MAMMARY GLAND STRUCTURE AND FUNCTION

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					                                 MAMMARY GLAND STRUCTURE AND FUNCTION

 The objective of this module is to allow the student to gain an appreciation of the intricacies of mammary gland structure and growth,
                                                  and synthesis of milk components.

                                               Growth and Development
                                               Anatomy
                                               Vascular, Lymphatic, and Neural Systems
                                               Cytology of the Mammary Secretory Cell
                                               Hormones Involved in Lactation
                                               Milk Synthesis and Secretion
                                               References



GROWTH AND DEVELOPMENT OF THE MAMMARY GLAND


Mammary gland growth occurs during five distinct phases of development. These phases include Prenatal,
Prepubertal, Postpubertal, Pregnancy,and Early Lactation.
Development of the mammary gland begins early during embryonic and fetal growth. A thickening of ectodermal cells occurs when a
calf is 1.4 to 1.7 cm long (30 days after conception). The cells continue to grow and aggregate to form lines of mammary tissue on each
side of the midline (figure to appear later). Four structures called mammary buds form from differentiated cells which have progressed
through crest and hillock stages in distinct areas of the mammary lines. Mammary buds are precursors of the secretory protein of the
mammary gland, therefore, the number of buds determines the number of mammary glands. Buds divide into primary sprouts (figure to
appear later) which differentiate into teat and gland cisterns. Several secondary sprouts arise from primary sprouts to form mammary
ducts. The sprouts become hollow shortly before birth of the calf.


PREPUBERTAL
At birth, the number of cells needed to form connective tissue and fat (non-glandular portion) of the udder are established. The
glandular portion of the mammary gland is rudimentary at this stage. From birth to 3 months of age, growth of the duct system
continues at a rate equal to body growth. From 3 months of age to 9 months of age, growth of mammary tissue exceeds that of the body
by 3.5 fold.


POSTPUBERTAL



Growth of mammary tissue increases rapidly after puberty as estrogen and progesterone exert their influences on duct and secretory
tissue. While duct and lobular growth is occurring at this time, alveoli are not formed until pregnancy is established.


PREGNANCY



Growth of mammary tissue is very rapid during pregnancy. The increase in udder size becomes obvious after 3 to 4 months of
pregnancy in heifers, although it is not until secretions of mammary tissue cause major increases in size during the 7th to 9th month of
gestation that the udder is highly visible.


EARLY LACTATION



Lactation is associated with increased mammary cell numbers. Cell numbers increase until peak lactation, at which time, mammary cell
loss exceeds the rate of cell division and milk production decreases.
                                                                 Because most cows are bred during the first 40 to 90 days
                                                                 post-partum, a major portion of lactation co-exists with
                                                                 pregnancy. Initial stages of lactation have little effect on
                                                                 mammary cell numbers, however, a decrease in cell
                                                                 numbers occurs relative to non-pregnant cows after 5
                                                                 months of pregnancy.

                                                                 DRY PERIOD

                                                                 The dry period is a stage in the lactation cycle when cows are no longer
                                                                 being milked. This usually occurs when cows have been milked for 10 to
                                                                 12 months (7 months pregnant) and are about 2 months from next
                                                                 expected calving. The cessation of milking causes the udder to become
engorged with milk (a process taking several days depending on level of milk production). The subsequent decrease in metabolic
activity causes a degeneration and loss of alveolar epithelial cells. Myo-epithelial cells and connective and adipose tissues remain, with
the adipose and connective tissues becoming more prominent as the alveoli degenerate. Complete involution (degeneration) of the
alveoli occurs within 75 to 90 days in the non-pregnant cow. Pregnancy stimulates udder growth, therefore, complete involution is not
attained if cows are approximately 7 months pregnant at the start of the dry period. Involution of the mammary tissue is important for
subsequent lactation and a dry period of 45 to 75 days is necessary to maximize milk yield in the next lactation. The absence of a dry
period interferes with the increase in cell numbers that normally occurs during the early stages of lactation.


REFERENCES

   Schmidt, G.H. Biology of Lactation. W.H. Freeman and Company. San Francisco, California.
   Keenan, T.W., and D.P. Dylewski. 1985. Aspects of intracellular transit of serum and lipid phases of milk. J. Dairy Sci. 68:1025-
1040.
   Davis, S.R., and R.J. Collier. 1985. Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci. 68:1041-
1058.
   Kuhn, N.J., D.T. Carrick, and C.J. Wilde. 1980. Lactose synthesis: The possibilities of regulation. J. Dairy Sci. 63:328- 336.
   Smith, S. 1980. Mechanism of chain length determination in biosynthesis of milk fatty acids. J. Dairy Sci. 63:337-352.
   Khorasani, G.R., P.H. Robinson, G. de Boer, and J.J. Kennelly. 1991. Influence of canola fat on yield, fat percentage, fatty acid profile,
and nitrogen fractions in Holstein milk. J. Dairy Sci. 74:1904-1911.
   Grummer, R.R. 1991. Effect of feed on the composition of milk fat. J. Dairy Sci. 74:3244-3257.
   Jensen, R.G., A.M. Ferris, and C.J. Lammi-Keefe. 1991. Symposium: Milk fat - composition, function, and potential for change. The
composition of milk fat. J. Dairy Sci. 74:3228-3243.
   Neville, M.C., and C.W. Daniel. 1987. The Mammary Gland: Development, Regulation, and Function. Plenum Press, New York.




    ANATOMY OF THE BOVINE MAMMARY GLAND


Mammary glands of mammals (animals with hair and mammary glands) are located on the underside or front of both males and females
in positions ranging from just between the front legs (elephants), along the midline (cats, dogs, pigs), to between the hind legs (cattle).
The word "mammal" arises from "mamma" which means breast or milk gland. Mammary glands are modified sudoriferous (sweat) glands
which secrete milk (exocrine gland) and serve as accessory glands to the reproductive system.
                              Knowledge of mammary gland structure will aid in the understanding of the
                              mechanisms of milk secretion and removal from the udder and of the mechanisms by
                              which cows respond to changes in feeding, milking procedures, and environmental
                              conditions.

                                EXTERNAL FEATURES OF THE MAMMARY GLAND



                              The udder of the dairy cow is comprised of four separate mammary glands clearly demarcated into right
                              and left halves by a longitudinal furrow called the intermediary or intermammary groove (figure to
appear later). Sometimes, but not always a transverse furrow is seen between the front and rear halves; a characteristic not desired.
Viewed from the side, the floor of the udder should be flat and show a strong attachment to the underside of the abdominal wall and a
wide and high attachment at the rear. Individual quarters of the udder should be symmetrical. Empty weights of the udder will range
from 12 to 30 kg, however, udder weight is not correlated with milk production. The rear quarters tend to be larger than the front
quarters and tend to produce more milk (60% vs 40%). The teats vary in shape from cylindrical to conical, however, shorter and smaller
teats are preferred for machine milking. Udders should be large enough to produce large volumes of milk, yet at the same time, not be
too large or have poor attachments to create management problems.


The udder is supported by the skin, and lateral and median suspensory ligaments. The skin, while providing only
limited support to the udder, provides protection from abrasions and from invasion by microorganisms. Fine
connective tissues connect the skin to the udder and coarser connective tissue (superficial facia) attaches the
front quarters to the abdominal wall.
                                    The lateral suspensory ligaments (a ligament is a supportive structure connecting
                                    one organ to another) are one of the main supporting structures of the mammary
                                    gland. The lateral suspensory ligament is a fibrous, non-elastic structure arising
                                    from the sub-pelvic tendons (tendons attach organs to bone). These tendons are
                                    above an posterior to the rear udder. The lateral suspensory ligaments extend
                                    along both sides of the udder (superficial) and also into the interior of the udder
                                    (deep).

                                The median suspensory ligament is the major supporting ligament of the udder and
is comprised of elastic tissue arising from the midline of the abdominal wall and extending between right and left
halves of the udder to join with the deep lateral suspensory ligaments. The median suspensory ligament forms
the centre of gravity of the udder and carries the majority of the weight of the udder. The ligament provides a
distinct internal separation between left and right halves of the udder. A separation exists between the front and
rear quarters, although this is not clearly defined by a membrane.

 INTERNAL FEATURES OF THE MAMMARY GLAND



The teat orifice, called the streak canal or teat meatus, (see figure) is the terminus of an extensive alveolar and tubular system which
synthesizes and collects milk. The streak canal is composed of 5 to 7 cornified epithelial projections that form a star-shaped slit. An
involuntary sphincter muscle serves to constrict the opening, thereby, inhibiting entry of microorganisms and also preventing leakage of
milk from the mammary gland. This muscle controls milk flow and ease of milking and because the trait is heritable, dairy cattle
breeding programs include selection for strength of the sphincter muscle. Located just above the streak canal is a series of folds known
as Furstenburg's Rosette. These folds radiate in all directions and flatten out when milk accumulates within the mammary gland to aid in
the retention of milk.


The teat cistern (sinus papillaris), located above the Furstenburg's Rosette, is a cavity within the teat capable of
holding 30 to 60 ml of milk. The teat cistern is lined with numerous longitudinal and circular folds in the mucosa
which tend to overlap and serve as storage places for bacteria during episodes of mastitis. The gland cistern
(sinus lactiferous) is located just above the teat cistern and is capable of storing 100 to 400 ml of milk. No
relationship exists between the size of cistern and the amount of milk secreted by the mammary gland. The gland
and teat cisterns are continuous, but in most cows a definite circular constriction of dense connective tissue
called the annular (cricoid) fold is seen between the two cisterns.

The gland cistern serves as a collection point of major ducts (10 to 20) which branch repeatedly to eventually
drain the alveoli (figure to appear later) which are the milk secreting tissue. The alveoli are composed of a single
layer of epithelial cells surrounded by connective tissue, myoepithelial cells, and blood vessels.
The epithelial cells are capable of extracting nutrients from blood, synthesizing milk components (fat, protein,
and lactose), and secreting milk into the lumen of the alveoli. The myo- epithelial cells are specialized muscle
cells which envelope the alveoli and small dusts and contract when milk "let-down" occurs. The alveoli are
arranged in small groups surrounded by connective tissue to form lobules. Each lobule is drained by a single duct
and groups of lobules when surrounded by connective tissue form lobes drained by intralobular ducts to
eventually drain into the major ducts.

The epithelial cell , the major component of the alveoli, is comprised of many intracellular elements:

       The nucleus which contains the genetic material (DNA) for cell division and function;
         The plasma membrane which regulates nutrient supply and waste excretion;
         The cytoplasm which is comprised of mitochondria for energy supply of the cell and the microsomal fraction for assembly of
       cell products (the endoplasmic reticulum synthesizes protein and esterifies fatty acids, the Golgi apparatus synthesizes lactose
       and processes protein); and
         The cytosol in which the cell components are immersed which provides precursors for the endoplasmic reticulum and Golgi
       apparatus, and which contains enzymes necessary for glycolysis, the hexose mono-phosphate shunt, activation of glucose for
       lactose synthesis, and fatty acid synthesis.



 VASCULAR, LYMPHATIC AND NERVOUS SYSTEMS OF THE MAMMARY
GLAND


 VASCULAR SYSTEM


Arteries (see figure) bring oxygenated nutrient bearing blood to the mammary gland from the heart via the aorta and external pudic
(pudendal) arteries (2) and caudal and cranial mammary arteries (major supplies). The perineal artery, a continuation of the internal
pudendal artery supplies a small amount of blood to the caudal portion of both halves of the udder. Arteries become smaller to form
arterioles which then become capillaries to provide nutrients to the alveoli.
                                                <>Blood flows through the capillaries to venules which form veins which
                                                drain the blood away from the udder toward the heart. Veins converge to
                                                form a venous circle before leaving the mammary gland. From the circle
                                                the blood can leave via the two external pudendal veins which parallel
                                                the pudendal arteries to eventually reach the vena cava, or via the
                                                subcutaneous abdominal or milk veins (the large veins seen on the lower
                                                abdominal wall of high-producing dairy cows) to eventually reach the
                                                vena cava. While the cow is standing, most of the blood returns to the
                                                heart via the abdominal veins, however, blood may return preferentially
                                                through other veins while the cow is lying down.

Blood flow in high producing cows can approach 800 litres per hour. Approximately 500 volumes of blood is
required to produce 1 volume of milk.

 LYMPHATIC SYSTEM



Lymph is a colourless fluid drained from tissue spaces by thin-walled lymph vessels. Lymph originates as a filtrate of blood serum and
contains no red blood cells and only half the amount of protein. The lymph vessels carry lymph from the tissue spaces to lymph nodes
(see figure) where the fluid is filtered to remove foreign material. The lymph is then transferred to larger lymph vessels to the thoracic
lymph duct which returns the lymph to the blood via the anterior vena cava.
                                                 Lymph nodes add lymphocytes (a type of white blood cell involved in
                                                 immunity) to the blood system. The udder contains two large
                                                 supramammary lymph nodes which collect lymph from most of the
                                                 mammary gland. Lymph enters the node via afferent vessels into a
                                                 network of sinuses and exits into efferent vessels. Lymph is moved by
                                          pressure changes within the body caused by breathing, heart beats and
                                          contraction of muscles. Flow of lymph is directed toward the heart via
a series of one-way valves. Udder edema can result at calving, because of the incomplete development of these
valves and/or because of the increased blood flow associated with the onset of lactation.

 NERVOUS SYSTEM



The udder is innervated by several nerves arising from the spinal cord (figure to appear later). The nervous system is comprised of
afferent sensory and efferent sympathetic nerve fibres arising from the autonomic nervous system (system that regulates involuntary
vital functions) and from the sympathetic nervous system (system that can accelerate heart rate, constrict blood flow to the gastro-
intestinal tract, increase blood flow to muscles and increase blood pressure for example in the fight or flight syndrome).
Parasympathetic system innervation is absent from the mammary gland.
                                          Nervous stimulation to the udder is received from three main nerves; the 1st
                                          and 2nd lumbar nerves which provide sensory nerves to the anterior and
                                          lateral surfaces of the front quarters, the 3rd and 4th lumbar nerves which
                                          join with the 2nd lumbar nerve to form inguinal nerves to provide the bulk of
                                          sensory and motor fibres to the udder and teats, and the perineal nerve which
                                          originates from the pudic, and 2nd, 3rd, and 4th sacral nerves to provide
                                          sensory fibres to the posterior part of the udder.

                                    The efferent sympathetic nervous system innervates the smooth muscles
which surround the blood vessels and collecting tubules and the teat sphincters. Excitation of the cow causes a
constriction of blood flow to the mammary glands and reduces milk production. Sensory nerve fibres within the
teats send afferent information to the central nervous system to initiate milk ejection. The teats have touch,
temperature, and pain receptors which can respond to the sucking of a calf, massage or washing. The afferent
signals sent by these receptors are received by the posterior pituitary to release oxytocin and by the anterior
pituitary to release other hormones. The oxytocin enters the blood to travel to the mammary gland where it
causes the myo-epithelial cells to contract, thereby forcing the milk out of the alveoli and into the small ductules.


 CYTOLOGY OF MAMMARY GLAND SECRETORY CELL


The following figure is a pictorial view of a typical mammary gland secretory cell
                                              The plasma membrane surrounds the entire secretory cell and is
                                             composed of a bilayer of lipid molecules in which is interspersed a
                                             number of pores which allow the entry of nutrients, the excretion of
                                             wastes and the secretion of milk components. Little is known of the
                                             mechanisms by which nutrients pass from blood through the plasma
                                                 membrane into the cell. The processes are likely to involve a
                                                 combination of active transport (requiring energy), passive transport
                                                 (diffusion, osmosis), and electrochemical forces (no direct expenditure
                                                 of energy). Because most compounds absorbed by the cells have a
low solubility in lipids, it is unlikely that simple diffusion is a major route of entry.

 The nucleus is encased by a double membrane having pores which allow for entry of compounds necessary for
proper functioning. The nucleus contains the genetic material (DNA) necessary for the synthesis of mRNA for
protein (milk proteins and enzymes) synthesis of the cell.

 The endoplasmic reticulum is an organelle comprised of a series of membrane-bounded tubules, vesicles and
cisternae that extend from the nuclear membrane to the plasma membrane. The endoplasmic reticulum serves as
a staging area for the ribosomes to combine with the mRNA and amino acids to synthesize protein. The presence
of ribosomes causes a rough appearance hence the term rough endoplasmic reticulum. Endoplasmic reticulum
devoid of ribosomes is called smooth endoplasmic reticulum and simply serves as a channel for intracellular
transfer of materials and endproducts.

 The Golgi Apparatus is a series of smooth membrane-lined tubules and cavities that are continuous with the
smooth endoplasmic reticulum. The Golgi Apparatus serves to package proteins for export into the lumen of the
alveoli. Within the Golgi Apparatus calcium and phosphorus are added to casein molecules. Lactose synthesis
also occurs here. The secretory contents of the Golgi Apparatus are discharged into the lumen of the alveoli by
the process of reverse pinocytosis. Pinocytosis is the process whereby cells can take in fluid and molecules too
large to be carried across the plasma membrane by active transport mechanisms. During pinocytosis, material
becomes adsorbed to the plasma membrane causing changes in surface tension and electrical property in the
membrane. These changes cause an invagination in the membrane to form a pocket. Eventually the pocket is
pinched off to float freely within the cell. Lysosomes later digest the vesicle to release the contents. For milk
protein secretion, the Golgi Apparatus forms vacuoles which migrate and become contiguous with the plasma
membrane. Through the reverse of the procedure just described the vacuole discharge it's contents without
rupture of the cell membrane.

 The mitochondria are sausage shaped organelles located throughout the cell which supply the cell with over
90% of it's energy requirements. The mitochondria supply energy to the cell by oxidation of substrates and the
conversion of released energy into the form of the bond energy of adenosine triphosphate ATP).

 Lysosomes are membrane bound particles which contain degradative enzymes. Lysosomes are especially active
during involution of the mammary gland.

 The microtubules are essential for cell division and serve to maintain shape of the mammary cells. The
microtubules assist in the movement of secretory vesicles to the apex of the cell.

 The cytoplasm is a fluid matrix which constitutes a large portion of the mammary cell. The cytoplasm contains
the enzymes, nutrients, etc. which are essential for cell function. Processes such as anaerobic breakdown of
glucose, synthesis of fatty acids, and activation of amino acids for the synthesis of proteins occur within the
cytoplasm.


 HORMONAL CONTROL OF MAMMARY DEVELOPMENT AND LACTATION


Growth and development of the mammary gland is under hormonal control. The list and description of the hormones presented below is
not meant to be complete but to serve as an introduction to the endocrine (hormonal) control of growth and metabolism of the bovine
mammary gland. While functions of the hormones are described as being somewhat specific, most hormones function in concert with
one another.
 ANTERIOR PITUITARY


The anterior pituitary gland (adenohypophysis) is attached to the base of the brain and rests upon the floor of the cranial cavity. The
anterior pituitary produces hormones which stimulate the ovary such as follicle stimulating hormone and luteinizing hormone, and which
stimulate most tissues such as somatotropin (growth hormone), prolactin, thyroid stimulating hormone, and adrenocorticotropic hormone.
Most hormones with the exception of prolactin stimulate their target tissues to release other hormones.


Prolactin is involved in the development of the mammary gland and initiation of milk secretion. Somatotropin and
prolactin are directly required for normal development and growth of the mammary gland, and also serve to
indirectly synergize the effects of ovarian hormones on mammary tissue. Mammary glands atrophy following
hypophysectomy (removal of the pituitary gland), however, growth of the mammary gland is restored by
somatotropin and prolactin therapy. Injection of somatotropin which can increase milk production by 10 to 40% is
now possible with the advent of recombinant DNA technology. To achieve the increase in milk production, the
hormone must be injected daily or biweekly depending on the preparation. Increases in feed efficiency of 5 to
20% are also seen. The effect of somatotropin is mediated by insulin-like growth factor-I which is secreted by
the liver and mammary gland. As of 1994, recombinant somatotropin is available commercially in the United
States. Injections are to be given every 14 days beginning at 60 days postpartum.

 POSTERIOR PITUITARY



The posterior pituitary (neurohypophysis) lies adjacent to the anterior pituitary and produces vasopressin (antidiuretic hormone) and
oxytocin. Vasopressin serves to maintain blood volume through it's effects on the kidney. Oxytocin functions primarily in a
neuroendocrine reflex to allow milk "letdown" during lactation. The release of oxytocin is caused by mechanical stimulation of the teats.
The oxytocin is then carried to the mammary gland via the blood stream where it acts on the myoepithelial cells to cause constriction.
The constriction expresses the milk from the alveoli into the lobular ducts and subsequently into the teat canal.


 ADRENAL GLAND



The adrenal gland secretes steroid hormones from it's medulla (epinephrine, norepinephrine)and it's cortex (aldosterone, and the
glucocorticoids (corticosterone, cortisol, cortisone)). Epinephrine is the hormone that is largely responsible for the "fight or flight"
reaction to environmental stimulation. An increase in epinephrine will reduce or inhibit milk let-down, therefore, overstimulation of
cows in the milk parlour is not recommended. Aldosterone is involved in electrolyte metabolism (ie. sodium and potassium balance) and,
like the glucocorticoids, is involved in carbohydrate metabolism (ie. gluconeogenesis). Large doses of glucocorticoids decrease milk
production.


 THYROID GLAND



The thyroid gland is stimulated by thyroid stimulating hormone to secrete the iodine containing hormones, Triiodothyronine and
Thyroxine. The thyroid hormones, when released into the blood stream, affect practically every organ in the body and serve to increase
metabolic rate and oxygen consumption. There is some indication that thyroid hormones will increase glucose transport into cells. Years
ago, iodinated casein (thyroprotein) was fed to dairy cows to increase milk production. The thyroprotein mimicked thyroxine and served
to increase the metabolic rate of the entire animal rather than the mammary gland specifically and caused a 10% increase in milk
production at peak lactation and a 15 to 20% increase in later lactation. Beneficial effects of the thyroprotein were lost after 2 to 4
months and negative effects on production began to appear.


 OVARY



The ovary produces the steroids, estrogen and progesterone. Estrogen is produced by theca interna and granulosa cells of the growing
follicle and progesterone is produced by the corpora lutea after ovulation. If the ovary is removed prior to puberty the mammary glands
fail to develop, and if removed after puberty, the mammary glands involute. Estrogen is primarily responsible for duct growth and
progesterone is primarily responsible for lobulo-alveolar development.


 GROWTH FACTORS
The liver, kidney and mammary gland produce a variety of growth factors such as epidermal growth factor and insulin-like growth
factor I. These growth factors are partly responsible for the growth and maintenance of mammary tissue prior to and during lactation.
Insulin-like growth factor I appears to be the intermediary for the somatotropin stimulated increase in milk yield.



 SYNTHESIS AND SECRETION OF MILK


Milk is synthesized from nutrients provided to the mammary gland secretory cell by the blood supplying the mammary gland. These
supplies arise from dietary sources directly or after modification in animal tissues prior to reaching the mammary gland. The following
topics are briefly discussed here.
         General Comments
         Lactose Synthesis
         Fat Synthesis
         Protein Synthesis
         Secretion of milk components




       The principal components of milk are:

         water, 86 - 88 %
 total solids 12.0 - 14.0 %
 fat, 3.5 - 4.5 %
 protein, 3.2 - 3.5 %
 lactose, 4.6 - 5.2 %
 minerals, 0.7 - 0.8 %
 vitamins,
 bacteria, leucocytes (white blood cells), mammary secretory cells


A generalized scheme of the steps involved in the conversion of dietary components to milk components is
shown in the following figure.
                                                                                                    <>




 BIOSYNTHESIS OF MILK LACTOSE



Lactose is the main carbohydrate found in milk and is also almost exclusively found in milk and mammary gland tissue. Small
amounts are occasionally found in plants but in very low concentrations. The synthetic mechanisms in plants are also different.
Lactose concentrations are very constant in milk (4.6 to 5.2%). Lactose is a disaccharide comprised of one glucose molecule and
one galactose molecule joined by a beta linkage between the carbon 1 atom of galactose and the carbon 4 atom of glucose.
The biochemical pathways for lactose synthesis are demonstrated in the following graphic.




Step 1 in the sequence is an irreversible reaction. Steps 1 to 4 occur in the cytosol. Steps 2 to 4 are at
equilibrium and are reversible depending on the concentrations of the precursors and endproducts. Step 5
may be the rate limiting step and occurs in the lumen of the Golgi Apparatus. Glucose and UDP-Galactose
are transported into the lumen of the Golgi Apparatus and are acted upon by Lactose Synthase to form
lactose. Lactase Synthase contains two components: galactosyl transferase which is membrane bound and
alpha-lactalbumin which is free floating. Galactosyl transferase is capable of transferring UDP-galactose
to carbohydrate groups of glycoproteins in the Golgi Apparatus membrane. The alpha-lactalbumin mediates
the specificity of the galactosyl transferase for glucose such that the affinity for glucose increases by a
factor of 1000. The activity of galactosyl transferase is augmented by cations at 2 different sites. These
cations help to stabilize the conformation of the enzyme (Mn, Cu, Zn, Cd) and also to form a bridge
between the enzyme and substrate (Ca). Lactose synthase activity increases markedly during the onset of
lactation. During pregnancy the activity is suppressed through an inhibition of alpha-lactalbumin formation
by progesterone.

The availability of glucose in the Golgi Apparatus appears to limit lactose synthesis. Lactose Synthase is
not saturated by glucose in mammary secretory cells. The Golgi Apparatus transports (diffuses) small
molecules such as glucose quite freely, however, larger molecules such as UDP-galactose must be actively
transported, therefore, UDP-galactose concentrations may also limit lactose synthesis. Concentrations of
UTP and calcium may also limit substrate supply and the activity of the enzyme.

 PRECURSORS OF MILK LACTOSE



Quantitatively, glucose is the sole precursor of lactose, although some of the carbons of lactose, especially galactose, may
originate from acetate and glycerol. Glucose cannot be formed in the mammary gland because the mammary cells lack glucose-
6-phosphatase. Glucose within the mammary cell is used for lactose synthesis, for NADPH formation, for ribose-5- phosphate
formation (needed for RNA synthesis), and for glycerol formation (needed for triglyceride synthesis). NADPH and ribose- 5-
phosphate are synthesized in the cytosol through the Pentose Phosphate Shunt.


 REGULATION OF LACTOSE SYNTHESIS



Lactose appears in the mammary gland well before the end of pregnancy, however, major increases occur around the time of
                   parturition. Lactose concentrations increase to maximum concentrations until peak milk production has been
                   reached and then decrease thereafter. Lactose synthesis decreases rapidly during starvation, but returns to
                   near normal upon refeeding. The onset of lactose synthesis is correlated with the appearance of alpha-
                   lactalbumin. The synthesis of alpha-lactalbumin occurs under the influence of prolactin and glucocorticoids,
                   but these effects are overridden by progesterone during pregnancy. Factors that affect the general nutrition
                   of the dairy cow such as decreased protein or energy content in the diet may limit glucose production as well
as protein synthesis within the cow.




 BIOSYNTHESIS OF MILK FAT



<> Fat is one of the major components of milk and also the most variable. Milk fat is composed primarily of triglycerides (97-
98%). Bovine milk fat contains approximately 1% other lipids (0.28 - 0.59% diacylglycerides; 0.2 - 1.0 phospholipids; 0.02 -
0.04% monoacylglycerides). The general form for a triglyceride is demonstrated opposite.


R1, R2, and R3 vary in carbon length and also in degree of unsaturation. Milk of ruminants contains higher
proportions of short chain fatty acids and lower proportions of unsaturated fatty acids than non-ruminant.

 PRECURSORS OF MILK FAT



Milk fat can arise from the diet directly or from de novo synthesis within the animal. Approximately fifty percent of milk fat
arises directly from dietary long chain fatty acids (LCFA), from microbial synthesis of fatty acids, or from body stores. The
remaining 50% arises from de novo synthesis from volatile fatty acids (acetate and beta-OH butyrate which arise from microbial
fermentation within the rumen).




                                                                                                  <>


Most of the free fatty acids of plants in the diets of ruminants are LCFA and are usually highly unsaturated.
The rumen microorganisms are capable of quickly saturating these unsaturated fatty acids which largely
explains the high degree of saturation of the fat in milk of ruminants. Long chain fatty acids are absorbed
from the small intestine into the lymph system where they become bound to proteins to form lipoproteins.
These lipoproteins move into the blood and are absorbed from the blood by the mammary gland secretory
cell. Blood contains approximately 100 mg lipoproteins/litre of which 92% are high density lipoproteins, 7%
are low density lipoproteins, and 1% are very low density lipoproteins. Approximately 60 to 80% of the
triglycerides are removed from the lipoproteins and absorbed by the mammary tissue. This is accomplished
by lipoprotein lipase which is located on the capillary cell membrane and which is capable of hydrolyzing
triglycerides from very low density lipoproteins and chylomicrons and of facilitating uptake of hydrolyzed
fatty acids by the secretory cell. No significant uptake of free fatty acids, esterified sterols, or
phospholipids occurs in the mammary gland. Feeding whole canola or flax seed can alter the fatty acid
composition such that the proportions of unsaturated fatty acids are increased in milk.
 DE NOVO SYNTHESIS OF MILK FATTY ACIDS



De novo synthesis of milk fatty acids involves the provision of acetyl-CoA derived from glucose (in non-ruminants) and from
acetate (in ruminants) which acts as a primer for the reaction, as a substrate for the synthesis of malonyl-CoA which is required
for fatty acid elongation , and for provision of NADPH for the reductive steps of the pathway. The pathways to describe the
synthesis of fatty acids are illustrated in the following figure.
Glucose is the major source of acetyl-CoA and NADPH in non- ruminants and it also supplies the glycerol
backbone for triglyceride synthesis. Glucose is first degraded to pyruvate via glycolysis which then enter
the mitochondria to be converted to acetyl-CoA. Because acetyl-CoA cannot diffuse out of the
mitochondria it combines with oxaloacetate to form citrate which readily diffuses out of the mitochondria.
Within the cytosol, the citrate is acted upon by citrate lyase to form oxaloacetate and acetyl-CoA. NADPH
in non-ruminants is produced by the pentose phosphate pathway, the pyruvate/malate cycle, and the
Isocitrate dehydrogenase shuttle.

In ruminants, glucose is not used effectively for free fatty acid synthesis. The activity of ATP citrate lyase
is low, therefore, little acetyl-CoA is derived from extramitochondrial citrate. The pyruvate/malate cycle
and isocitrate dehydrogenase enzymes appear to be in adequate supply in ruminant tissue. In addition,
acetyl-CoA synthase activity is increased in ruminants and appears to be a more effective means of
generating acetyl-CoA from acetate produced by rumen fermentation of dietary nutrients.

The first committed step in the synthesis of fatty acids is the conversion of acetyl-CoA to malonyl-CoA by
acetyl-CoA carboxylase. The enzyme requires biotin, Mg/Mn as cofactors and it's activity is proportional
to the lipogenic capacity of the mammary gland. Elongation of the fatty acid requires the addition of more
acetyl-CoA molecules which have been activated to malonyl-CoA. The reaction is catalyzed by fatty acid
synthase. Palmitate (16 carbons) is the main product of de novo synthesis of fatty acids, as the presence of
acylthioesterase limits further elongation. Acetate provides 35 to 45% of the carbon of total milk fatty
acids or approximately 80% of de novo synthesized fatty acids.

Beta-hydroxybutyrate can be directly incorporated into milk triglycerides as butyryl-CoA (4 carbon unit)
or converted into acetyl-CoA in the mitochondria for elongation through malonyl- CoA. Beta-
hydroxybutyrate provides approximately 8% of the total fatty acids in milk, or 16 to 20% of de novo
synthesized fatty acids and seems to be the major source of carbons for short chain fatty acids.
Medium chain fatty acids (8 to 12 carbons) are produced by the same process as for palmitate, however, in
non-ruminants, an enzyme called medium chain acylthioesterase cause premature termination of elongation.
This enzyme is not found in ruminant mammary gland tissue. In ruminants, medium chain fatty acids are
released as acyl-CoA esters which can be incorporated directly into triglycerides (triacylglycerols) without
need for activation.

 TRIGLYCERIDE SYNTHESIS



Triglycerides are synthesized from fatty acids extracted from blood or from de novo synthesis, and from glycerol. Glycerol,
required as a backbone for triglycerides, is provided by lipolysis (30 to 50%) of absorbed triglycerides, and by glucose through
glycolysis (50 to 70%). Glycerol is activated to glycerol-6-phosphate by the enzyme glycerol-3-phosphate dehydrogenase.
Fatty acids not activated to CoA thioesters, must be activated by an ATP dependent acyl CoA synthetase. Formation of
triglycerides is associated with the endoplasmic reticulum.


Triglycerides are formed by 2 separate pathways. The alpha- glycerol-phosphate pathway involves the
esterification of 2 molecules of acyl-CoA to glycerol-3-phosphate to form phosphatidate
thiolysophosphatidate. Removal of phosphate by phosphatidate phosphatase causes a conversion to 1,2-
diacylglycerol. This is followed by the addition of a third acyl-CoA to from a triacylglycerol.
The monoacylglycerol pathway, on the other hand, esterifies 2 molecules of acyl-CoA to 2-monoglycerol
to form a triacylglycerol, however, the importance of this pathway has not been established.

Esterification of fatty acids on the glycerol molecule does not occur in a random order. Fatty acids of 4, 6
and 8 carbons are almost exclusively (95%) positioned on carbon 3 of the glycerol molecule, but
occasionally do occur on carbon 1. Fatty acids of 10 carbons are primarily positioned on carbons 1 and 3 of
the glycerol molecule in low molecular weight triglycerides (32 to 46 carbons in total) and on carbon 3 for
higher molecular weight triglycerides. Fatty acids of 12 to 16 carbons are primarily positioned on carbon 2
                       of the glycerol molecule. Fatty acids with 18 carbons (stearic 18:0, oleic 18:1, and
                       linoleic 18:2) are primarily found on positions 1 and 3.

                       Removal of hydrogen atoms (desaturation) results in double bond formation, hence the
                       term unsaturation. Mammary tissue of lactating cows contains desaturation enzymes
                       which are capable of converting stearate to oleate and palmitate to palmitoleate.
                       Desaturation may ensure sufficient fluidity of milk fat for efficient secretion from the
                       mammary gland.

 SYNTHESIS OF OTHER LIPIDS



Bovine milk contains 20 major fatty acids yet over 400 fatty acids have been detected. In addition to these, the mammary gland
is also capable of synthesizing phospholipids and cholesterol. Phospholipids, synthesized de novo, comprise less than 1% of total
milk lipids but are essential components of the milk fat droplet and membranes of the secretory cell. Cholesterol comprises less
than 0.5% of total milk lipids and can originate from de novo synthesis and from direct uptake from blood. Cholesterol is found
associated with the droplet membrane.




 BIOSYNTHESIS OF MILK PROTEIN



Milk protein generally comprises 3.2 to 3.5% of milk volume and is composed of specific proteins with casein being the most
important. Of the many proteins in milk, casein, beta-lactoglobulin, and alpha- lactalbumin make up approximately 95% of the
total protein.


These proteins are found only in milk and are synthesized in the mammary gland. Milk proteins contain
more essential amino acids than any other natural protein, and can be considered as nature's most ideal
protein.

Casein is found in various forms. Each form has a different function such as the stabilizing effect of kappa-
casein on micelles in milk to prevent curdling. Other proteins in milk such as blood serum albumin, immune
globulins, and epsilon-casein are not synthesized in the mammary gland but arise from blood.

 PRECURSORS FOR MILK PROTEIN



Milk protein can be synthesized from precursors provided by peptides, plasma proteins, and amino acids. Peptides and plasma
proteins provide approximately 20% of the amino acids for protein synthesis in the mammary gland, with free amino acids
providing the other 80%. Essential amino acids for incorporation into milk protein must be provided from dietary sources or
synthesized by rumen microorganisms. The essential amino acids are absorbed in sufficient quantities to account for all the
amino acids incorporated into milk protein. Non-essential amino acids are synthesized from other amino acids or from
carbohydrates. Amounts of some non-essential amino acids absorbed from blood are not sufficient to account for the quantities
incorporated in milk proteins suggesting that the mammary gland synthesizes these, however, the mechanisms are not well
understood.
 SYNTHESIS OF PROTEIN



Protein synthesis is very complex and beyond the scope of this course. Readers are encouraged to consult a recently published
Biochemistry textbook for more details. The capability of individual cows to synthesize protein will vary, however, an individual
cow will synthesize the same proteins throughout her productive life.


A generalized scheme for milk protein synthesis is as follows:

 Passage of precursors from blood capillaries to extracellular fluid.
 Cellular uptake of nucleic acids and amino acids. This process requires energy and may involve pinocytosis.
 Activation of amino acids by transfer RNA's to form specific amino acid - tRNA complexes.
 Intracellular synthesis of milk proteins involving transcription of DNA to mRNA and the translation of the mRNA to protein
using the amino acid - tRNA complexes.
 Translocation of the protein to the apical membrane. This involves membrane flow to the Golgi Apparatus and the processing
of casein within the Golgi Apparatus.
 Passage of protein across the apical membrane into the lumen via reverse pinocytosis.


Most of the protein is synthesized on the rough endoplasmic reticulum because of the association of
ribosomes (mRNA containing compounds) within the endoplasmic reticulum.

 REGULATION OF MILK PROTEIN SYNTHESIS



The amount of protein in milk is relatively constant, therefore, some form of control must be present in the mammary gland. The
control of protein synthesis is not well understood, although it is likely to involve feedback inhibition directly on enzyme function
as well as enzyme synthesis within the cell.




 SECRETION OF MILK COMPONENTS



Milk is secreted principally during the intervals between milkings. The rate of milk secretion and, therefore, the level of milk
production depends on the:
 availability of precursors in blood
 rate of blood flow to mammary gland
 uptake of precursors from blood by mammary gland
 the rate at which the mammary gland secretory cell transforms the blood precursors into milk components and discharges them
into the lumen of the alveoli.


 SECRETION OF MILK LACTOSE



Synthesis of lactose has been more difficult to localize within the mammary secretory cell because of it's small molecular weight,
therefore, much of what is known arises from biochemical evidence. Synthesis of lactose occurs in the Golgi Apparatus. The
Golgi Apparatus is also the major site for two proteins which, when combined, form the functional lactose synthetase enzyme
system. Galactosyl transferase is membrane bound and is located within the Golgi Apparatus. Alpha-lactalbumin is synthesized
by the rough endoplasmic reticulum and then transported to the Golgi Apparatus where it associates with galactosyl transferase.
The active Lactose Synthetase Complex catalyzes the reaction between glucose and UDP-galactose to form lactose. Because
the lactose is synthesized within the Golgi Apparatus, it is believed that lactose is secreted by reverse pinocytosis.


 SECRETION OF MILK FAT



Fat present is milk is found primarily in the form of fat droplets. Small amounts of free fatty acids may also be found.


Within the mammary gland secretory cell, the smallest droplets of fat are present near the basal membrane
and the largest droplets appear near the apex. Fatty acids are esterified within the endoplasmic reticulum
and contribute to the small droplets as the droplets migrate to the apex of the cell. The increase in size of
droplets may also result from the aggregation of separate droplets.

Near the vicinity of the apical membrane, strong attractive forces (Landon - Van-der-Walls forces) cause
the droplets to be enveloped by plasma membrane. At a certain point, the droplets become completely
surrounded by plasma membrane and are released into the lumen of the alveoli. During the enveloping
process, small quantities of cytoplasm may be trapped, although cytoplasm is not always trapped. When
cytoplasm is trapped, the droplets are called signets.

 SECRETION OF MILK PROTEIN



Proteins are synthesized by the rough endoplasmic reticulum and pass to the Golgi Apparatus. The means by which this
movement occurs is not clearly defined. Possibly the peptide chains pass through the lumen of the rough endoplasmic reticulum
directly into the Golgi apparatus, or the pinching off of endoplasmic reticulum may form vesicles which either migrate to and
merge with the Golgi Apparatus or which become a Golgi Apparatus directly. The Golgi apparatus migrates to the apical
membrane where it fuses to the plasma membrane. Then through the process of reverse pinocytosis, the proteins are released
into the lumen of the alveoli. At this point, the Golgi Apparatus becomes part of the plasma membrane and serves to replace, at
least in part, the plasma membranes lost during the formation and secretion of fat droplets.


 SECRETION OF MAJOR MINERALS



The minerals in milk are found in varying proportions and forms.
                   Na, 0.05%, 100% soluble
                   K, 0.15%, 100% soluble
                   Ca, 0.12%, 25% soluble
                   P, 0.10%, 45% soluble
                   Cl, 0.11%, 100% soluble
                   Mg, 0.07%, 20% soluble Calcium and magnesium are insoluble because of their association with caseinate,
                  phosphate and citrate.


                    Milk has an increased concentration of potassium and a decreased concentration of
                    sodium which is the reverse of what is found in extracellular fluid. An increased K/Na
ratio in alveolar cells could mean that these ions are derived from intracellular fluid rather than a leakage
of extracellular fluid between alveolar cells. The presence of tight junctions and hemedesmosomes
between alveolar cells as detected by electron microscopy greatly impede the leakage of extracellular fluid
into the lumen of the alveoli. The high K/Na ratio may be explained by the need to maintain an electrical
gradient between the cell interior (negative) and the extracellular fluid (positive).

Carrier systems pump Na outward and potassium inward using energy in the form of ATP to accomplish the
electrical gradient. Sodium pumps have been detected on the basal and lateral surfaces of the alveoli cell
but not on the apical surface. Enzyme systems (Na - K - ATPase) associated with the Na pump have also
been found on the basal and lateral surfaces. The lack of active carrier systems on the apical surface
translates into a mechanism which lacks the ability to conserve K and to extrude Na. Ions, therefore, can
diffuse freely across the apical membrane into the lumen down their respective concentration gradients. In
fact analyses of the interior of the mammary secretory cells and milk show identical K/Na ratios.
Chlorine diffuses down a concentration gradient from intracellular fluid to milk, however, a carrier system
which exists at both the basal and apical surfaces actively pumps Cl back into the cell. The mechanisms by
which Ca, P, and Mg are transported into milk are not well understood. An increased concentration of these
minerals above those found in blood indicate the presence of active transport systems. The formation of
mineral complexes with casein (to stabilize the micelle) may decrease the soluble concentration within the
cell and allow for some flow of these minerals down a concentration gradient.

 SECRETION OF WATER



Milk is comprised of 86 to 88% water. Because milk is isotonic with blood, water transport across the apical membrane is
governed by osmotic pressure associated with solutes secreted into milk. Fat and protein molecules are too large to exert
osmotic pressure. Lactose (MW of 342) and free ions exert high osmotic pressure and largely determine the movement of water
into the lumen. Lactose is believed to be the primary osmotic regulator of milk volume in the dairy cow.


 SECRETION OF COLOSTRUM



Colostrum is characterized by increased concentrations of serum proteins (immunoglobulins) and Na, and decreased
concentrations of casein, lactose, and K. The serum proteins are believed to enter milk as a result of conditions which inhibit the
removal of products of secretion of the alveolar cells from the mammary gland, and the breakage of the tight junctions.
Equilibration of milk with extracelluar fluid accounts for the high Na/K ratio and the increased concentrations of serum proteins
in colostrum.
        REABSORPTION OF SECRETORY PRODUCTS



      The mammary gland is unlike most other exocrine glands (eg. salivary glands) because it's duct system is not capable of
      reabsorbing secreted products. The cells of the duct system show diminutive microvilli, few mitochondria, and sparse
      endoplasmic and, therefore, have minimal capacity to absorb nutrients from the lumen of the ducts.


                                 MAMMARY GLAND STRUCTURE AND FUNCTION

 The objective of this module is to allow the student to gain an appreciation of the intricacies of mammary gland structure and growth,
                                                  and synthesis of milk components.

                                               Growth and Development
                                               Anatomy
                                               Vascular, Lymphatic, and Neural Systems
                                               Cytology of the Mammary Secretory Cell
                                               Hormones Involved in Lactation
                                               Milk Synthesis and Secretion
                                               References



GROWTH AND DEVELOPMENT OF THE MAMMARY GLAND


Mammary gland growth occurs during five distinct phases of development. These phases include Prenatal,
Prepubertal, Postpubertal, Pregnancy,and Early Lactation.
PRENATAL



Development of the mammary gland begins early during embryonic and fetal growth. A thickening of ectodermal cells occurs when a
calf is 1.4 to 1.7 cm long (30 days after conception). The cells continue to grow and aggregate to form lines of mammary tissue on each
side of the midline (figure to appear later). Four structures called mammary buds form from differentiated cells which have progressed
through crest and hillock stages in distinct areas of the mammary lines. Mammary buds are precursors of the secretory protein of the
mammary gland, therefore, the number of buds determines the number of mammary glands. Buds divide into primary sprouts (figure to
appear later) which differentiate into teat and gland cisterns. Several secondary sprouts arise from primary sprouts to form mammary
ducts. The sprouts become hollow shortly before birth of the calf.


PREPUBERTAL



At birth, the number of cells needed to form connective tissue and fat (non-glandular portion) of the udder are established. The
glandular portion of the mammary gland is rudimentary at this stage. From birth to 3 months of age, growth of the duct system
continues at a rate equal to body growth. From 3 months of age to 9 months of age, growth of mammary tissue exceeds that of the body
by 3.5 fold.


POSTPUBERTAL



Growth of mammary tissue increases rapidly after puberty as estrogen and progesterone exert their influences on duct and secretory
tissue. While duct and lobular growth is occurring at this time, alveoli are not formed until pregnancy is established.


PREGNANCY



Growth of mammary tissue is very rapid during pregnancy. The increase in udder size becomes obvious after 3 to 4 months of
pregnancy in heifers, although it is not until secretions of mammary tissue cause major increases in size during the 7th to 9th month of
gestation that the udder is highly visible.
                                                                EARLY LACTATION



                                                                Lactation is associated with increased mammary cell numbers. Cell
                                                                numbers increase until peak lactation, at which time, mammary cell loss
                                                                exceeds the rate of cell division and milk production decreases.


                                                                Because most cows are bred during the first 40 to 90 days
                                                                post-partum, a major portion of lactation co-exists with
                                                                pregnancy. Initial stages of lactation have little effect on
                                                                mammary cell numbers, however, a decrease in cell
                                                                numbers occurs relative to non-pregnant cows after 5
                                                                months of pregnancy.

DRY PERIOD



The dry period is a stage in the lactation cycle when cows are no longer being milked. This usually occurs when cows have been milked
for 10 to 12 months (7 months pregnant) and are about 2 months from next expected calving. The cessation of milking causes the udder
to become engorged with milk (a process taking several days depending on level of milk production). The subsequent decrease in
metabolic activity causes a degeneration and loss of alveolar epithelial cells. Myo-epithelial cells and connective and adipose tissues
remain, with the adipose and connective tissues becoming more prominent as the alveoli degenerate. Complete involution (degeneration)
of the alveoli occurs within 75 to 90 days in the non-pregnant cow. Pregnancy stimulates udder growth, therefore, complete involution
is not attained if cows are approximately 7 months pregnant at the start of the dry period. Involution of the mammary tissue is important
for subsequent lactation and a dry period of 45 to 75 days is necessary to maximize milk yield in the next lactation. The absence of a
dry period interferes with the increase in cell numbers that normally occurs during the early stages of lactation.


REFERENCES

   Schmidt, G.H. Biology of Lactation. W.H. Freeman and Company. San Francisco, California.
   Keenan, T.W., and D.P. Dylewski. 1985. Aspects of intracellular transit of serum and lipid phases of milk. J. Dairy Sci. 68:1025-
1040.
   Davis, S.R., and R.J. Collier. 1985. Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci. 68:1041-
1058.
   Kuhn, N.J., D.T. Carrick, and C.J. Wilde. 1980. Lactose synthesis: The possibilities of regulation. J. Dairy Sci. 63:328- 336.
   Smith, S. 1980. Mechanism of chain length determination in biosynthesis of milk fatty acids. J. Dairy Sci. 63:337-352.
   Khorasani, G.R., P.H. Robinson, G. de Boer, and J.J. Kennelly. 1991. Influence of canola fat on yield, fat percentage, fatty acid profile,
and nitrogen fractions in Holstein milk. J. Dairy Sci. 74:1904-1911.
   Grummer, R.R. 1991. Effect of feed on the composition of milk fat. J. Dairy Sci. 74:3244-3257.
   Jensen, R.G., A.M. Ferris, and C.J. Lammi-Keefe. 1991. Symposium: Milk fat - composition, function, and potential for change. The
composition of milk fat. J. Dairy Sci. 74:3228-3243.
   Neville, M.C., and C.W. Daniel. 1987. The Mammary Gland: Development, Regulation, and Function. Plenum Press, New York.

				
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