly) and improve outcome for baby and
     mother
      Salbutamol
              β-agonist – tocolytic (contractions )
              problems: Na+ , glucose , chest pain, SOB, ECG changes, hypotension, pulmonary oedema
              only used for 24-48 hours because of side effects
              unequivocally delays delivery but no beneficial effect on perinatal mortality/morbidity
      Oxytocin Antagonists
              side effects: injection site reaction, chest pain, tachycardia, foetal distress
              insufficient information, but delivery not delayed and fetal deaths 
      PG inhibitors
              side effects: constriction of DA, reduction in cerebral blood flow  persistent pulmonary
               hypertension of neonate
              no properly controlled trials
              COX 2 inhibitors may prove to be more helpful
              delays labour but no reduction in mortality or RDS

Hadley Wickham                                           42                                                16/06/11
      What is Effective?
           treatment with glucocorticoids
           transfer to tertiary care facility
           β-agonists may give time

Hormonal Regulation
  don‟t understand very well at all, most messengers are paracrine and very difficult to measure
  many complex interacting processes
  P4/E2 
      uterine receptors                  (OT, PG, ET, GJ)
      uterotonins                 (CRH, PGs, cytokinin, OT)
      catabolic enzymes                   (PGDH)

Causes
  infection is major cause
  infection  inflammation  cytokinins  PGs  contractions   labour  delivery
  PTL + infection  PDGH  PG   labour  delivery

Post Natal Growth
  always important to measure and plot child on growth chart, regardless of what speciality you are
     practising
           should always be first part of any examination
           need to plot accurately – don‟t just round to nearest year
           also need to measure accurately (head in Frankfort plane etc.)
  can‟t say just because they are below 5th percentile that something is wrong with them
  of much more physiological importance is growth velocity (50th percentile velocity mean that child
     stays at about same height percentile)
  sex steroids (especially E2) cause maturation of epiphysial plates leading to cessation of growth
      if produce too much: maturation will occur early and growth stops early
      if produce too little: maturation will occur late and growth stops later
  measuring bone age radiographically (by looking at epiphysial fusion in hand and wrist) can give an
     indication of how much growth potential is left
  seasonal differences in growth rate (highest in summer) means that you need to take measurements
     over whole year, not just over half as favoured by some practitioners
  there are two basic phases of growth that occur postnatally:
      age < 2: almost asymptotic, very rapid carryover from intrauterine growth, more determined
            by nutrition than (both post- and prenatal) than hormonal
           age > 2: childhood growth pattern, decreasing curve, most sensitive to genetic endowment (ie.
            height of parents) and hormonal factors




Hadley Wickham                                      43                                               16/06/11
Clinical
      Familial Short Stature
           annual growth rate normal
           height at or below 3rd percentile
           no systemic or endocrine malfunctions
           pubertal growth spurt at normal age
           skeletal age = chronological age
           ancestors relatively short
      Constitutional Growth Delay
           height at or below 3rd percentile
           annual growth rate < 5 cm per year
           no systemic or endocrine malfunction
           pubertal and bone age delay
           positive family history (more often occurs in males than females)
      Taking Account of Genetic Potential
           because there is an average height difference of 13cm between males and females you must
            take account of that when estimating expected height (average of adjusted parental heights)
      Non-Endocrine Causes of Short Stature
           genetic endowment
           target tissue defects
             intrauterine growth retardation (20% won‟t catch up)
             bone and cartilage disorders
           chromosomal defects
             Turner‟s (most important)
             thyroid hormone deficiency
             GH deficiency

Lactation
Development
    develop on ventral surface of all mammals, most with a line of nipples (up to 18)
    some women have extra/ectopic breasts along the embryological milk line
    glands develop in both sexes, but are very poorly developed in males
    same pattern of development in all mammals
           paracrine influences responsible for growth
           resemble sweat glands and develop in a similar manner (branched ducts with secretory and
            myoepithelial cells)

Resting Gland
  very little development as gland until puberty

Hadley Wickham                                      44                                               16/06/11
  surge in E2 at puberty triggers growth (duct growth , connective tissue , breast growth )
      many females experience a cyclic change because of hormones of menstrual cycle
      testosterone inhibits in males, but sometimes high levels of E2 in males stimulates
            gynacomastia
  only becomes fully developed after parturition
  described as a tubuloalveolar gland
      15-20 irregular lobes radiate back towards and one duct opens onto mamillary papilla (nipple)
      further subdivided into lobules by dense connective tissue and, in younger women, adipose
            tissue
           composed of rather loose connective tissue (allows expansion; from papilla) around branches
           lactiferous duct lined with cuboidal epithelium (2-layered in ductal region, 1-layered in
            lobules)
           just prior to opening, sinus/ampulla stores milk prior to release
           myoepithelial cells are flattened cells with flattened nuclei wrapped around the outside of
            glands

Growth During Pregnancy
  glands , connective tissue , lobules become more defined
  hormones act synergistically, actions of individual hormones are uncertain
      E2 + GH + steroids  duct growth
      E2 + P4 + PRL + GH + steroids  lobuloalveolar growth
      PRL + steroids  milk secretion
  PRL is dominant hormone, with a regulatory effect
  hormones also affects foetus, occasionally causing milk production (witch‟s milk)
  secretory cells start to become quite active by 3rd trimester and secretory product starts to
     accumulate

Milk
       Colostrum
           1st type of milk produced for the first few days
             proteins and vitamins , Na+ , Cl- , lactose , lipids , K+ 
             has laxative property (important in activating gut)
             also contains a few lymphocytes which aid in protection
       Normal Milk
           lipid , alveolar cells become packed with lipid
           myoepithelial cells change shaped according to stage
           ducts , connective tissue , alveolar cells 
       Galactopoeisis
           dependent on PRL (acts directly on cells at transcription level)
           milk protein mRNA 
           P4 inhibits production of α-lactalbumin, essential cofactor for lactose synthetase

Hadley Wickham                                      45                                                    16/06/11
      Post Partum
                E2 , P4 , milk production 
                initially high levels of PRL which decrease with time, but increase with suckling
                over time response decreases, but sensitivity to PRL increases
      Milk Removal
                “let down” controlled by oxytocin (released by hypothalamus)
                can be stimulated by sucking (reflex) and conditioning

Milk
  humans are the only species that drinks another species milk
  usually baby is weaned (immediate transition from milk to solids) when it starts to grow teeth and
     never drinks milk again
  baby normally survives on its on fat stores until mother starts producing milk, at about day 3
  number of differences between human and cows milk
                                    Human           Cow
        Ca2+/P                   2.5         1.5
        (5x more in cow)

        Lactose                  70 g/L      50 g/L
        Protein                  10 g/L      30 g/L
        Fat                      40 g/L      40 g/L

                protein is much higher than in human milk, due to the much higher concentration of casein
                 (proteins that precipitate under acidic conditions, whey, carries Ca2+ and P)
                                                   Human          Cow (g/L)                                     Notes
                                                    (g/L)
                           casein            4                25
                           α-lactalbumin     1                3
                           β-lactoglobulin   0                3                can be source of allergy
                           albumin           0.45             0.55             comes directly from plasma, where level in 50g/L
                           lactoferrin       2                0                binds iron very tightly, bacteriostat
                           IgG               0                0.6
                           IgA               1.1              0                absorbed by neonates, very important in resistance to infection,
                                                                               colostrum contains very high quantities
                           lysozyme          0.4              0                breaks down bacterial cell walls, bacteriostat
                           hexosamine        small quantity   0                essential nutrient for growth of Lactobacillus bifidus, which converts
                           polysaccharide                                      lactose to lactic acid in the neonatal gut protecting against many
                                                                               pathogenic organisms

                  caseins form into micelles using non-covalent bonding, with one molecule of κ-casein in
                           the centre
                  contain very high proportion of phosphorylated SER and THR
                  generally species that need to grow fast have high Ca2+ and P, therefore high casein
                           content,
                  humans don‟t require as much protein, Ca2+ and P because growth is much slower (eg. cow
                           reaches sexual maturity at 1, humans at 13)
                  immature liver can‟t cope well with high protein content
                not really any difference in fat composition (although cows do have a higher percentage of
                 SCFA‟s)
                lactose only sugar found in milk, and lactose only found in mammalian milk
                  sucrose about 5x sweeter than lactose
                  formed by GAL and GLU linked by β(14) linkage
Hadley Wickham                                                                46                                                                        16/06/11
             specific enzyme system (ie. β galactosidase) required for degradation

Milk Lactose
  lactose is main CHO in milk
      Synthesis
           synthesised by galactosyl transferase: UDP-galactose + glucose  lactose + UDP
           UDP-galactose is made from glucose in plasma
      Induction of Synthesis
           lactose not synthesised in non-lactating mammary tissue, despite presence of adequate levels
            of galactosyl transferase
           normally, galactosyl transferase has low affinity for glucose (Km = 1.4 M) but high affinity for
            N-acetyl galactosamine
           after start of lactation, α-lactalbumin production is induced in mammary tissue by prolactin
           α-lactalbumin forms a loose, dissociable 1:1 complex with galactosyl transferase increasing
            its affinity for glucose (Km = 5 mM)
           results in rapid switching on of lactose synthesis
           most of α-lactalbumin spills over into milk, forming one of components of whey
      Lactose Utilisation in Neonate
           lactose hydrolysed to galactose and glucose by lactase (=β-galactosidase) in brush border of
            jejunum
           both glucose and galactose can then be metabolised to glycogen in the liver, but galactose is
            preferentially used because UDP-glucose is on the glycogen synthesis pathway
           hypothesised that galactose is to provide long-term energy sources, while glucose provides
            short-term energy
      Lactose Intolerance
           mutation arose about 60,000 in most dairying cultures, high selective advantage led to high
            population occurrence
           adult humans belonging to races that were historically dairying in lifestyle are mostly lactose
            tolerant
           lactase is either present or can be induced in these subjects
           in lactose-intolerant subjects lactase is absent and lactose remains undigested
             it passes into the large bowel where it causes diarrhoea via its osmotic effects
             is also fermented by bacteria into lactic acid causing flatulence and discomfort
           widespread in Pacific Island nations
           lactose common in foodstuffs, as milk is major ingredient in many convenience foods

Lipids in Milk
  triglycerides make up most of the fats in milk
       about 50% are synthesised de novo in mammary tissue
       remaining 50% come from fats in the diet
  lipoprotein lipase activity increases in lactation


Hadley Wickham                                      47                                                  16/06/11
      Lipid Digestion in Neonate
           60-70% of fats are digested in the stomach of the neonates
           Pancreatic Lipase
             activity low at birth, due to incomplete pancreatic development
           Lingual Lipase
                secreted by serous glands of tongue
                induced by suckling
                relative specificity to SCFA‟s which can be absorbed across stomach
                completely hydrolyses triglycerides
           Gastric Lipase
             produced by gastric mucosa
             may be identical to lingual lipase (both have broad specificity, required no cofacters and
                 have acid pH optimum)
           Bile-salt Stimulated Lipase
                produced by mother, high concentration in milk (100 mg/L)
                found only in primate milk
                activity against water-soluble, micellar and emulsified lipids
                activated by bile salts with 7α-hydroxy group
                completely hydrolyses triglycerides

Hormones and Contraception
  factors that determine the fertile period are the survival of sperm (~6 days) and the survival of eggs
     (~24 hours)

Factors Influencing Sperm Survival
    endocrine changes
    cervical mucus
    sperm number and quality
    contractile activity of reproductive tract
    presence of antibodies against sperm
    infection
      Cervical Mucus
           E2  (after menstruation) mucus , viscosity , sperm penetration 
           E2  mucus , viscosity  (water content , changes in content, channel diameter)
           E2 , P4  (prior to menstruation) profound, sudden change, volume , viscosity 
           sperm can only move through and survive in oestrogenic mucus
           used as natural family planning method
      Phases of Sperm Transport
           Phase 1 – rapid transport (5 min, no role in fertility)
           Phase 2 – formation of sperm stores in cervical crypts


Hadley Wickham                                         48                                              16/06/11
           Phase 3 – slow release of sperm from crypts

Contraception
  the pill has two main functions: decrease FSH & LH and make hostile mucus
  both progestagens and oestrogens used

Puberty
  puberty = phase of bodily development during which gonads secrete hormones in amount sufficient
     to cause accelerated growth of genital organs and appearance of secondary sexual characteristics
      Events
           growth and maturation of primary sexual characteristics
           secondary sexual characteristics appear
           growth spurt
           psychological changes
           fertility ultimately achieved
      Terminology
             Term                                Meaning
        Adrenarche       rise in adrenal androgen secretion
        Pubarche         onset of pubic hair development
        Thelarche        onset of pubertal breast development in female
        Menarche         onset of menstruation
        Spermarche       onset of completed sperm production
        Gynaecomastric   palpable or visible breast tissue in male


Regulation of Onset of Puberty
  during fetal life and early infancy, few constraints on axis
  by early childhood HPG axis is effectively inhibited by CNS inhibitory centre
       become very sensitive to small amounts of sex steroids
  by early puberty hypothalamus is released from restraint (mechanism unclear)
       hypothalamus and pituitary also become less sensitive to effects of sex steroids
  early puberty characterised by pulsatile LH and FSH secretion at night
  mid puberty both baseline and pulsatility increase
  late puberty results in increased pulsatility and secretion during the day and night
      Inhibin and Activin
           stimulated by FSH and have a negative feedback effect
           inhibin is a dimer with α and β chains, and has two forms A & B
           increases in early puberty (only inhibin B in pubertal males)
           activin A & B are dimers of inhibin subunits, stimulated by FSH
      Gonadostat
           sex steroid inhibitor
           GABA appears to be potent inhibitor of GnRH pulsatile secretion
           in prepubertal monkeys GABA-blockers  GnRH


Hadley Wickham                                                       49                                 16/06/11
           GAD catalyses conversion of GLU to GABA, and is found in median basal hypothalamus
      Leptin & Puberty
           leptin is an adipostatic hormone (leptin  with fat mass)
             limits obesity through CNS
           may be signal to influence timing of puberty
           obese children have early puberty, anorexic children have delayed puberty
           levels peak just prior to onset of puberty in females
           leptin deficient mice never achieve puberty, and leptin treatment induces puberty

Male Puberty
  increased testicular size first physical evidence of puberty in boys (11.5 – 12 years)
  sperm found in urine of normal boys in early puberty, even long before sexual maturation complete
  maturation of genitalia divided into 5 stages
      penile enlargement begins at 11.5 years
      scrotum begins to enlarge and increase in rugosity with darkening of skin
      development of genitals may take between 2 – 5 years to complete
      boys with delayed puberty tend to have slower progression
  pubic hair usually begins to appear shortly after genital development begins
  axillary and facial hair occurs later
  voice beings to break at 14.5 years and is definitely broken 1 year later
  sperm production usually occurs when 13
      precedes peak of growth spurt, achieve at testicular volume of < 10 mls
      first detectable in first morning urine specimen

Female Puberty
  vagina beings to increase in length before secondary sex characteristics appear, continue to grow
     until menarche or later
  during premenarchal period mucosa of both vulva and vagina becomes softer and thicker
  with approaching puberty mons pubis increases in size by gradual deposition of fat, labia major
     enlarge
  prepubertal uterus has body:cervix ratio < 1, reverses with puberty
  first clinical evidence of puberty in girls is breast development
       mean age of onset is about 11 years
       breast growth may being asymmetrically and takes between 2-6 years to complete
  pubic hair development usually beings within bout 6months of breast development
       due to increased secretion of androgens from adrenals and ovaries
  menarche usually late event, about 2 years after onset of breast development
       age determined by environmental and genetic factors
       improved nutrition though to have been factor in declining age at menarche
       commonly adolescent girls have irregular menses for a year or more after menarche
Hadley Wickham                                      50                                                 16/06/11
Body Composition
  prepubertal males and females have same lean, skeletal and fat mass
  after puberty:
      lean mass of males 1.5x that of females
      fat of females (generalised) 2x that of male (truncal)
      hips enlarge in female
       BMD at puberty, peak bone mass reached at end of puberty

Infertility
  infertility = failure to conceive after 12 months of regular unprotected sex
  affects 1 in 6 heterosexual couples
  45% of problems are female, 25% male, and 10% unexplained/combined

Investigations
      Female
            ovulating? (hormone levels etc)
            tubes open? (laparascopy/histosalpinography)
            PCT (sperm survival in mucus after intercourse)
      Male
            semen analysis (number, motility, morphology, antisperm antibodies)

Causes
      Poor Sperm
            varicose veins in testicle (poor heat dispersion)
            drugs, chemicals, insecticides
            stress, smoking, alcohol (dubious)
            blocked tubes, infection, injury
            genetic
            surgery
            antibodies
      Ovulation Failure
            genetic
            body weight (only need to lose/gain 3 kg to restore)
            stress
            unknown
      Blocked Fallopian Tubes
            IUD
            abdominal surgery
            pelvic infection (eg. appendicitis, PID)


Hadley Wickham                                          51                         16/06/11
      Fertilisation Failure
            antibodies (to sperm head, prevent acrosome reaction)
            mucus
            egg/sperm binding
            subtle interactions
      Uterine Problems
            endometriosis
            miscarriage (insufficient endometriosis(

Treatment
      IVF
            drugs (FSH/LH)  hyperovulation (~10 eggs released)
            given hCG when 3 follicles are >18 mm to trigger release (~36 hours later)
            eggs collected transvaginally and incubated for 6 hours
            eggs inseminated by about 50,000 sperm/egg
            18 hours later pronuclei should be visible, and 1 day later should be at 2-8 cell stage
            maximum of 3 (usually 2 if <38) transferred back into uterus
      ICSI
            better pregnancy rates if mid piece struck
      Female
                        Cause                               Treatment
        anovulatory                          drugs (GnRH, FSH)
                                             lifestyle change
                                             weight gain/loss
        polycystic ovarian syndrome (PCOS)   drugs
        blocked tubes                        surgery, IVF
        cervical problems                    AIH (no good for male infertility)
        endometriosis                        drugs/IVF (pregnancy rate low)
        premature menopause                  donor egg
        uterus absent                        surrogacy

      Male
                        Cause                               Treatment
        azoospermia                          donor insemination
        oligospermia                         ICSI
        CAVD                                 ICSI

      Unexplained
                        Cause                               Treatment
        unknown aetiology                    mild stimulation and IUI
                                             (intrauterine insemination)
                                             IVF/GIFT

      Alternatives
            adoption (number decreasing)
            “moving on” and accepting childlessness

Menopause
  menopause = last episode of natural menstruation a woman experiences

Hadley Wickham                                                  52                                     16/06/11
           commonly occurs between 50-52
             but 5% enter before 39 and 5% after 50
             if occurs before 38, termed premature menopause and is pathological
           timing independent of racial background in well-nourished populations
           poorly nourished women experience early menopause
           cigarette smokers tend to have an early menopause (~2 years) and shorter transition period
  consequence of ovaries running out of follicles
      quota of follicles established in fetal life
      atresia and ovulation subsequently results in exhaustion of reservoir of follicles and ovarian
            function ceases
  menopause usually preceded by period of menstrual irregularity with increase incidence of
     anovulatory cycles
           terms used for this period and for other events associated with menopause are:
           premenopausal – 40+, menstruates regularly
           postmenopausal – period after menopause
           menopausal transition – period between first irregular menstrual cycle and menopause
           perimenopause – time of erratic hormone fluctuations which starts at beginning of
            menopausal transition and continues through out ovarian senescence and ends about 1 year
            after menopause
  entry in menopausal transition normally occurs about 4 years before menopause
  between 5-10% don‟t experience transition phase

Hormone Changes
  depletion of follicular reserves associated with profound changes in hormone production by ovary,
     and secondarily changes in pituitary gonadotrophin secretion
  ovarian secretion of inhibin begins to decline well before menopause starts (in late 30‟s)
      accompanied by rise in circulation FSH
      suggested that higher amount of FSH stimulates more primordial follicles to enter growing
            pool, accelerating depletion of reserve
  onset of transition marks progression to stage where normal processes of follicular maturation and
     ovulation become disturbed
           extensively investigated in NZ women
           most characteristic feature is sporadic appearance of high levels of FSH and LH
           high levels occur with increasing frequency with approach of menopause
           cycles longer than 50 days invariable bean with prolonged period of high FSH/LH levels
            usually associated with low E levels
             no evidence that women wasn‟t post menopausal until E levels began to rise
             if E sustained, ovulation subsequently followed
             if E transient, menstruation delayed until sustained rise
           also some cycles paradoxically have both raised FSH/LH and E
  cycle length has been shown to be related to ovulation status of cycle

Hadley Wickham                                        53                                             16/06/11
             in 200 women aged 40-55, 89% of 20-35 days, 32% of 36-49 days, 26% 50+ days
             in 322 women age 10-24, 64% of 20-35 days, 62% of 36-49 days, 43% 50+ days
  no simple endocrine test for menopause
      marked fluctuations in hormone levels can continue during at least 1st 6 months after
              menopause
             as general guideline, a women over 45 years who has been amenorrhaic for at least 12 months
              in 95% likely to be postmenopausal (over 50 years: 99%, over 55 years: 100%)
             on average a 40 year old women has ¼ of fertility of 25 year old and 4x miscarriage rate

Postmenopausal Oestrogen Production
  by ~1 year post-menopause ovary has essentially ceased producing E
      amount of E produced daily less than 1/10 of pre-menopausal woman
      circulating E2 levels accordingly very low and show little day-to-day variation
  E in post-menopausal woman arise almost entirely from aromatisation of androstenedione in adipose
     tissue
             E3 main product but some is converted to E2
             women with more fat are likely to produce more E and suffer fewer E-withdrawal side effects

Perimenopausal Syndrome
  most women experience variety of clinical symptoms of E and perhaps P deprivation
     usually follow onset of menopausal transition but may precede it and sometimes continue for
              a time after
             include vasomotor (especially hot flushes and night sweats), genito-urinary (reflecting
              atrophic changes) and muscular symptoms
  disturbed emotional and psychological conditions such as nervousness, anxiety, irritability,
     depression, forgetfulness and decreased libido may also be experienced
             may not be directly attributable to hormonal deprivation but also stress from family and social
              problems commonly experiences by middle-aged women
  term climacteric relates to physical and emotional symptoms associated with perimenopause
      Osteoporosis
             E has important role in bone metabolism (osteoblasts ) and after menopause bone density
              and strength decrease
              Risk Factors
                 white, elderly female
                 thin, slender physique
                 early menopause
                 familial incidence
                 smoking, alcohol
                 sedentary, low Ca intake




Hadley Wickham                                       54                                                  16/06/11
Ageing
Characteristics
    progressive, universal, irreversible
    for humans, probability of dying doubles every 8 years
    mean life span is distributed normally
    in past 100 years health care has reduced mortality in 1st 30-40 years but has not increased total
     lifespan
      Diseases that cause death and related to ageing
           diseases part of ageing process (eg. atherosclerosis, HT)              PUI
           diseases that show increasing incidence with age (eg. neoplasm)              not PUI
           diseases that have increasing consequences with age (eg. accidents)not PUI
      Diseases that are PUI (but need not be fatal and not always associated with age)
           degenerative joint disease (osteoarthritis)
           emphysema
           osteoporosis

Physiological Changes
  over entire population general run down of all systems (GFR , systolic bp )
      peak at 15-20 years old
      must be careful looking at population extrapolations because data is very variable
      looking at individuals there are populations with different rates of decline
  age  moisture  fibrousness  collagen cross-linking  permeability  perfusion 
  only proven way to reduced ageing is through caloric restriction (<30% normal calories)
      Theories
           programmatic: ageing due to inherent genetic damage
                genetic pleiotropy – ageing in evolutionary and selection terms
                neuro-endocrine-ontogenetic – OK for vertebrates
                rate-of-living – fixed amount of energy used by member of species
                immunobiological – age-dependent decline in thymus dependent system
           stochastic: ageing results from environmental damage
             free-radical
             amino-acid racemisation, non-enzymatic glycosylation
             somatic mutation and error accumulation
           in reality ageing probably involves both




Hadley Wickham                                          55                                                16/06/11
Metabolic Endocrinology
Adrenal Cortex
  adrenal glands play vital role in maintaining body homeostasis and reaction to acute and chronic
    stress
  each gland consists of inner medulla (catecholamines) and outer cortex (steroids)
  adrenal cortex composed of three zones
      zona glomerulosa (outer)
      zona fasciculata (middle)
      zona reticularis (inner)
  differences in steroidogenic enzymes results in production of different hormones in different zones
                Zone       Secretory Product
           ZG          aldosterone
           ZF          cortisol
                       DHEA (minor)
           ZR          DHEAS
                       cortisol (minor)

  cortisol and aldosterone essential for life

ACTH
            regulates growth and function of adrenal cortex
            increases steroid production by stimulating conversion of cholesterol to pregnenolone
            simple peptide (only 25 of 39 aa necessary for biological activity, contains all essential aa‟s)
            regulated by cortico-releasing hormone (CRH), which has diurnal rhythm with peak just
             before waking
            most types of stress stimulate production of ACTH

Cortisol
  biosynthesis and secretion of cortisol regulated by ACTH, tightly regulated by negative feedback
  has circadian, episodic rhythm
      rhythm determined by rhythm of CRH
      circadian rhythm gets disturbed if travelling and takes about 1 day/hour to readjust
      peak just before waking prepares body for stresses of day
  part of protective mechanism against stress
      Levels
           Production rate                         22 – 70 μmol/day
           Serum levels                            200 – 700 nM
           Urinary free cortisol                   60 – 300 nmol/day
           Urinary 17 OHCS                         5 – 28 μmol/day
           Urinary 17 ketosteroids                 Men:      25 – 90 μmol/day
           (including metabolites of cortisol,
           androstenedione and testosterone)       Women:    14 – 50 μmol/day

      Hormonal Actions
           gluconeogenesis 
           lipolysis 
           K+ secretion 
           stabilises cell membranes
           anti-inflammatory (inhibits phospholipase A2, which attacks membranes for arachidonic acid)
           immunosuppresive
           white blood cells  T & B cells 
           bone formation 

Aldosterone
  principle function is maintenance of Na+ and K+ balance
  Na+ excretion  K+ excretion 
  controlled by renin-angiotensin system
      kidney sensitive to changes in Na+ and K+, blood volume, arterial pressure and renal flow rate
      responds by secretion of renin
      renin catalyses conversion of angiotensinogen to angiotensin I
      angiotensin I converted to angiotensin II in lungs by angiotensin converting enzyme (ACE)
      angiotensin II acts on zona glomerulosa to stimulate synthesis of aldosterone
      aldosterone synthesis also stimulated directly by elevated serum K+
      Levels
           Production rate: 140 – 700 nmol/day
           Serum levels:                        140 – 560 pM
           Urinary aldosterone: 14 – 50 nmol/day

Dehydroepiandrostrone Sulphate (DHAS)
    quantitatively largest steroidal product of adrenal cortex
    no specific hormonal action yet identified
    may be used in very minor way as precursors for testosterone and oestradiol production in gonads
    have maximum levels in late teens/early 20‟s then slowly decline
      Serum Levels
           DHEA:                                7 – 40 nM
           DHEAS (men):                         3 – 9 μM
           DHEAS (women):                                2 – 7 μM
           Androstenedione: 3 – 11 nM
Hadley Wickham                                                            58                        16/06/11
Adrenal Medulla
  SNS always activated as a whole (A released from adrenal medulla and NA released at individual n.
     endings)
  A involved with “fight or flight” reaction
  necessary for development (congenital absence = death, replacement only leads to survival until
     weaning)
  80% of adrenal medulla is chromaffin cells (so named because of affinity for chromaffin) dedicated
     to secreting A (80%) and NA (20%)
           very committed for hormone storage (20% dry weight)
           high ATP concentration in granules
             complexes with catecholamines to reduce osmotic pressure
             when released with NA and A has extracellular effects (like neurotransmitter)
           also common opioid compounds (enkephalins)
           nerve stimulation (via Ach) increases production (via  mRNA) and activity (via
            phosphorylation) of tyrosine hydroxylase (rate limiting step)

Synthesis
    TYR is taken into the cell form bloodstream
    converted to DOPA by tyrosine hydroxylase
    converted to dopamine by dopa decarboxylase
    transported into chromaffin granule by active transport using proton antiport
    converted to NA by β-hydroxylase (process stops hear for sympathetic neurons)
    leaks out into cytoplasm, and converted to A by PNMT
    transported back into granule by same transporter as dopamine

Release
    Ach  - Ca2+ , granules move from cytoplasm to plasma membrane
    membranes fuses
    contents released into bloodstream
    vesicles reinternalised and reused (otherwise cell surface area would dramatically increase)

Regulation
  tyrosine hydroxylase and dopamine β-hydroxylase induced by nerve stimulation
  PNMT has different properties dependent on location
      adrenal medulla blood supply very rich in glucocorticoids, which hugely upregulate PNMT
      A produced instead of NA

Removal/Degradation
      Specific Reuptake
           Na+ dependent family of transporters specific to uptake of dopamine, serotonin and NA
           found close to sites of release


Hadley Wickham                                     59                                                16/06/11
             number of antidepressant work by blocking catecholamine reuptake
      Specific Metabolism
             most urine metabolites have been metabolised by two enzymes
             might request analyses for VMA (vanillyl mandelic acid) if looking for phaechromatosis
             Catechol-o-methyl transferase (COMT)
               methylates catecholamines at 2nd hydroxyl group on benzene
               found in cytoplasm of most cells
             Monoamine Oxidase (MAO)
               oxidises terminal carbon to carboxylic acid, removing NH2
               found inside mitochondria

Effects
  variety of adrenergic receptors
      two classes: α and β
      typical G-coupled receptors with 7 membrane spanning regions
           α1 – phospholipase C 
           α2 – adenyl cyclase , cAMP 
           β – adenyl cyclase , cAMP 
                     α1                                  α2                               β1                         β2
       glycogenolysis                     smooth muscle relaxation         lipolysis                 gluconeogenesis 
       smooth muscle contraction           smooth muscle contraction        myocardial contraction    glycogenolysis 
                                           lipolysis                                                  insulin , glucagon , renin 
                                           renin release                                              smooth muscle relaxation
                                           insulin secretion 
                                           platelet aggregation 

  tissue may have apparently contradictory responses due to different receptor subtypes present
  much more complicated in the CNS (involved with subtle blood pressure regulation and appetite)

Calcium Metabolism
Regulation of Calcium
  calcium levels in blood are very tightly regulated, even though only very small amount of total body
     calcium is present in them
                Location              Amount
         Bone                      99% (1300 g)
         Muscle                    0.3%
         Extracellular fluids      0.7%
         Plasma                    0.05%

  important type of calcium in plasma (2.3-2.6 mM) is ionised, others aren‟t involved in biological
     activity to any extent
             Calcium in Plasma               Amount
         Bound to albumin                  35-50%
         Ionised                           50-60%
         Organic complexes                 5-10%

  ionised calcium maintained at a very constant level with only 4% overall variation (cf. Na+ 9%,
     glucose 35%)

Hadley Wickham                                                         60                                                               16/06/11
  several pools of calcium exist within bone, can be divided into two basic pools:
      slowly exchanging (99%)
      readily exchanging (1%, ~13g)
  bone histomorphometry = give fluorescent derivative of tetracycline (laid down in bones), wait a
     couple of weeks then give another which fluoresces at a different wavelength, take bone biopsy and
     look
  about 1% of skeleton turns over per year (40 mg/day)
      variable between bone (ribs: very high, long bones: moderate, skull: minimal)
      turns over more rapidly in areas of stress
  plasma calcium turns over 3-4x per day
      Ca2+   tetani (spontaneous muscle contractions, especially of thumb)
      Ca2+   malaise, dehydration, generally non-specific symptoms
  poorly understood pseudoequilibrium between Ca2+ and PO43-
      [Ca2+][PO43-] = constant
      equilibrium maintained at expense of bone
  controlled via parathyroid hormone (PTH)

Parathyroid Hormone
  84 aa protein (two structured domain, 1-30 and 50-84 linked by region with little tertiary structure)
      secreted as 120 aa. protein but N-terminal cleaved to activate it
      only first 30-34 aa. required for biological activity (short derivatives used clinically)
  very rapidly cleaved in the circulation by liver and kidney (shown with radio-iodinated bovine PTH)
      C-fragment hands around for a while, no-one knows what it does but it is highly conserved
            and has its own receptor
           N-fragment (active) rapidly lysed (20x more rapidly than C-terminal)
             in radioimmuno assay, antiserum may bind preferentially to one fragment or the other
             means you can‟t really compare PTH levels between two different labs
  PTH-R has 7 transmembrane domains (ie. classical G-protein coupled receptor)
      activates two G proteins (adenyl cyclase and phospholipase C)
  takes about one day to work (but G-protein coupled receptors only take ms)
           thought to be through cAMP gene regulation
  very limited stores (~1 hours), synthesised as needed
  regulation independent of hypothalamus and pituitary
  malfunction usually related to incorrect set-point
      Actions
           acts to increase plasma Ca2+ levels
             bone breakdown 
             urinary excretion of Ca2+ and PO43- 
             activation of vitamin D 


Hadley Wickham                                    61                                                  16/06/11
           Actions on kidneys
                instantaneous: urinary cAMP from kidney  (through  PIP3)
                8 min – 2 hr: 10-fold increase in excretion of PO34-
                3 – 6 hr:      activation of 1α-hydroxylase, 1:25 DHCC 
                12 hr – 2 days: Ca2+ excretion  (through increased tubular resorption)
           Actions on bone
                instantaneous: cAMP in osteoblasts 
                5 – 30 min:    uptake of Ca2+ 
                6+ hrs:        hypercalcaemia (through increased osteoclastic resorption)
                days – weeks: low levels elevated chronically enhance bone growth
                PTH has no direct actions of osteoclasts which have no PTH receptors so must be mediated
                 by osteoblasts
      Hyperparathyroidism
           high levels cause massive bone resorption  osteitis fibrosa cystica
           Symptoms of Hypercalcaemia
                often asymptomatic
                general malaise, fatigue, psychoneurosis, weight loss, pruritus
                renal colic, polyuria-polydipsia
                constipation, epigastric pain, anorexia, nausea, vomiting
                lethargy, muscular weakness, confusion, psychosis, stupor, coma
           Causes
             most common (primary): tumour of parathyroid
             hypercalcaemia of malignancy: generation of PTH-rP by tumour
             secondary: chronic renal disease, pseudohypothyroidism)
      Hypoparathyroidism
           idiopathic: defective secretion or PTH, low Ca2+, episodes of tetany, treated effectively with
            vit D
           pseudohypoparathyroidism: defect in PTH receptor or receptor coupling to Gs

Parathyroid Hormone Related Peptide (PTH-rP)
    141 aa protein, striking homology with PTH at N-terminus (although not past 34th aa)
    homology corresponds to biological activity, and PTH-rP has very similar actions to PTH
    null mice are very small with overdeveloped bones (rib cage so small that they can‟t breath)
    hypothesise to prevent the too rapid mineralisation of cartilage to bone
    mice only expressing PTH-rP in epiphyseal plates mice develop normally, except for a failure of
     mammary tissue to grow
  often secreted by tumours (unlike PTH)
      Physiological Role
           found in normal skin (in keratinocytes and inner root sheath of hair follicle), role unknown
           acts as calciotrophic hormone in fetus, may maintain fetal Ca2+ levels


Hadley Wickham                                       62                                                16/06/11
Vitamin D
  there is circulation of sterols in the skin (ensuring see sunlight even in heavily pigmented skin)
      5-dehydrocholesterol converted to pre vitamin D3 by sunlight
      most vitamin D present as 25-OH Vit D3 (no biological activity), carried by a special protein
  vitamin enhances Ca2+ uptake into bones
      wide seams of unmineralised bone seen especially in spongy bone very characteristic of
            osteomalacia
  calbindins increase by vitamin D
      Renal Metabolism
           only small amount (in order of ng/day) of 25-OH DHCC converted to 1:25 DHCC by
            hydroxylation in kidney
           most is converted to 24:25 and 25:25 DHCC, which have no biological activity
           relative activity of 1α-hydroxylase is increased by
                 plasma Ca2+
                PTH 
                PO34- 
                1:25 DHCC 
      Actions
           Nuclear Mediated
             binds to 427 aa receptor present in almost all cells (especially intestine and bone)
             induce transcription of specific genes
             specific Ca2+ binding proteins (calbindins) induced in both intestine and bone, along with
                 Ca2+-dependent ATPase and alkaline phosphatase
             oesteocalcin is induced in osteoblast
           Non-nuclear Mediated
             enhanced generation of ceramide (?), stimulating short-term stimulation of Ca2+ and PO34-
                 uptake
             other less specific effect described including growth of intestinal villi, calcification of
                 cartilage, amd immunomodulation acitivities
      Rickets
           Ca2+  epiphyseal plate growth , bone density 
           99% due to  vitamin D
           also caused by hydroxylation enzyme or nuclear receptor defects
           diagnosed through the form of Vitamin D that cures it

Calcium Clinical
  deviation of plasma Ca can cause/be result of a number of diseases:




Hadley Wickham                                       63                                                     16/06/11
              Generalised (Metabolic)                     Localised/Multifocal
       osteomalacia (±rickets)                  10 bone tumour (benign/malignant)
       osteoporosis                             metastatic disease
                                 0
       osteitis fibrosa cystica (1 hyper-PTH)   Paget‟s disease (osteitis deformans)
       renal osteodystrophy

  osteoblastic activity can be monitored through alkaline phosphatase levels, osteoclastic activity
     through breakdown products of bone

Hypocalcaemia
      Signs/Symptoms
             hypertonia
             anxiety, paraesthesia, cramps, diarrhoea
             laryngeal spasm (laryngismus stridus in children)
             opisthotonus (spasm of external muscles of spine), fits, hypertonia
             creatinine  (kidney‟s damaged by excess Ca)
             serum alkaline phosphatase (osteoblastic activity trying to compensate)
             Chvostek’s sign – relatively non-specific, tap facial n. observe response (1=twitch at corner
              of mouth, 2=twitch at eye, 3=twitch of side of face), demonstrates muscle hyperexcitability
             Trousseau’s sign – use blood pressure cuff to make limb ischaemia  tingling, thumb
              crosses palm (obstetrician‟s hand), hallmark of tetany, specific but rare
      Mechanism
             disturbances to muscle and nerve function
      Causes
             10 hypoparathyroidism
             Mg2+ deficiency
             vitamin D deficiency (eg. osteomalacia)
      Differential Diagnosis
             hysteria
             epilepsy
             hyperventilation (blood pH , Ca2+ )
      Treatment
             IV Ca2+
             PTH
             Vitamin D

Hypercalcaemia
      Signs/Symptoms
             nil
             thirst, polyuria (H2O and Na+ not flushed out)
             fatigue, depression, confusion
             anorexia, weight loss, constipation
             muscle hypotonia

Hadley Wickham                                                       64                                  16/06/11
           band keratopathy (long term) – rim of Ca around edge of cornea, interrupted and dense-
            looking
      Mechanisms
           disturbances in muscle and renal function
      Complications
           renal – Na+ loss, GFR  (reversible), nephrocalcinosis (irreversible), hypertension/CVS
           bone – rarefaction depending on aetiology
      Causes
           malignant bone disease (metastatic or humoral)
           10 hyperparathyroidism
           vitamin D intoxication
           granulomas (sarcoid, TB, leprosy), lymphomas (convert 25 MHCC to 1:25 DHCC)
           milk alkali syndrome

Bone Diseases
      Osteomalacia
           mineralisation of bone impaired
           affects bones and muscles (lack of vitamin D disturbs specific muscle functions)
           low Ca, low PO43- (indicative  PTH)
           impaired Ca absorption due to low Vitamin D
           may be caused by lack of sunlight
      Osteoporosis
           bone formation in balance between factors promoting growth and factors promoting
            breakdown
                            Bone Formation                    Bone Breakdown
                 External
                 sunlight/vitamin D                 no sunlight
                 calcium phosphate                  alcohol
                 exercise
                 Hormonal
                 GH, thyroid hormones, insulin,     PTH
                 vitamin D
                 sex hormones, calcitonin (reduce
                 breakdown)

           low post-menopausal oestrogen levels shift balance towards bone breakdown
           results in decreased bone density (quantitative abnormality of trabecular and cortical bone)
           Ca intake during childhood influences later bone mass (ie. low Ca  osteoporosis later)
           clinical definition of osteoporosis = bone density two standard deviations below mean for
            young people
           clinical definition of osteopenia = bone density between one and two standard deviations
            below mean for young people
           Histopathology
             thin trabeculi with trabecular perforations (microfractures  fragility )
             cortical bone affected to a lesser extent

Hadley Wickham                                                65                                        16/06/11
             wrist, then vertebrae, then hip affected
           Clinical Assessment
                family history (genetic factors involved)
                fracture history
                risk factors (low Ca intake in childhood, low exercise)
                bone density
           DEXA
             DEXA = dual energy X-ray absorptionometry (uses 2 x-rays with different intensities)
             obtains precise measurement of Ca (precision: 0.7% in vitro, 3% in vivo)
           Treatment
             why treat? – responsible much of morbidity and mortality in elderly, 5%  in BMD would
                 give 50% reduction in fracture rate
             calcium – (1200 mg/day), retards bone loss, shuts off PTH, take before bed-time (no Ca
                 intake during night)
             oestrogens – increases BMD 10% over 10 years but 5% increase in breast cancer (low
                 grade)
             bisphosphonate – impairs osteoclastic ability, additive effect when combined with
                 oestrogens
             vitamin D
             thiazide diuretics (decrease Ca urine loss)
             fluoride (bone strenght , resorption )

Glucose Homeostasis
Digestion, Absorption, Interconversion
    amylase then dissacharidases in gut break down complex CHO into component sugars
    absorbed and passed into portal blood stream and then travel to the liver
    major products are glucose (80%), fructose and galactose
    much of fructose and most of galactose concerted to glucose
    glucose is final common pathway for transport of almost all CHO to tissues of body

Transport Through Cell Membrane
  glucose able to pass through lipid cell membrane by facilitated diffusion down concentration
     gradient
  mediated by glucose transporters (GLUT), characterised at molecular level as family of proteins
  important forms include GLUT-2 (on liver and β-cells) and GLUT-4 (insulin sensitive, on skeletal
     muscle and adipose tissue)
  phosphorylation of glucose prevents it from leaving

Pattern of Overall CHO utilisation
  under normal circumstances most of dietary CHO stored in skeletal muscle as glycogen


Hadley Wickham                                         66                                            16/06/11
           implies that most glucose passes through liver to system circulation, from which it is taken up
            by skeletal muscle (via GLUT-4), phosphorylated and incorporated into glycogen
  transfer of glucose from UDP-glucose to glycogen subject to important hormonal regulation by
     insulin, glucagon and adrenalin
  20% of glucose are stored in liver as glycogen (direct pathway)
  very little glucose used directly by adipose tissue
  muscle lacks glucose 6-phosphatase so cannot release glucose back into the circulation
      energy is instead released through 3-C compounds, especially ALA and lactate
      pass to liver via bloodstream and are taken up and used for gluconeogenesis or fatty acid
            synthesis
           known as indirect (Cori) cycle and is thought be important in mechanisms leading to NIDDM

Role of Skeletal Muscle
  effect of insulin to lower blood glucose are mediated though two actions:
      stimulation of glucose clearance in skeletal muscle
      inhibition of glucose output from liver
  insulin acts by binding to a cell surface receptor
      activates intrinsic tyrosine kinase
      phosphorylates and activates series of further enzymes (IRS-1 and IRS-2)
      leads to complex phosphorylation cascade
  insulin though to act on skeletal muscle via receptor-mediate mechanisms to stimulate insertion of
     GLUT 4 molecules into surface membrane, increasing capacity of muscle to clear glucose from
     blood
  also stimulates incorporation of glucose into glycogen via stimulation of glycogen synthase
  in IDDM, lack of insulin means that these processes are not simulated, leading to in ability of
     muscle to clear glucose form blood
  in NIDDM, skeletal muscle becomes resistant to actions of insulin for reasons that are poorly
     understood, so insulin fails to normally stimulate uptake and clearance of glucose
           important clinically as it is sometimes necessary to give large doses of insulin in order to
            overcome resistance

Secretion of Insulin
  secreted from β-cells of pancreas in response to stimulation by glucose
      IDDM due to complete destruction of β-cells, leading to an absolute insulin failure
      NIDDM worsened by failure of nutrient coupled insulin secretion
  other nutrients such as ALA and LEU and some drugs (such as sulphonylureas) also stimulate
     secretion
  initial release of insulin by cephalic phase prior to meals
      seems to prime tissues to further effects of insulin, in response to raised blood glucose etc
      mediated by brain and ANS, important in maintaining normal tissue sensitivity to glucose
      one of the reasons insulin injected 20 minutes prior to eating


Hadley Wickham                                      67                                                     16/06/11
  molecular basis of stimulus-response coupling complex, but known to involve mobilisation of
     Ca2+and seems to be sensitive to intracellular glucose metabolism
  molecular mechanisms coupling insulin release to glucose include ATP-sensitive β-cell K+ channel
     and glucose-mediated stimulation of cyclic ADP-ribose

Role of Liver
  glucose uptake into liver occurs via liver-type GLUT and not stimulated directly by insulin
  driven by concentration gradient
  liver serves as major source of glucose production through:
       glycogen breakdown
       gluconeogenesis
  constant supply of glucose critical to survival of CNS, heart and RBCs and completely dependent on
     ongoing supply from blood
  glucose production increases in hypoglycaemia and starvation
      due to stimulation by glucagon and decreased activity of insulin
      in severe hypoglycaemia adrenergic agonists (A, NA) also play a role
      NA thought to protect against hypoglycaemic effects of exercise via direct effect to decrease
            insulin sensitivity of skeletal muscle an din starvation by stimulating lipolysis and supply of
            FFAs to tissues
  major pathway responsible for acute increase in hepatic glucose production is glycogenolysis
     if hepatic glycogen stores depleted hypoglycaemia will occur readily and be prolonged and
            severe
  if hypoglycaemia persists gluconeogenesis comes to play a part
  glucagon stimulates acute hepatic glucose production, through direct stimulation of glycogenolysis
     and inhibition of glycogen synthesis, and indirectly by counteracting effects of insulin
           also stimulates gluconeogenesis
           action mediated through cell surface receptor and subsequent receptor mediated stimulation of
            cAMP
  adrenergic agonists also stimulate hepatic glycogenolysis and glucose production both through
     cAMP and mobilisation of intracellular Ca2+ stores

Diabetes Mellitus
  commonest (~200 million) severe metabolic problem
  two major forms: insulin (IDDM) and non-insulin dependent (NIDDM)
  many other secondary forms (~400)

Therapy
      Insulin
           2 chain peptide hormone, produced by β-cells in pancreas
           must be administered subcutaneously otherwise it will be digested
             IM administration leads to much more rapid onset of action
             PK changes varying on injection site, best seems to be periumbilical region

Hadley Wickham                                      68                                                   16/06/11
           two-forms are produced commercially:
             short-acting: 10-15 min onset, 1 hr peak
             intermediate-acting: 4-5 hr onset, 12-20 hr peak, bound to slowly releasing semi-
                 insoluble complex
           standard replacement dose = 21-24 U/day (~25% more than normal due to suboptimal
            placement)
      Long-term Management
           education
           insulin and self blood glucose monitoring
           lifestyle modification (diet, exercise)
           support groups
           regular follow-up and complication screening
           special issues (childhood, adolescence, pregnancy)

Complications
  diabetes therapy aims to restore blood glucose to normal to prevent chronic complications, often
     difficult or impossible to reverse with medical therapy
      Vascular
           lead to most of morbidity and mortality associated with diabetes
           also associated with other diseases (eg. atherosclerosis)
           thought to be caused by exposure to high glucose levels (probably in small blood vessels
            through non-enzymatic glycosylation)
           Kidney: diabetic nephropathy
             proteinuria (especially albumin)
             nephrotic syndrome
             chronic renal failure
           Retina: diabetic retinopathy
                largest cause of blindest in age 15-60
                background retinopathy
                proliferative retinopathy (new vessel formation, intraocular haemorrhage, blindness)
                exudative retinopathy (macular oedema or exudates)
           Nerves: diabetic neuropathy
             frequently bilateral socking distribution
             progressive tingling, pain and numbness in feet associated with loss of temperature and
                 vibration sensation
             numerous less common forms
      Other
           increased incidence of infections (cellulitis, UTI, balanitis, vaginitis)
           diabetes associated skin lesions
           eye disease (glaucoma, cataracts)


Hadley Wickham                                        69                                                16/06/11
      Diabetic Ketoacidosis (DKA)
           end result of complete lack of insulin
           can occur either as 1st presentation of IDDM (sufficient for diagnosis) or in a patient where
            insulin isn‟t working
           “starvation in the midst of plenty” – body unable to use glucose so has to breakdown TG
            instead
           ketone bodies formed as by product
                5% acetone (characteristic odour recognisable by ~80%)
                15% acetoacetate
                80% β-hydroxybutyrate
                acetone and acetoacetate can be detected by reaction with nitoprusside (urine dipstick 
                 purple)
           Osmotic Diuresis
             hyperglycaemic results in osmotic diuresis with loss of water and electrolytes in urine
           Ketone Body Generation
             large amounts of ketone acids generated
             can‟t be cleared efficiently due to rate of production, accumulate in blood and contribute to
                 osmotic diuresis
             can get accumulation of large amounts of lipoprotien in blood with resultant excluded
                 volume effects
           Metabolic Acidosis
             accumulation of organic acids produced metabolic acidosis
             elicits respiratory compensation (tachyopnoea) [HCO3-]  pCO2 
             initially buffered by intracellular buffering with K+ and Mg2+ leading to loss in urine
           Progressive Dehydration
             with progressive dehydration fall in blood volume and onset of prerenal failure
             effectiveness of respiratory compensation falls, and patients become progressively
                 exhausted
             leads to 20 respiratory acidosis
             reduction in consciousness, followed by death with occur if process not reversed
           Treatment
                IV fluid and electrolyte replacement, + insulin and HCO3- in severe acidosis
                response usually good, but need for frequent monitoring of electrolyte levels
                raised blood levels of intracellular ions can mask severe whole body depletion
                when therapy starts to take effect, pH falls and renal output pick, can be rapid shifts of ions
                 (particularly K+) back into intracellular compartment
             may lead to life-threatening hypokalaemia (arrhythmia) unless replaced
             2nd metabolic complication may be presence of insulin resistance, so insulin levels need to
                 be lowered slowly and glucose monitored

Diagnosis
  based on demonstration of pathologically elevated serum glucose levels
Hadley Wickham                                        70                                                    16/06/11
  usually sufficient to demonstrate raised fasting or post meal blood glucose levels of 2+ separate
     occasions
           be careful of patient with IGT due to external factors (eg. thiazide diuretics, infection etc.)
  oral glucose tolerance test (oGTT) only necessary when diagnosis is unclear (~10%)
                           Fasting         2 hr
        Normal        <6             <8
        Diabetes      >8             >11
        Pregnant      <9             <9

  more stringent definition applied in pregnancy because milder elevation can cause fetal damage
  diagnosis is sometimes obvious (eg. DKA coma) but is often obscure especially in type 2
      Risk Factors
           obesity
           older age
           positive family history
           racial
           random finding of glucosuria
           present with known complication
           woman with history of large babies (>4.5 kg)
      IDDM (Type 1)
           previously called juvenile onset diabetes
           10% of diabetes mellitus
           die within 24-72 hours without insulin
           peaks of incidence in Caucasians at age 5 and early adolescence
           familial
           interaction between poorly defined environmental factors (eg. viruses)
           molecular genetic basis beginning to be understood
      NIDDM (Type 2)
           previously called adult or mature onset diabetes
           90% of diabetes mellitus
           about half take insulin, but it is not required for life
           IGT develops into NIDDM in ~25% of cases
             associated with prevalence of large vessel disease but not diabetes
           IGT considered as gestational diabetes mellitus (GDM) during pregnancy
             doesn‟t exist before, starts during and because of pregnancy and ends ~6 weeks after birth
             associated with increased risk of maternal (eg. pre-eclampsia) and fetal (eg. macrosomia)
                   complications
             30% develop into NIDDM
             risk and severity increases with successive pregnancies
           Disease Mechanisms
             mechanisms remain obscure, but characterised by 3 pathophysiological findings:

Hadley Wickham                                        71                                                      16/06/11
             insulin resistance (muscle, liver) – not due to structurally abnormal insulins, insulin
                 receptors, glucose transporters
             abnormal secretion – loss of 1st phase of secretion, selective glucose blindness of islets,
                 possible role of defective glucokinase
             islet amyloid – present in majority, abnormal deposits of amylin adjacent to β-cells,
                 probably disrupt architecture and late deterioration of insulin secretion
           Treatment
             similar to IDDM
             5-10% successfully treated with diet/exercise alone
             use sulphonylureas to increase sensitivity to glucose
           Complications
             non-ketotic hyperosmolar coma – similar to DKA but no acidosis due to residual insulin
                 production allowing metabolism of ketone bodies, severe complication requiring intensive
                 hospital treatment
             lactic acidosis – rare complication of metformin therapy, usually associated with
                 alcoholism, liver or renal disease
      Secondary Diabetes
           heterogenous group of 300+ disorders which can give rises to DM
           only accounts for ~1% of all diabetes

Hypoglycaemia
    potentially life threatening situation
    may occur as complication of insulin therapy or number of de novo circumstances
    frequent cause of presentation in coma
    brain has absolute requirement for glucose (2-2.5 mM)
    with extreme hypoglycaemia (<1 mM) brain rapidly (~5 mins) damaged, worse than hypoxia
    treated with glucose (IV, caustic if administered directly to tissues) and glucagon if severe

Symptoms
      Adrenergic
           usually major form
           pallor, sweating, tremor, palpitations, dry mouth
      Neuroglyopaenic
           lack of concentration, slow movements, slurred speech, double vision, personality changes,
            transient stroke, fits, coma
           not infrequently mistaken for alcohol intoxication
      Sequelae
           usually no obvious permanent effects
           prognosis for recovery declines with length of coma, outcome worse if associated with
            hypoxia/hypotension




Hadley Wickham                                        72                                                16/06/11
Classification
      Fasting
           provoked by starvation and sometimes exercise
           always indicated identifiable underlying disease
           Excess insulin-like Activity
                insulin overdose (occurs repeatedly in most IDDM patients)
                insulinomas
                factitious hypogylcaemia (surreptitious intentional overdosage)
                sulphonylurea overdose
                insulin agonists (rare, eg. autoimmune)
           Hepatic Dysfunction
                severe infections
                ethanol toxicity
                malaria
                severe parenchymal liver disease
                drugs
                hormone deficiency
      Reactive hypoglycaemia
           related to operation which have produced excessive rate of gastric emptying, with
            consequently elevated stimulation to insulin secretion
           occasionally seen early in development of NIDDM, discoordinated excessive insulin secretion
            3-5 hrs after a meal
           occasionally in otherwise “normal” patient

Thyroid
  main products of thyroid are T3 and T4 (iodinated products of tyrosine) and calcitonin
     enough thyroid hormones stored to last 2-3 months
     approximately 50x more T4 than T3 in plasma (T4: 8 μg/dL, T3: 0.12 μg/dL)
     T3 is major hormone active in target cells
  migrates from back of tongue to neck during development
     lingual thyroid may occur if migration doesn‟t occur properly

Function
  very important in development, especially of CNS, linear growth and regulation of metabolic rate
  even though structurally related to tyrosine, not directly manufactured from
  only reason that body requires iodine (150 mg/day)
      in New Zealand, soil used to be deficient in iodine so it was added to salt, but now sufficient
            because of chemical run off from pesticides/herbicides etc.

Histology
  each lobe consists of many spherical follicles, about 50 - 500 μm in diameter
Hadley Wickham                                      73                                              16/06/11
  each follicle composed of single layer epithelial cells, surrounding cavity in which thyroid hormones
     are stored as integral components of thyroglobulin
           thyroglobulin is very large (Mr ~ 600,000) protein, with very high tyrosine content
           synthesised in follicular cells and secreted into follicular cavity

Synthesis
  thyroid actively concentrates iodide to level normally 25x higher than that of plasma
  iodide oxidised by peroxidase to atomic iodine, which immediately iodinates tyrosine residues
     contained in thyroglobulin
  iodinated tyrosines undergo coupling reaction to form T3, rT3 or T4 (thyronines)
      same enzyme catalyses both iodination and condensation
  proteolytic degradation of thyroglobulin within follicular cells causes liberation of MIT, DIT and
     thyronines
           iodine removed from MIT and DIT recycled
           T3 and T4 released into blood stream
      Summary
           iodide uptake and concentration                         (blocked by thiocyanate and perchlorate)
           oxidation of I- to I                              (blocked by propylthiourical and carbimazole)
           iodination of tyrosine residues in thyroglobulin(also blocked by propylthiourical and carbimazole)
           coupling of either 2 DIT to form T4, or MIT & DIT to form T3

Transport and Protein Binding
  99% carrier protein bound in plasma
      thyroxine binding globulin (TBG) (70%)
      thyroxine binding pre-albumin
      albumin
  T4 not usually measured directly, but instead free plasma binding sites are measured with
     radiolabelled T4
           need to be aware of treatments that can affect number of sites eg. E2, TBG; androgens,
            TBG
  5‟ deiodinases remove iodine from 1st or 2nd ring of T4 giving T3 or rT3
      T3 10x more potent than T4
      rT3 has no biological effect

Function
  general effect on metabolism, including CHO, lipid and protein
  mediated through direct effect on gene expression
       Na+/K+ ATPase , metabolic rate , fuel consumption 
  at normal level there is balance between break down and build up – this becomes disturbed when
     levels are abnormally high or abnormally low



Hadley Wickham                                       74                                                 16/06/11
Clinical
      Hypothyroidism
           ankle reflex is good test for thyroid hypofunction – normally rapid response becomes
            dispersed and delayed
           in I deficient goitre thyroid produces mostly T3 (normally produced peripherally) in order to
            maximise iodine available
           fetus is dependent on mother‟s thyroid hormones until it starts to produce it‟s own at about 18
            weeks (T3 receptors start appearing at around 10 weeks)
           if hypothyroidism (neurological) occurs early: deaf-mutism and spasticity of legs results
           if hypothyroidism (cretenic) occurs later: intellectual development stunted  cretinism
             if treatment can begin within 1st few weeks of birth intellectual development will be
                 normally
           hypothyroidism occurs after birth: growth , upper/lower segment ratio , cold sensitive
           middle-age: tends to occur slowly, feel tired and lethargic, brain function , cold intolerance,
            mucopolysaccharides accumulate, CO , tendon reflexes slowed
           myxodema is result of severe iodine deficiency, with retardation, atrophy of thyroid and
            mucopolysaccharide invasion of skin
           Auto-immune Thyroiditis
                most common cause of goitre in NZ
                T cells set up inflammatory response, B cells produce antibodies against thyroid
                5-7% of Aucklander‟s have antibodies against thyroid
                results in T3/T4 , TSH , thyroid size 
                50% shrink after treatment with T4
                fibrosis and nodules (only 5% detectable by physical examination) develop with long-term
                 inflammation
             sometimes nodule becomes autonomous and produces to much T4
           Hashimoto’s Disease
             antibodies against thyroid
      Hyperthyroidism
           irritability and nervousness, pounding heart, arrhythmias, heat intolerance, weight loss,
            muscle mass
           thyrotoxicity = muscle wasting, palpitations, heat intolerance, intermittent diarrhoea, heart
            failure, atrial fibrillation
           Plumber‟s diseases = nodular thyroid enlargement, treated with radioactive I
           Grave‟s disease = auto-immune, treated with T4 antagonist
             Congenital thyrotoxicosis = occurs if mother has hyperthyroidism while pregnant, usually
                 resolves within few weeks after birth




Hadley Wickham                                        75                                                    16/06/11
Development of Reproductive System
  although chromosomal and genetic sex of embryo determined at fertilisation, male and female
     morphological characteristics don‟t develop until 7th week
  early genital systems in two sexes are similar, therefore initial period of development is called
     indifferent stage of sexual development

Development of Gonads
  derived from three different sources:
      mesothelium (lining post abdominal wall)
      mesenchyme
      primordial germ cells
             Mature                Embryonic Tissue
      spermatogonia       germ cells
      oogonia
      Sertoli cells       epithelial cords of cells
      granulosa
      Leydig cells        interstitial tissue between cords
      theca interna
      Myoid cells         interstitial tissue between cords


Indifferent Gonads
  initial stages occur during 5th week, when thickened area of mesothelium develops on med
     mesonephros
    proliferation of ep and underlying mesenchyme produces bulge, gonadal ridge
    finger-like ep cords (10 sex cord) soon grow into underlying mesenchyme
    indifferent gonad now consists of external cortex and internal medulla
    in embryos with XX sex chromosome, cortex differentiates into ovary and medulla regresses
    in embryos with XY sex chromosome, medulla differentiates into testis and cortex regresses

Primordial Germ Cells
  large round primitive sex cells visible in 4th week among endodermal cells of yolk sac, near origin of
     allantois
  during folding, dorsal part of yolk sac incorporated into embryo
      primordial germ cells migrate along dorsal mesentery of hindgut to gonadal ridges
  during 6th week, primordial germ cells enter underlying mesenchyme and incorporate into primary
     sex cords

Sex Determination
  Y chromosome required for development of male phenotype
      has testis-determining effect on medulla of indifferent gonad, causing primary sex cords to
              differentiate into seminiferous tubules
            gene for testis-determining factor (TDF) has been localised in short arm of Y chromosome
  two X chromosomes required for development of female phenotype
  type of gonads determines type of sexual differentiation that occur in genital ducts and external
     genitalia
             testosterone determines maleness
             primary female sexual differentiation occurs independently of hormones

Development of Testes
  embryos with Y chromosome usually develop testes
  coordinated sequence of genes induces development of testes (gene for TDF acts as switch)
  TDF induces 10 sex cords to condense and extend into medulla where they branch and anastomose
     to form rete testis
  connection of sex cords (seminiferous cords) with surface ep lost when tunica albuginea develops
      characteristic and diagnostic feature of testicular development in fetus
  gradually enlarging testis separates from degenerating mesonephros and becomes suspended by own
     mesentery, mesorchium
  seminiferous cords develop into seminiferous tubules, tubuli recti and rete testis
  seminiferous tubules become separated from mesenchyme that gives rise to interstitial cells (of
     Leydig)
  by about 8th week these cells start to secrete androgenic hormones (eg T and DHEAS)
       production stimulated by CG, which reaches peak amounts during 8th to 12th week
       induce masculine development and external genitalia
  fetal testes also produces anti-müllerian hormone (AHM or MIH or MIF or MIS)
       produced by sustenactular cells (of Sertoli) which continue to until puberty
       suppresses development of paramesonephric (müllerian ducts)
  seminiferous tubules remain solid until puberty
  during later development, surface ep flattens to form mesothelium of ext surface of adult testis
  rete testes becomes continuous with 15 to 20 mesonephric tubules that become efferent ductules

Development of Ovaries
  occurs slowly in female embryos
  not identifiable histologically until about 10th week
  primary sex cords don‟t become prominent but extend into medulla and form rudimentary rete
     ovarii
  secondary sex cords extend from surface ep into underlying mesenchyme during early fetal period
  as primary sex cords increase in size primordial germ cells are incorporated into them
  at about 16 weeks cords begin to break up into isolated cell clusters (primordial follicles) each
     consisting of oogonium (from primordial germ cell) surrounded by single layer of flattened follicular
     cells (from sex cord)
             proliferative phase terminates before birth, by which time all oogonia have entered into their
              first division, thereby becoming primary oocytes
             termination of mitosis and entry in meiosis evoked by meiosis initiation factor (from
              mesonephros)



Hadley Wickham                                       78                                                   16/06/11
           during progress through first meiotic prophase, oocytes organise surrounding mesenchymal
            cells to form granulosa cells of primordial follicles
           follicular cells secrete basement membrane and oocytes arrest meiosis at diplotene
           remain at dictyate phase until signal to resume development which occurs at some random
            time after puberty
  active mitosis of oogonia occurs during fetal life, producing ~7 million primordial follicles
      although many degenerate before birth, 2 million remain, enlarging to become 10 oocytes
            before birth
  after birth surface ep of ovary flattens to single layer of cells continuous with mesothelium of
     peritoneum
  surface ep becomes separated from follicles in cortex by thin fibrous capsule, tunica albuginea
  as ovary separates from regressing mesonephros it is suspended by mesovarian which is its
     mesentery

Development of Genital Ducts
  both male and female embryos have two pairs of genital ducts
      mesonephric (wolffian) ducts play important part in development of male reproductive system
      paramesonephric (müllerian) ducts play important role in development of female reproductive
            system
  during 5th and 6th weeks, genital system is in indifferent stage where both male and female ducts are
     present

Development of Male Genital Ducts and Glands
  T stimulates mesonephric ducts to form male genital ducts and MIS causes paramesonephric ducts
     to regress
  as mesonephros degenerates some mesonephric tubules persist and are transformed into efferent
     ductules
           ductules open into mesonephric duct, which has transformed into ductus epididymis
  distal to epididymis, mesonephric duct acquires a thick layer of smooth muscle and becomes ductus
     deferens
  lateral outgrowth from caudal end of each mesonephric duct gives rise to seminal vesicle
  part of mesonephric duct between seminal vesicles and urethra becomes ejaculatory duct
      Prostate
           multiple endodermal outgrowths arise from prostatic urethra and grow into surrounding
            mesenchyme
           glandular ep of prostate differentiates from these cells and associated mesenchyme
            differentiates into dense stroma and smooth muscle of the prostate
      Bulbourethral Glands
           develop from paired outgrowths of spongy urethra
           smooth muscle fibres and stroma differentiate from adjacent mesenchyme

Development of Female Ducts and Glands
  paramesonephric ducts form most of female genital tract

Hadley Wickham                                    79                                                  16/06/11
  uterine tubes develop from unfused cranial parts of paramesonephric duct
  caudal fused portions form uterovaginal primordium
      gives rise to uterus and sup part of vagina
      endometrial stroma and myometrium are derived from adjacent mesenchyme
  fusion of paramesonephric ducts also brings together two peritoneal folds that form the right and left
     broad ligaments and two peritoneal compartments rectouterine pouch and vesicouterine pouch
  along side of uterus, mesenchyme proliferates and differentiates into parametrium, composed of
     loose ct and smooth muscle
      Development of Vagina
           vaginal ep derived from endoderm of urogenital sinus, fibromuscular wall from surrounding
            mesenchyme
           contact of uterovaginal primordium with urogenital sinus (sinus tubercle) induces formation
            of paired endodermal sinovaginal bulbs
             extend from urogenital sinus to caudal end of uterovaginal primordium
             sinovaginal bulbs fuse to form a vaginal plate
             later central cells apoptose forming the lumen of the vagina
           until late fetal life lumen of vaginal is separated from cavity of urogenital sinus by hymen
             formed by invagination of post wall of urogenital sinus
             usually ruptures during perinatal period and remains as a thin fold of mucous membrane
      Auxiliary Genital Glands in Female
           buds grow from urethra into surrounding mesenchyme, forming urethral and paraurethral
            glands
             correspond to prostate gland in male
             outgrowths from urogenital sinus from greater vestibular glands, homologous to
                 bulbourethral glands in male

Development of External Genitalia
  from 4th to 7th week external genitalia are sexually undifferentiated
  distinguishing sexual characteristics begin to appear during 9th week but are not fully apparent until
     12th week
  early in 4th week proliferating mesenchyme produces genital tubercle at cranial end of cloacal
     membrane
  labioscrotal folds and urogenital folds develop on each side of cloacal membrane
  genital tubercle elongates to form a phallus
  when urorectal septum fuses with cloacal membrane at end of 6th week, it divides cloacal membrane
     into dorsal anal and ventral urogenital membrane
  urogenital membrane lies in floor of median cleft, urogenital groove, bounded by urogenital fold
  anal and urogenital membranes rupture a week or so later forming the anus and urogenital orifices
  in female fetus, urethra and vagina open into a common cavity (vestibule of vagina)

Development of Male External Genitalia
  masculinisation of indifferent external genitalia induced by T produced by fetal testes
Hadley Wickham                                      80                                                     16/06/11
  as phallus enlarges and elongates to form penis, urogenital fold forms lat walls of urethral groove on
     ventral surface
            lined by proliferation of endodermal cells, the urethral plate
  urogenital folds fuse with each other along ventral surface of penis to form spongy urethra
  surface ectoderm fuses in median place forming penile raphe and enclosing spongy urethra within
     penis
  at tip of gland penis ectodermal ingrowth forms cellular cord, glandular plate, which grows toward
     the root of penis to meet spongy urethra, then canalises
  during 12th week a circular ingrowth of ectoderm occurs at periphery of glands penis
      when this ingrowth breaks down it forms the prepuce
      for some time prepuce is adherent to glans and is not usually retractable at birth
      break down of adherent surface normally occurs during infancy
      corpora cavernosa and spongiosum develop from mesenchyme in phallus
  labioscrotal swellings grow towards each other and fuse to form scrotum, line of fusion clearly
     visible as scrotal raphe

Development of Female External Genitalia
  feminisation of indifferent gonad not fully understood, but oestrogens produced by placenta and
     ovaries are important
  growth of phallus gradually ceases and becomes clitoris
      still relatively large at 18 weeks, develops like penis, but urogenital folds don‟t fuse (except
             post where they form the frenulum of the labia minora)
            unfused parts form labia minora
            labioscrotal folds fuse post to form posterior labial commissure and ant to form ant labial
             commissure
            most parts of labioscrotal folds remain unused and form two large fold of skin, labia majora

Disorders of Sexual Differentiation
  prior to 7 weeks fetal gonad, internal and external genitalia are undifferentiated with potential to
     develop into male or female structures
  two factors are now known to be necessary for formation of urogenital ridge and bipotential gonad:
     steroidogenic factor 1 and Wilm’s tumour 1
      Steroidogenic Factor 1 (SF1)
            from large family of orphan nuclear transcription factors
            found in UGR, adrenals, gonads, hypothalamus and pituitary gonadotropes
            appears vital for normal formation of HP-adrenal and HPG axis
            no mutation described in humans, but when knocked out in mice phenotype is always female
             and only streak gonads formed
              mullerian structures present, no adrenal development and mice die of adrenal insufficiency
              no GnRH, LH or FSH produced
      Wilm’s Tumour 1 (WT-1)
            putative tumour suppressor gene

Hadley Wickham                                       81                                                    16/06/11
            found in UGR around time of formation of bipotential gonad
            deletions in humans lead to Denys-Drash syndrome (gonadal dysgenesis, congenital
             nephropathy, and subsequent development of Wilm‟s tumour)
  downstream genes from SF1 and WT-1 involved in evolution of embryonic mesoderm to bipotential
     gonad are still unclear
  several other genes are now known to be involved directing bipotential gonad to either testes or
     ovary
      Sex-determining Region of Y (SRF)
            nuclear transcription factor identified and cloned in 1990
            resides on short arm of Y chromosome
            specifically found in UGR in pre-Sertoli cells
            loss of function mutations in SRY gene (1/100,000 females) leads to XY females
             (phenotypically female with normal female internal and external genitalia but with streak
             gonads)
            46 XX men almost always have SRY gene on a X chromosome
              appear phenotypically normal with palpable testes
              go into puberty noramlly, but subsequently often have pubertal arrest with gonadal failure
              testes are small with Leydig cell hyperplasia, azoospermic
      Dosage-sensitive sex reversal-adrenal hyperplasia congenita critical region on X chromosome
            similar to SF1, orphan nuclear transcription factor
            co-localised with SF1 in most tissues including hypothalamus, pituitary gonadotropes,
             adrenals and gonads
            loss of function point mutations lead to adrenal hypoplasia congenita in males and
             hypogonadotropic hypgonadism
            affected boys may have small penis, but are always unambiguously male
            however, double dose of DAX-1 represses SRY and leads to ovarian development and female
             phenotype
      SRY homeobox-like gene 9 (SOX9)
            recently identified as one of genes involved in testis determination
            further downstream than SRY and exact function in unclear
            loss of function point mutations results in XY sex reversal with streak gonads
            all affected individual also have a server skeletal dysplasia called camptomelic dysplasia
             usually resulting in death in first 2-3 years
  unlike gonadal development sexual differentiation of genitalia in female is default process that
     automatically occurs in absences of male sexual differentiating hormones
  predicably female external genitalia closely resembles undifferentiated genitalia
  differentiation in female also lags being active male differentiation

Clinical Evaluation
      History
            maternal drugs
            maternal infection

Hadley Wickham                                       82                                                   16/06/11
           family history of CAH, genital abnormalities or infertility
      Genital Examination
           length
           weight
           dysmorphic features
           visual fixing
      Investigations
           pelvis USS
           urgent karyotype
           serum 17-OH progesterone
           electrolytes
           testosterone
           if boy has small penis and palpable gonads also do
                GH
                T4
                TSH
                cortisol




Hadley Wickham                                     83                     16/06/11
              Histology of Endocrine Glands
                    Blood Supply                  Encapsulation                   Part             Organisation                   Secretion         Cont                Identification                                   Notes
                                                                                                                                                     rol
               sup thyroid .: from ext.      outer capsule loose ct       thyroid         composed of irreg. spheroid       produce T3 and T4      TSH     active follicles small, colloid         hormones are only know iodine-containing
               carotid                                                                    follicles                                                                                                 compounds with biological activity
                                             inner capsule fibroelastic                                                                                    inactive follicles large, colloid ,
               inf thyroid .: subclavian .   (septa divide gland into                     simple cuboidal ep                                               cells flattened                          crucial determinant of normal developmental
                                             lobules)                                     lumen – colloid material                                                                                  processes and maintain metabolic stability
Thyroid




                                                                          calcitonin      single cells (scattered)          calcitonin             Ca2+   larger, pale cytoplasm
                                                                          (clear cells)   small clumps (intrafollicular
                                                                                          spaces)


                                             thin fibrous capsule,        chief                                             PTH                    Ca2+   most abundant                            regulate serum Ca2+ and PO43-
Parathyroid




                                             septa divide into dense                                                                                                                                bone – osteoclastic resorption of bone 
                                                                          oxyphil                                           ??                             larger, smaller nuclei, strongly
                                             cord-like masses
                                                                                                                                                           eosinophilic                             kidney – tubular reabsorption of Ca2+ 
                                                                                                                                                           often clumped                            small intestine – Ca2+ absorption 


               >3 arterioles, >6 vv.,        supported by fine            glucagon        clumps within pancreas                                           20%, usually near periphery (pink
Islets




               many fenestrated              collagenous network                                                                                           with special stain)
               capillaries                   delicate capsule             insulin                                                                          70%
                                             surrounds
               three groups form plexus      dense fibrous ct invests     Cortex: ZF      irreg. clumps                     mineralocorticoids             secretory cells have strongly staining   similar origin to gonads
               within capsule                gland                                        separated by capillaries and      (esp. aldosterone)             nuclei and little cytoplasm
               network of sinusoids          provides external                            fine ct
               supplied by capsular          support
               plexus, desc between
               cords of ZF into deep                                      Cortex: ZG      narrow cords of epithelia         glucocorticoids (esp   ACTH    have dense nuclei and strongly
               plexus in ZR before                                                        separated by capillaries and      cortisol)                      staining cytoplasm
               draining into central v.                                                   fine ct                           small amounts of
Adrenal




               of medulla                                                                                                   androgens
               medulla supplied from                                      Cortex: ZR      irreg network of branching        androgens              ACTH    cells smaller than ZF and cytoplasm
               capsular plexus drain                                                      cells and clumps                  small quantities of            stains more strongly
               into central v.                                                            separated by numerous wide        glucocorticoids
                                                                                          capillaries
                                                                          Medulla         closely packed clumps             NA & (pulsatile)       SNS     large granular nuclei, basophilic        similar origin to SNS
                                                                                          supported by fine trabeculae of   enkephalins (?)                cytoplasm (pale)                         medulla receive fresh blood and blood rich in
                                                                                          connective tissue                                                NA secreting cells have much stronger    corticosteroids
                                                                                                                                                           chromaffin reaction

				
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