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Ovarian androgen in parabiotic mice.

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Ovarian Androgen in Parabiotic Mice
JEAN C. DESCLIN, JR.
Department of Histology, Faculty of Medicine, Brussels University,
Brussels, Belgium
Human ovaries, as well as those of many
animal species, seem to produce male
hormone. Spontaneous virilization of female mammals has been described. Under
experimental conditions, the administration of gonadotrophins to females, or the
implantation of ovaries into castrated
males developed or maintained a functional state of the male accessory sex organs of laboratory rodents. Parkes (’50)
and Ponse (’55) have ably reviewed the
literature concerning these problems.
Androgen is present in pregnant rats
(Price, ’41; Burrill and Greene, ’42) and
mice (Takewaki, ’41, ’51; Desclin, ’58).
During lactation the ovaries of these rodents secrete male hormone (Price, ’47;
Desclin, ’58).
The ovaries of rats parabiotically united
with a castrated partner are stimulated by
the pituitary of the castrate; they secrete
great quantities of male hormone, and they
contain numerous corpora lutea (Johnson,
’58). In the mouse, however, parabiotic
union with a gonadectomized partner of
either sex rapidly brings about permanent
oestrus; the ovaries of the intact parabiont
contain large follicles, but no corpora lutea
(Takewaki, ’36).
In the present experiment female mice,
both with normally located ovaries, and
with ovaries transplanted into the submandibular glands, were united in parabiosis with a spayed or castrated animal,
and ovarian androgen production was evaluated on histological examination of the
submandibular glands. The mandibular
salivary gland is a very sensitive target
organ for male hormone (Lacassagne, ’40;
Desclin, ’60).
MATERIAL AND METHOD
Adult male and female mice of the C5,BL
strain, and of the (C5,BL X CBA)F1 and
(C5,BL X DBAJF, hybrids were used. Thirty-
eight mice were divided into 4 groups as
follows: 13 control females between 13 and
19 weeks old, sxrificed without previous
treatment (group I ) ; 7 females spayed at
8 weeks with their ovaries autografted into
their submandibular glands, sacrificed 6
weeks after the operation (group 11); 9
females parabio tically united at 6 weeks
with a spayed female of the same age, according to the method of Bunster and
Meyer (’33); thl: pairs were sacrificed 28
days later (group 111); 9 females spayed at
6 weeks, with their ovaries transplanted to
the submandibu lar glands, were parabiotically united at 10 weeks with castrated
males of the same age; the pairs were
sacrificed 26 da y~slater (group IV).
The submandibular glands and ovaries
of all the animds were removed at necropsy, fixed in Susa fluid, embedded in
paraffin, and sectioned for histological examination. The ovaries were sectioned
serially. All sections were stained by the
periodic acid-Sc hiff technique and counterstained with E:hlrich’s haematoxylin and
ponceau red.
The increased ratio of the surface of
serous tubules to the surface of acini in
the submandibular gland indicates the
presence of androgenic hormone.
Chalkley’s method (’43 ) , slightly modified however, was used to establish this
ratio. Instead of the microscope eye-piece
devised by this author, the “Lanameter”’
apparatus was used. The picture of the
submandibular gland was projected at a
magnification OF X 500 on the opaque
glass screen of :his instrument. A transparent sheet with 25 points distributed at
random over a given area was placed over
the projected image, and the coincidence
of the points wi1.h the various tissue com1 Commercial narie of the projecting microscope
devised and dist -ibuted by Reichert, Wien,
Austria.
305
306
JEAN C . DESCLIN, JR.
ponents was noted. The ratio of serous
ducts to acini was calculated on the basis
of 500 points incident on serous tubules.
Results obtained by this method were compared with those obtained by planimetry
on similar material, previously reported
(Desclin, ’58, ’59, ’60). This method
proved to be very satisfactory as compared
with planirnetry, and is far more accurate.
RESULTS
Submandibular glands
Single animals with ovaries autografted
into their submandibular glands (11) did
not differ significantly from the controls
( I ) ; (see table 1 and fig. 1 ) . The submandibular glands of the animals united
with a castrated partner (I11 and IV) exhibited a definite masculinization as compared with the controls, both when the
ovaries were left in situ, and when they
were transplanted into the submandibular
glands (fig. 2 ) .
___
~
~~
The submandibular glands of the intact
partners of parabiotic pairs exhibited less
masculinization when the ovaries were
transplanted than when they were left in
situ (tables 1 and 2).
Ovaries
Transplanted ovaries in single animals.
Ovarian activity resumed in all animals 10
to 14 days after the operation, as evidenced
by daily vaginal smears. During the first
few days of detectable activity, while the
graft was still “taking,” oestrus was prolonged, but from the 20th day on, the cycles
were regular in all the animals. At necropsy, histologically the ovaries were essentially normal. Corpora lutea were present
along with follicles of various size. No
detectable changes could be found in the
interstitial tissue.
Ovaries in situ in the intact members of
the parabiotic pairs. Vaginal smears evidenced permanent oestrus appearing 5 to
TABLE 1
Mean ratios of serous ducts to acini in the submandibular glands
~
_
_
_
Experimental group
-~
I
II
13
7
~~
Number of animals
-~
__ ._- 20.28 & 1.06
__
IV
__-___
I11
~-
~
9
9
44.3 ? 1.56
35.2? 1.23
.-
Mean’ +- S.E.M.2
Mean
?
S.E.M. (Log)3
20.03 2 0.56
1.30554i-0.0118
1.30597i0.0224
1.64455+-0 01425
1.54477*0.01531
between I and 11: not significant
between I , 11, and 111, I V : p less than 0.01
between I11 and I V : p less than 0.01
p values4
__
-.
-.
Ratio (serous ducts/acini) :
:100.
Standard error of the mean.
Since the variance increased with the mean, the measures were replaced by their logarithms.
p values for the significance of the difference between means were established by the test of
Duncan (’55).
TABLE 2
Analysis of variance’
Source of variation
~
_
Between experimental groups
(experiment )
Within experimental groups
(error)
Totals
_
Sum of
squares
~
Degrees of
freedom
~
Mean
squares
F
127
840,342.40
3
280,114.13
74,561.18
34
2,192.94
914,903.58
37
All measures were replaced by their logarithms.
OVARIAN ANDROGEN I N PARABIOTIC MICE
7 days after parabiotic union, and persisting in all the animals until the end of the
experiment. At necropsy, the ovaries were
greatly enlarged, and the ovarian capsules
and uterine horns were distended with
fluid. On histological examination, the
ovaries contained numerous large follicles
and cysts, some of which were haemorrhagic. No corpora lutea were seen. The
interstitial tissue was highly developed,
consisting of cords of clear acidophilic
cells filled with large lipid droplets. In
two cases, remnants of corpora lutea were
observed as small clusters of cells with
dense, deeply acidophilic cytoplasm. These
clusters of acidophilic cells were surrounded by large masses of clear vacuolated cells (see fig. 5). Granulosa cells
exhibited numerous mitoses, and in some
follicles, they looked like lutein cells.
Ovaries transplanted into the submandibular glands in parabiotic pairs. Permanent oestrus after parabiotic union was induced as rapidly as in the former group,
and lasted during the entire experiment in
all the mice. At necropsy, the uterine
horns were greatly enlarged and distended
with fluid; the ovarian grafts were large,
and protruded from the surface of the
salivary glands. In 6 cases out of 9, the
grafts were filled with blood, and corpora
lutea were seen as white dots on the dark
surface of the graft. Histologically, the
ovaries differed from those seen in the
former group. In the 6 animals mentioned
above, large true corpora lutea were present along with cystic follicles (fig. 4).
These corpora lutea were formed by large
vacuolated cells with a clear cytoplasm.
The interstitial tissue was fairly well developed, consisting of numerous and large
clusters of polyhedral cells with a dense
acidophilic cytoplasm (fig. 6). These cells
resembled those of corpora lutea. In the
three remaining animals of this group, the
ovaries exhibited a picture similar to that
observed in the intact partner of parabiotic
pairs with normally located ovaries (fig. 3 ) .
DISCUSSION
In single female mice, the ovaries autografted into the submandibular glands
failed to masculinize this target organ. The
influence of a low temperature on the production of androgen by grafted ovaries has
307
been both asserted (Hernandez, '43; Hill,
'37; Lampton and Miller, '40) and denied
(Deanesly, '3El). In our short-term experiments, in spite of the fact that the transplanted ovaries were under the skin, and
therefore, at a lower temperature than in
their normal abdominal location, no masculinization ensued. In the parabiotic
pairs also, the transplantation of the ovaries did not enhance masculinization. Thus
temperature did not seem to influence
androgen prod xtion.
In the parabiotic pairs, increased gonadotrophin secretion by the pituitary of
the castrated co-twin induced permanent
oestrus and masculinization of the submandibular glz nds in the female partner.
This last effect cannot be due to continuous oestrogen secretion : long lasting treatment with oestrogens does not affect the
structure of tht: serous tubules of the submandibular glands (Lacassagne, '40;
Chamorro, '46). Our observations are in
agreement with those recently reported by
Johnson ('58) 3n the prostates of parabiotic female and of castrated, ovarian graftbearing male rats.
The adrenals cannot be excluded as a
possible source of androgens. Unfortunately, the adrc nals of our animals were
not weighed, but no enlargement of this
organ was noticed at autopsy in either
member of the parabiotic pairs. Furthermore, under the present experimental conditions, important changes primarily mvolved the ova pies. Cyst-formation and
interstitial tissue hypertrophy were accompanied by high aestrogen and androgen secretion. The absence of corpora lutea reported in earlier work by Takewaki ('36)
was confirmed. Takewaki also observed
the disappearance of the adrenal X-zone of
female mice united with a gonadectomized
parabiotic partntr. He did not ascribe this
effect to androgenic hormone, though it is
now considered hy most authors as indicative of the prer;ence of male hormone
(Chester Jones, 57).
With regard to the origin of the androgens within the ovary, though ovaries containing active corpora lutea may be masculinizing (Desclin, ' 6 0 ) , we may assume
that corpora lutea are not necessary for the
production of ovarian male hormone. The
most striking changes that occurred in nor-
308
JEAN C . DESCLIN, JR.
mally located ovaries, as well as in transplanted ones, involved the interstitial tissue. In normally located ovaries, the
hypertrophied interstitial tissue consisted
chiefly of large vacuolated acidophilic
cells. In some of these ovaries remnants
of corpora lutea appeared as small clusters
of dense acidophilic cells. In most transplanted ovaries, on the other hand, the
interstitial tissue was formed predominantly of cells with a dense acidophilic
cytoplasm devoid of detectable droplets.
Both types of cells, the acidophilic vacuolated ones and the unvacuolated ones have
been well described by Pfeiffer and Hooker
('42) in ovarian grafts in A-strain mice
injected with pregnant mare's serum.
These authors attributed the androgen secretion to the vacuolated cells. Our observations support this view, since transplanted ovaries containing predominantly
eosinophilic cells with a dense cytoplasm
were not as masculinizing as ovaries with
vacuolated cells predominating. The origin
of these cells is still obscure, though in
other strains, they seem to arise from old
corpora lutea (Pfeiffer and Hooker, '42).
At present, only a hypothetical explanation for the difference in appearance between transplanted ovaries and normally
located ones can be advanced. The sex of
the gonadectomized parabiont has no influence on the structure of the ovary of the
female co-twin (Takewaki, '36) ; therefore,
the use of castrated males in one experimental group, and of spayed females in
the other one, provides no explanation for
the difference between ovarian responses
in both groups. Mechanical disturbance
could possibly interfere with the graft's
response to intense gonadotrophic stimidation, though in single females the cyclic
activity of the transplanted ovary appeared
to be normal.
SUMMARY
1. Spayed mice, bearing ovaries autografted to the submandibular glands, and
which exhibited regular vaginal cycles,
failed to exhibit any masculinization of
the serous ducts of their submandibular
glands.
2. Female mice, united in parabiosis
with a gonadectomized partner, exhibited
a permanent oesirus, and virilization of
their submandibular glands developed,
both when their ovaries were transplanted
to the salivary glands, and when they were
left in situ. Transplanted ovaries, however,
exerted a less virilizing action.
3. Ovaries in situ, subjected to intense
gonadotrophic stimulation by the pituitary
of a gonadectomized parabiotic partner,
underwent cyst-formation, interstitial tissue hypertrophy; the interstitial tissue
consisted chiefly of large, clear vacuolated
cells. Corpora lutea were absent.
4. In 6 cases out of 9, transplanted ovaries in mice parabiotically united with a
gonadectomized partner contained corpora
lutea; the hypertrophied interstitial tissue
consisted predominantly of dense acidophilic cells.
5. The ovary seems to be the only important source of male hormone appearing
in female mice united in parabiosis with a
gonadectomized partner.
6. The site of production of male hormone within the ovary is discussed.
ACKNOWLEDGMENTS
The experimental part of this work was
performed at the Laboratory of the Antoni
van Leeuwenhoekhuis, The Netherlands
Cancer Institute, Amsterdam, Holland.
The author is deeply indebted to Professor
Dr. 0. Muhlbock and to Dr. L. M. Boot for
their kind hospitality and generous supply
of material. Professor L. Martin's (Brussels) help in the statistical analysis of results is gratefully acknowledged.
LITERATURE CITED
Bunster, E., and R. K. Meyer 1933 An improved
method of parabiosis. Anat. Rec., 57: 339.
Burrill, M. W., and R. R. Greene 1942 Androgen production during pregnancy and lactation
in the rat. Ibid., 83: 209-227.
Chalkley, H. W. 1943-1944 Method for the
quantitative morphologic analysis of tissues.
J. Nat. Cancer Inst., 4: 47-53.
Chamorro, A. 1946 L'action androghne stimulante des extraits gonadotropes sur l'ovaire.
C. R. SOC.Biol., 140: 25-27.
Chester .Tones, I. 1957 The adrenal cortex.
Cambridge University Press.
Deanesly, R. 1938 The androgenic activity of
ovarian grafts in castrated male rats. Proc.
Roy. SOC. London Series B., 126: 122-135.
Desclin, J. C., Jr. 1958 Masculinisation des
glandes sous maxillaires chez la souris femelle
pendant la gestation et la lactation. C. R. SOC.
Biol., 152: 1844-1848.
OVARIAN ANDROGEN IN PARABIOTIC MICE
1959 Masculinisation spontanee et experimentale des glandes sous maxillaires chez
la souris femelle. C. R. Acad. Sc., 248: 597-600.
1960 Recherches sur la masculinisation spontande et experimentale des glandes
sous maxillaires chez la souris femelle. Arch.
Biol. (LiGge), 61; 235-268.
Duncan, G. 1955 Multiple range and multiple
F tests. Biometrics., 11: 4.
Hernandez, TH. 1943 Hormonal ambisexuality
of ovarian grafts i n female rats. Am. J. Anat.,
73: 127-151.
Hill, R. T. 1937 Ovaries secrete male hormones. 11. Temperature control on the male
hormone output by grafted ovaries. Endocrinology, 21: 633-636.
Johnson, D. C. 1958 Ovarian androgens i n
parabiotic rats. Endocrinology, 62: 340-347.
Lacassagne, A. 1940 Mesure de l’action des
hormones sexuelles sur la glande sous maxillaire de la souris. C. R. SOC.Biol., 133: 227229.
Lampton, A. K., and A. J. Miller 1940 The influence of the temperature on the internal secretory activity of transplanted ovaries in the
female rat. Endocrinology, 26: 519-522.
309
Parkes, A. S. 15150 Androgenic activity of the
ovary. Recent Progress in Hormone Research,
5: 101-114.
Pfeiffer, C. A., and C. W. Hooker 1942 Experiments on the scurce of ovarian androgen in the
mouse. Anat. F,ec., 83: 543-571.
Ponse, K. 1955 La fonction androgene de
l’ovaire chez l’animal. Rapports de la IIIe
RBunion des Endocrinologistes de Langue
Frangaise, pp. 89-138. Masson editor, Pans.
Price, D. 1941 :3at prostate and seminal vesicle
graft i n relation to the age and sex of the host.
Physiol. Zool., 11: 145-161.
1947 The influence of maternal hormone on the reproductive organs of suckling
rats. Anat. Rec., 97: 519-545.
Takewaki, K. 1936 Sex gland and adrenal of
parabiotic mice. J. Fac. Sc. Imp. Univ. Tokyo,
4: 263-275.
1941 M, totic activity in seminal vesicle
cells transplantzd to female mice. Ibid., 5:
291-303.
1951 The source of the substance stimulating cell divjsion in seminal vesicle transplants in pregnant mice. Annot. Zool. Japon.,
24: 85-88.
-
PLATE 1
EXPLANATION OF FIGURES
1 Submandibular gland of adult female mouse. No treatment. Narrow
red stain. X 600.
serous tubules. P.A.S.-Haematoxylin-ponceau
310
2
Submandibular gland of adult female mouse parabiotically unite3
with a spayed female. Very large serous ducts. Marked secretory ac
red. X 600.
tivity. P.A.S.-Haematoxylin-ponceau
3
Ovary grafted into the
abiotically united with
Numerous large cystic
toxylin-Fonceau
red.
submandibular gland of a spayed mouse para castrated male; 26th day of the parabiosis.
follicles. No corpora lutea. P.A.S.-HaemaX 40.
OVARIAN ANDROGEN IN PARABIOTIC MICE
Jean C. Desclin, Jr.
PLATE 1
311
PLATE 2
EXPLANATION OF FIGURES
4
312
Ovary grafted into the submandibular gland of a spayed mouse parabiotically united with a castrated male; 26th day of the parabiosis.
Numerous corpora lutea. Dark masses of eosinophilic interstitial
cells. P.A.S.--Haematoxylin-ponceau red. X 40.
5
Ovary of a mouse parabiotically united with a spayed female; 28th
day of the parabiosis. Numerous hypertrophied interstitial cells with
vacuolated cytoplasm. On the lower left corner: remnants of old
corpus luteum. P.A.S.-Haematoxylin-ponceau
red. x 600.
6
Ovary grafted into the submandibular gland of a spayed mouse
parabiotically united with a castrated male; 26th day of the parabiosis.
At the top of the figure: interstitial cells with dense acidophilic cytoplasm; on the bottom of the photograph: a part of a persisting corpus
luteum; between both structures: vacuolated interstitial cells appear
red.
as small clusters of clear cells. P.A.S.-Haematoxylin-ponceau
x 600.
OVARIAN ANDROGEN IN PARABIOTIC MICE
Jean C. Desclin, Jr.
PLATE 2
313
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