The changes in the cytology of the adrenals of Rana esculenta

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					Zoology. - The changes in the cytology of the adrenals of Rana esculenta
      following hypophysectomy. By J. W. SLUITER. J. C. A. MIGHORST
      and G. J. VAN OORDT. (Zoological Laboratory. Dept. of Endo-
      crinology. University of Utrecht.) (Communicated by Prof. CHR.
      P. RAVEN.)

              (Commwlicated at the meeting of November 26. 1949.)

   1. Introduction.
   Following investigations into the regulation of spermatogenesis in the
Erog. the influence of the pituitary on the adrenals of the green frog.
Rana esculenta. has now been studied.
   The literature on this subject relates particularly to experiments with
hypophysectomized Rana-Iarvae. in which a regression of the interrenal
tissue has been observed (SMITH. 1920). Af ter the administration of
adrenocorticotrope hormone the adrenals soon recover (SMITH and SMITH.
1923; ATWELL. 1935).
   Similar results have been obtained in adult Amphibians. According to
ADAMS and BoVD (1933) the size of the nuclei of the interrenal cells of the
adrenals.as weIl as their lipoid content. decreases in Triturus viridescens.
and PORTO (1940) has described a hypofunction in the adrenal lipoid cells
(the interrenal ceIls) af ter removal of the anterior lobe of the pituitary in
Bufo arenarum. Following injections of extracts of the anterior lobes of
the pituitaries of both toads and mammals these cells resume their normal
function.
   It is our intention to trace the regression of the adrenal cells af ter
hypophysectomy in adult specimens of Rana esculenta during a period of
2-9 months following the operation. Before describing these changes.
however. it may be usefuI. in view of the KonsuloH-test. which is used
more and more for clinical purposes. to describe at length MIGHORST'S
method of hypophysectomy (MIGHORST et al.. 1949). This method is
somewhat simpIer than that used by SERVANTIE et al. (1947) and other
workers.

   2. Material and methods.
   On act. 11 and 13. 1948 more than 100 specimens of Rana esculenta
were hypophysectomized in the following manner.
   Af ter the frog has been anaesthetized by means of saturated ether-
vapour and has been laid on a cork-plate. its mouth is opened as widely
as possible with the help of two curved pins. which are hooked behind the
upper and lower jaw respectively. The pin holding the lower jaw is connected
to a string. fastened on the cork-plate behind the anima 1. Then the mucous
                                          1215

membrane of the palate is cut open in the median line from A to Al
(fig. 1) l) and the margins of the cut are pulled aside. Next, the para~
sphenoidal bone is superficially incised with a pair of scissors a t the points
I , 2 and 3, indicated in fig . I , and, using a pair of strong tweezers , is




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                                            -;---;fff7---+--\+--   a jJa/ö I
                                             ""'-''+r-Il<'----;-      -H-- jJarasph


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     Fig. 1.   R ana esculenta. The pa rasphenoidal bone, as se en before excision.

broken oH here and at its joint with the first vertebra. The musculu5'
retractor bulbi is cut, wh ere it joins the parasphenoida l bone. Of course it
is important not to damage the arteries.
   Under a dissection microscope a horseshoe-shaped section (the opening
of the hors es hoe directed forward) is made in the cartilage, lying under
the parasphenoidal bone. That part of the cartilage cut out is bent forward
and kept in this position with the aid of a preparation needIe. Now the
brain is visible (fig. 2 in which , however, this cartilage is not shown).
The entire pituitary is removed with the help of a small pipette (the
diameter of which is of about the same size as the pituitary) and a narrow
rubber tube by means of the adjustable sucking force of the operator's
mouth.
   After hypophysectomy the situation is as in fig. 3. The cartilaginous
lid is th en replaced in its natural position, and the wound in the mucous
membrane in the palate is sewn up with surgical thread. Next the animals
are placed separately into glass bowls with about 3 cm water, in which they
rapidly recover.

  1) To c1a rify the situation cuts perpendicular to line AAl a re also draw n in fig . 1.
but it is not necessary to make these cuts during the operation.
                                          1216




Fig. 2.   R,ana esculenta. The pituitary as seen af ter the removal of the parasphenoidal
                 bone and the partial removal of the underlying cartilage.




    Fig. 3.   R,ana esculenta. The base of the brain as seen af ter hypophysectomy.
                                          1217

   In Rana esculenta the mortality following the operation is about 10-20
per cent; during the breeding season, however, it is very high and of ten
reaches 100 per cent. Thus, it is ohvious that during the breeding period the
pituitary has a much greater influence on the frog's body than at other
times.
   The hypophysectomized frogs were kept together with controls in a
large vivarium at room temperature and fed copiously with mealworms,
which were taken actively. Some experimental frogs were kept alive until
July 1949 and so survived the operation for about 9 months. Experimentals
and controls were killed at regular intervals and their adrenals fixed in
Kolster's fIuid, sectioned (3,t) , and stained with Altmann's acid-fuchsin
combined with brilliant-cresyl-blue.

  3.   Results.
   The normal adrenal. The adrenal of the frog is composed of tissue
strands which are separated by large blood lacunae. In these strands many
interrenal and few suprarenal cells are to be found. Thus a true cortex
and medulla are absent (fig. 4a).




Fig. 4. Rana eseulenta. Cross sections of adrenal strands. The size of each fig. is
approximately proportional to the size of the adrenals. Fig. 4a: Control-specimen, killed
Dec. 7, 1949. Figs 4b and 4e: experimentals, hypophysectomized on Oct. 13, 1948, and
killed respectively on Dec. 7, 1948 and May 23, 1949. Fixation: Kolster's fluid ; Altmann-
                                   staining. (X 2000.)

   As in the higher vertebrates the interrenal cells are characterized by
lipoid droplets which fill these cells totally, while the suprarenal ce lis
possess a chromophil plasm in which lipoid droplets are lacking. In addition
to these two cell types, so-called "summer-cells" are scattered in the frog's
adrenal. These are filled with numerous fuchsinophil granules.
                                          1218

   From October till July only insignificant fluctuations in the lipoid content
take place in the interrenal cells of frogs caught in the wild and of normal
control frogs kept in confinement in the laboratory.
   The adrenals following hypophyseetomy. The first differences between
adrenals of norm al controls and of hypophysectomized frogs were visible
about 2 months af ter the operation. On Dec. 7. 19i8 some of the experi-
mentals possessed adrenals in which several interrenal cells showed
shrinkage of the nuclei and an almost total absence of lipoid droplets. but
the suprarenal and summer-cells were still normal (fig. ib).
   On Jan. i, 1949 the nuclei of all 3 cell types in the experimental frogs
were distinctly smaller than those of con trol specimens: lipoid droplets
were scarce and the whole gland had shrunk.
   In March the adrenals showed no distinct deviations from those in
January. but during May 1949 the regression of the adrenals had
proceeded much further (fig. ie). Then most of the cells possessed still
smaller nuclei and still less protoplasm in which the mitochondria were
strongly concentrated; lipoid droplets were al most entirely lacking.
Summer-cells and interrenal cells were difficult to distinguish from each
other; the suprarenal cells were less affected.
   The cytology of the adrenals in the experimental frogs in July was about
the same as that of the adrenals in the experimental frogs in May. The
cells were reduced in si ze. but nevertheless some still contained small
numbers of lipoid droplets. Consequently it is evident that the adrenal
regression following hypophysectomy in October reached its maximum
in May of the following year.

  Summarizing, we can conclude that af ter hypophysectomy the cytology
of the adrenals shows several typical features, suggesting a hypofunction
of the cells. First. about 2 months af ter the operation the interrenal cells
are affected; later the two other celI types (suprarenal and summer-cells)
also show a distinct regression . Maximum adrenal regression does not
occur until 7 months af ter hypophysectomy.

   Aeknowledgement. We wish to express our sincere thanks to Or W. S.
BULLOUGH   (Sheffield) for correcting the English text of the present paper.

                                     REFERENCES.

ADAMS, A. E . and E. M. BOYD: Changes in ad ren als of newts following hypophysectomy
         or thyroidectomy. An. Rec., 57, Suppl. p. 34 (1933).
A-rwELL, W . J.: Effects of thyrotropic and adrenotropic principle on hypophysectomized
         Amphibia. An. Rec., 62 (1935).
MIGHORST, J. C. A .• L. A. M. STOLTE, P. H. M. DE Roo and F. CREUTZBERG: The
         value of the melanophore reaction in Rana esculenta (Konsuloff-reaction) as a
       . pregnancy test. Acta Endocrinol., 2 (1949) .
PORTO. J.: Relaciones entre la hipófisis y la adrenal en el sapo. Rev. Soc. Argent. Biol..
         16 (1940) .
                                         1219

SERVANTIE, L.. R. CAMBAR, G. F. MORETTI et R. BONNAL: Technique d·hypophysec-
        tomie chez la grenouiIIe. Mise en application du test des mélanophores. Causes
        d'erreur, intérêt. Biol. méd., 34 (1947) .
SMITH, P. E. : The pigmentary growth and endocrine disturbances induced in the Anuran
        tadpole by the early ablation of the pars buccalis of the hypophysis. Amer. Anat.
        Memoirs, 11 (1920).
SMITH, P. E . and I. P. SMoITH : The function of the lobes of the hypophysis as indicated
        by replacement therapy with different portions of t!1e ox gland. Endocrinology,
        7 (1923) .




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