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The effects of antigonadotropic serum on the early postnatal development of the reproductive system of the male rat.

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T H E E F F E C T S OF ANTIGONADOTROPIC SERUM ON
T H E EARLY POSTNATBL DEVELOPMENT OF
T H E REPRODUCTIVE SYSTEM O F T H E
MALE R A T 1
HORACE N. M A R V I N * AND ROLAND K. MEYER
Department of Zoology, University of Wisconsin, Madison
TWO TEXT FIGURES ANP TWO PLATES (TWELVE FIOURES)
The atrophy of the gonads and the accessory organs following hypophysectomy has been repeatedly observed for mammals and lower vertebrates. The dependence of the gonads
of infantile mammals upon the pituitary gonadotropic substances has not been demonstrated. This has resulted, in
part at least, from the difficulties of surgical procedures in
very young animals.
With the advent of the antihormone concept, reviewed by
Collip et al. ( ' 3 5 ) and Thompson ( '41), a method was suggested for hypophysectomizing an animal immunologically
without surgical procedures. An animal could be injected
over long periods of time with a serum containing aspecific
antihormones, thus passively neutralizing the animal's own
pituitary secretions. Sera containing aspecific gonadotropic
antihormones have been produced in animals by several investigators as a result of repeated injections of gonadotropic
hormones. Rowlands ( '37) and Fluhman ( '36) showed that
injections of these sera lead to atrophy of the reproductive
systems of older animals similar to that following surgical
Supported in part by a grant-in-aid from the Wisconsin Alumni Research
Foundation.
' Present address : Department of Anatomy, University of Arkansas School
of Medicine, Little Rock.
177
178
H. N. MARVIN Ah’D R. K. MEYER
hypophysectomy. Thus it was thought that similar treatment of infantile male rats might reveal some relationships
between the gonads and pituitary gland hitherto undemonstrated for mammals of this age.
M A T E R I A L S AiYD METHODS
Litters of healthy newborn rats were selected on the day
of birth and the males divided into two groups. One group
from each litter served as experimentals and the other group
as littermate controls. The experimental rats were marked
by tying the tails near the base with thread and then amputating them just distal to the thread.
The serum used was obtained from the blood of a pony
which had been injected with aqueous extracts of sheep pituitary glands for 30 to 38 months. I n some cases, the blood
was allowed to clot normally and the serum expressed from
the clot was separated by centrifugation. I n other cases, the
blood was drawn into sodium citrate solution to prevent
clotting. The corpuscles were separated either by centrifugation o r by allowing them to settle while in the refrigerator.
The plasma thus obtained was made to clot by the addition of
an amount of calcium chloride solution equivalent to the
sodium citrate originally used. The coagulated fibrin was
removed and the serum was dialyzed against distilled water to
remove the salts which had been added. The euglobulin protein precipitating during the dialysis was removed, and the
remaining solution was dialyzed against physiological saline
t o render it again isotonic. Regardless of the method of
obtaining the serum, it was Seitz-filtered and aseptically stored
in serum bottles. These bottles of serum were kept frozen
in the refrigerator except for the short time necessary to
remove some serum for use.
The antigonadotropic effect of the serum was such that
2.0 nil. inhibited an amount of sheep pituitary extract which
produced 80 mg. ovaries when assayed in 21-day-old female
rats. The serum was aspecific since it inhibited the gonadotropins of the pituitary glands of other species, including
ANTIHORMONE EFFECTS I N MALE RATS
179
the rat (unpublished data). When tested for antithyrotropic
activity, the serum failed to produce any inhibition of the
increased oxygen consumption of adult rats resulting from
the injections of the extract of sheep pituitary glands.
The serum was injected subcutaneously each day for 10
or 20 days. The volume of serum each day was gradually
increased with the growth of the rats so that a total volume
of 2.2 ml. and 6.1 ml. was given during the 10- and 20-day
periods respectively. The rats were autopsied on the day
following the last injection. The organs removed were first
weighed on an analytical balance and then fixed in Bouin’s
fluid. As a variation from the above procedures, one group
of rats was injected from the day of birth t o the tenth day
and was then allowed to go for 10 days without injection
before being autopsied on the twenty-first day of life.
Control data consisted of two types : data from uninjected
littermate rats autopsied at the same time as the experimental
rats, and data from rats injected with normal horse serum
having no antigonadotropic effect. Since the tails of the
injected rats were amputated as a means of marking them,
the effects of tail amputation alone were determined.
RESULTS
The data summarized in table 1 are largely self-explanatory. The weights of certain organs of day-old rats were
not determined because of the difficulty of uniformly complete removal and the doubtful significance of such small
weights.
I f one compares the organ weights of rats with amputated tails and the organ weights of untreated littermates,
no significant differences are seen. Thus it can be concluded
that amputation of the tails for the purpose of marking
the rats had no effect upon the organ weights. Since no
result of amputation was observed in 10 days, it seems
highly improbable that one would appear in a longer time.
Injections of normal horse serum resulted in an apparent
inhibition of body, testis and thymus growth. When the testis
1
41 (19)
46 (13)
None
Antiseruni 1st 10days
37 (30)
44 (54)
None
Antiserum
38 ( 6 )
48 ( 4 )
None
Antiserum
20 (21)
24 (15)
None
Antiscriim
21 (14)
23 ( 9 )
None
Xormal serum
18 ( 5 )
19 ( 6 )
Tail amputation
5.4 (10)
R'one
Body
None
TltEATMENT
177 (19)
195 (13)
62 (30)
192 (54)
88 ( 6 )
208 ( 4 )
25 (21)
45 (15)
39 (14)
50 ( 9 )
36 ( 5 )
33 ( 6 )
5.1 (10)
Testes
14.7 (19)
7.6 (13)
3.8 (30)
6.9 (46)
4.5 (6)
7.6 ( 4 )
~
1.6 (21)
3.4 (15)
3.2 ( 1 4 )
3.3 ( 9 )
3.2 ( 5 )
3.4 ( 6 )
1.0 (10)
$;:A1
~~~~~~
3.3 (21)
40 (19)
30 (13)
20 (30)
3 1 (42)
17 (6)
32 ( 4 )
~~
5.3 (15)
5.9 (14)
5.8 ( 9 )
4.6 ( 5 )
4.9 (6)
1.0 (10)
Prostate
WEIGHT OF
Thymus
ddrenal
172 (22)
140 (6)
217 (4)
~
60 ( 2 1 )
71 (15)
55 (14)
73 ( 9 )
53 ( 5 )
59 ( 6 )
14.3 (22)
14 ( 6 )
16 ( 4 )
5.5 (21)
6.2 (15)
4.9 (14)
5.3 (9)
4.1 ( 5 )
4.1 ( 6 )
5.3 (19)
5.8 (13)
149 (19)
190 (13)
13.0 (19)
15.9 (13)
......................
5.2 (22)
5.3 ( 6 )
6.5 ( 4 )
2.2 (21)
3.1 (15)
2.8 (14)
3.1 (9)
2.7 ( 5 )
2.8 (6)
-
Pituitary
gland
2.2 (19)
2.4 (13)
2.4 (30)
2.2 (42)
3.1 (6)
2.5 ( 4 )
1.5 (21)
1.6 (15)
1.2 (14)
1.3 (9)
1.5 ( 5 )
1.5 ( 6 )
.............................
Thyroid
l The body weights are g i w n in grams; all other weights are in milligrams.
The numbers in parentheses indicate the numbers
of rats from which the averages were obtained.
2 T h e experimental and control rats of this group were not littermates.
21
21
21
11
11
11
-
AUTOPSY
A O l IN
DATE A T
TABLE 1
The e f f e c t s o f carious treatments on the body and endocrine gland weights of immature male rats.
00
0
CI
ANTIHORMONE EFFECTS IN MALE RATS
181
and thymus weights were calculated as percentage of body
weight, these differences disappeared. Whether this inhibition
of body growth is merely incidental to disturbance of the
young o r is a general response to the injected serum proteins
has not been determined.
When an actively antigonadotropic serum is injected for
10 days, a real inhibition of testis, seminal vesicle and prostate
weights is produced. It should be pointed out at this time
that the depressions in thyroid and adrenal weights of all
groups are of doubtful significance, and no attempt will be
made to interpret the changes in thymus weights.
If the injections are continued for 20 days, the depression
of the testis, seminal vesicle and prostate weights of treated
animals is in the same direction but is of greater magnitude.
The pituitary glands of the treated rats, however, are increased
in weight.
I n order to test whether or not the hypertrophied pituitary
gland was in a hypersecretory state, rats were injected for
10 days and autopsied at 21 days of age. The data in table 1
show that the seminal vesicles and prostates were larger than
those of controls, but the testes and pituitary glands were
slightly smaller than normal. These results appear contradictory since the seminal vesicle data support the hypersecretory concept and the data on testis growth do not. An
interpretation is offered later whereby these two groups of
data can be brought into agreement.
Although the amount of partial descent of the testes in
11-day-old rats and the age of complete descent of the testes
in the 21-day-old rats were determined, the data are not
given in tabular form. It suffices to say that the variations
between individuals of the same litter made the small differences obtained quite insignificant.
If the data are graphically represented, some interesting
facts appear. In figure 1 are given the curves which result
when the data are plotted as graphs. It can be seen that the
body growth of untreated rats is rather uniform for at least
21 days. When rats are injected with antigonadotropic serum,
182
H. N. MARVIN AND R. B. MEYER
I
I
ru
I
P
I
Q)
I
a,
I
0
I
6
SEMINAL VESICLE WEIGHT IN MILLIGRAMS
Fig. 1 Effects of aiitigoiiadotropic serum on the body and endocrine gland
weights of 11-day-old and 21-day-old male rats.
A N T I H O R M O N E E F F E C T S I N M A L E RATS
183
the growth is uniform but slightly depressed. The rate of
normal testis growth is increased after the eleventh day of
age. This may be the result of an increased sensitivity of the
testis to the gonadotropic hormone, o r it may correspond to the
onset of secretion of this hormone by the pituitary gland.
The increase in growth rate does not occur if the animals are
injected with antigonadotropic serum. The seminal vesicles
increase in size at a fairly uniform rate, and this rate of
growth is depressed by the serum injections. This decrease in
rate of growth is more than would result from the slight
inhibition of body growth since corrections of the seminal
vesicle weights t o percentage of body weight show a relative,
as well as an absolute, depression of growth.
The data obtained from rats injected for only the first
10 days of life and killed on the twenty-first day are graphically shown in figure 2. The testes of the experimental rats
apparently grow during the 10 days without treatment at a
rate equal to that of control rats. This may mean that the
depressed androgen production during the 10 days of injection, as indicated by smaller seminal vesicles, has not resulted in a castration-like hypersecretion of the pituitary
gonadotropic hormone. An alternative explanation is that
the testes of the rats are growing at a maximum rate between
the tenth and twentieth days and cannot respond by increased
growth to an increased gonadotropin (FSH), even if present.
The latter hypothesis is supported by the curve of seminal
vesicle growth which indicates that an excessive amount of
gonadotropin (LH) is indirectly stimulating the accessory
glands during the last 10 days of the experiment. It seems unlikely that a castration-like hypersecretion of one factor of the
gonadotropic complex would occur without an increase in the
secretion of the other.
HISTOLOGICAL R E S U L T S
The tissues to be studied histologically were fixed in Bouin’s
fluid at the time of autopsy, embedded in paraffin, cut at 6 v
t o 10 p, and stained with hematoxylin and eosin. I n all cases
only tissues taken from littermate rats were compared.
184
H. N. MARVIN A N D R. K. MEYER
I
I
N
I
P
I
I
Q)
m
SEMINAL VESICLE WEIGHT IN
I
i
3
I
J
I
5
;
MILLIGRTMS
-
Fig. 2 The body and endocrine gland weights of 21-day-old male rats injected
from the first to the tenth day of life with antigonadotropie serum.
ANTIHORMONE EFFECTS I N MALE RATS
185
I n figure 3 are shown the tubules of the testis of an untreated 11-day-old rat. At this stage the lumen has not formed
and there is a definite tendency toward a doubling of the
number of cell layers in the tubular wall. X7hen compared
with figure 4, no very striking differences are seen. The
diameters of the tubules are much the same. Those of the
treated animals are more densely packed and this fact alone
may account for part of the difference in the weights of the
two organs. Comparison of figures 5 and 6 shows that treatment with antiserum results in an inhibition of histological
development as well as the gross weight of the seminal
vesicles. Those of treated animals lack the secondary and
tertiary branches of the secretory tubules. Two photomicrographs cannot show the full extent of the difference but serial
observation of the sections makes the differences quite
apparent.
I n figure 7 are shown the seminiferous tubules of a normal
21-day-old rat. The differentiation of the cell types in the
tubules has progressed with the increase in diameter. I n
figure 8 are the tubules of a littermate treated daily with antigonadotropic serum. The increase in diameter, formation of a
lumen and the cellular differentiation have been inhibited.
Here again the tubules appear to be much more compactly
arranged than in the testis of the normal littermate. The differences between the seminal vesicles shown in figures 9
and 10 are quite apparent. Those of the treated male, although somewhat larger in size, show little advancement
beyond the 11-day stage.
I n figures 11 and 1 2 are shown the seminiferous tubules
of a 21-day-old control and those of a littermate rat which
had been injected for the first 10 days of life with antigonadotropic serum. Although they are not identical, the similarity
is very striking. The cellular differentiation of the tubule in
the injected animal is practically at the same stage as in the
uninjected littermate. The difference in compactness of the
tubules is much less marked than in the preceding cases. The
seminal vesicle (fig. 14) of the treated rat is greatly advanced
186
€1. N. MARVIN A N D R. I<. MEYER
over that of the control (fig. 13). Secretion can be seen in
the lumina of the secretory tubules, although Moore et al.
( '30) state that such secretion normally does not appear until
the forty-fifth day of age.
Differential counts of the cells of the pituitary glands were
made by Dr. John C. Finerty, and the relative percentages of
acidophilic, basophilic, and cbromophobic cells were determined f o r three rats from each type of experiment. There
was an increase in basophilic cells from an average of 5%
for normals to 21.7% in treated rats on the day following
the tenth daily injection. Untreated 21-day-old rats had an
average of 9.4% basophiles but 20 days of treatment increased
this to 18.7%. When the rats were injected for only the first
10 days, 7.8% of the pituitary cells were basophiles at 21 days
of age. I n addition to the basophilic changes, antiserum resulted in a definite reversible decrease in the acidophile cells
which cannot be interpreted at the present time.
DISCUSSTON
The various types of data given with the results can be
brought into agreement as a working hypothesis. For the
sake of convenience, the follicle-stimulating (FSH) and the
luteinizing (LH) factors of the pituitary gonadotropic complex
will be considered separately. The selective action of these
factors on seminiferous tubules and interstitial cells has been
well established by Evans et al. ('34), Smith et al. ('34) and
Greep et al. ( '36).
The LH from the pituitary gland stimulates the interstitial
cells of the infantile testis t o secrete androgen. Since Womack
and Koch ('32) have obtained androgen from the fetal testis,
this stimulation of androgen production may extend well into
prenatal life. When antigonadotropic serum is injected into
male rats, the LH is prevented from stimulating the interstitial cells and seminal vesicle growth is retarded. This
retardation is not complete and the finding is in agreement
with that of Price ( '36). The small amount of growth which
does occur map depend upon one or more factors. Howard
ANTIHORMONE EFFECTS I N MALE RATS
187
( '41) believes that this growth of accessories which occurs in
the absence of testicular influence is a result of stimulation
by androgens secreted by the adrenals.
An inspection of the testes growth curves indicates that an
increase in the rate of growth occurs about the eleventh day
of life. This suggests that the FSH may be first secreted at
this time from the pituitary gland, o r that there is a sudden
increase in the amount being secreted. Since Clark ('35)
finds gonadotropic activity present in the pituitary glands of
male rats at the day of birth, and that the pituitary potency
increases gradually until the rats are 50 days of age, these
two explanations appear to be untenable. Another explanation
is that the seminiferous tubules develop independently of the
pituitary FSH stimulation for about the first 10 days. After
that they become reactive to the FSH and begin to differentiate
at a maximum rate. That spermatogenesis is independent for
a time is supported by our evidence from the experiments
with antihormone. The antihormone injected during the first
10 days of life inhibited the testis weight and interstitial cell
activity, but failed to prevent the differentiation of the cellular
elements of the tubule. If the injections were continued
through the twentieth day of life, the tubules develop very
little beyond the 11-day stage. Although Moore ('36) presented evidence showing that spermatogenesis could not be
accelerated and suggested that spermatogenesis in the rat goes
on at a maximum rate, our evidence in favor of this is yet
only circumstantial.
It is also suggested that the androgen being secreted by the
infantile testis is acting as a brake on the pituitary gland
even during the early postnatal life. When the androgen
level is decreased by antihormone injections, the brake is
reduced and the pituitary gland becomes hyperactive and
increasingly basophilic. If the antihormone injections are
stopped after the tenth day, the androgen secreted from the
interstitial cells as a result of LH activity, stimulates the
seminal vesicles t o hypertrophy and also results in a reduction of the basophilism of the pituitary gland. It is tentatively
188
H. N. MARVIN AND R.
rc.
MEYER
suggested that the excessive amounts of FSH which may be
present are not obviously manifested because the maximally
growing testis cannot respond by excessive growth.
I n conclusion it may be said that the results obtained and
the conclusioiis derived bring into reasonable agreement the
several lines of evidence relating to this problem. Certain
aspects must be substantiated by additional and alternative
investigations. It is thought that gonadotropic antihormone
will be very useful in the solution of these problems.
SUMMARY
1. Injections of antigonadotropic serum into male rats from
the first to the eleventh day of life result in a small inhibition of
testis growth, and definitely retard the growth of the accessory
reproductive organs.
2. Continuation of these injections through the twentieth
day of life prevents the increase in testicular growth rate
which normally occurs, and continues to inhibit the growth
of the male accessory glands.
3. Histologically the differentiation of the seminiferous
tubules of injected rats is not materially inhibited during the
first 11 days of injection, and this suggests that the gonadotropic hormone is not necessary for this amount of development.
4. Differentiation beyond the 11-day stage, however, is
almost completely inhibited by the antihormone.
5. Antihormone treatment for the first 10 or the first 20
days of life results in about the same degree of pituitary
basophilism.
6. The gonadotropic secretion of the pituitary gland in the
male rat is under the control of gonadal hormones as early
as the tenth day of life.
7. It is suggested that the rate of tubular differentiation and
growth in the testis is maximal in the rat during the early
postnatal period.
ANTIHORMONE EFFECTS I N MALE RATS
189
LITERATURE CITED
CLARK,H. M. 1935 A prepubertal reversal of the sex difference in the gonadotropic hormone content of the pituitary gland of the rat. Anat.
Rec., vol. 61, pp. 175-192.
COLLIP,J. B., H. SELYEAND D. L. THOMSON 1940 The antihormones. Biol.
Rev., V O ~ . 15, pp. 1-34.
EVANS,
H. M., R. I. PENCHARZ
AND M. E. SIMPSON1934 On a selective gametogenic effect of certain hypophyseal extracts. Science, vol. 80, p. 144.
FLUHMAN,
C. F. 1936 Comparative studies of gonadotropic hormones. 4. Ovaries
and hypophyses of rats in chronic experiments. Proc. Soc. Exper.
Biol. Med., vol. 34, pp. 691-694.
GREEP,R. O., H. L. FEVOLD
AND F. L. HISAW 1936 Effects of two hypophyseal
gonadotropic hormones on the reproductive system of the male rat.
Anat. Rec., vol. 65, pp. 261-272.
HOWARD,
EVELYN1941 Effects of adrenalectomy and desoxycorticosterone substitution therapy on the castrated r a t prostate. Endocrinology, vol. 29,
PP. 746-754.
MOORE,C. R. 1936 Responses of immature r a t testes to gonadotropic agents.
Am. J. Anat., vol. 59, pp. 63-88.
MOORE,C. R., W. HUGHESAND T. F. GALLAGHER1930 Rat seminal vesicle
cytology as a testis hormone indicator and the prevention of castration
changes by testis extract injections. Am. J. Anat., vol. 45, pp. 109-136.
PRICE,
DOROTHY1936 Normal development of the prostate and seminal vesicles
of the r a t with a study of experimental postnatal modifications. Am.
J. Anat., vol. 60, pp. 79-128.
ROWLANDS,
I. W. 1937 The effect of antigonadotropic serum on the reproductive organs of the normal animal. Proc. Roy. Soc., London, s.B.,
V O ~ . 121, pp. 517-532.
SMITH,
P. E., E. T. ENGLE
AND H. H. TYNDALE1934 Gametokinetic action of
extracts of follicle-stimulating urine. Proc. SOC.Exper. Biol. Med.,
V O ~ . 31, pp. 745-746.
THOMPSON,K. W. 1941 Antihormones. Physiol. Rev., vol. 21, pp. 588-631.
WOMACK,E. B., AND F. C. KOCH 1932 Studies on the extraction of the
testicular hormone from tissues and on its quantitative distribution
therein. Endocrinology, vol. 16, pp. 267-272.
PLATE 1
EXPLANATION OF FIGURES
3
Seminiferous tubules of a n untreated 11-day-old r a t ( r a t no. 2, litter no. 5 2 ) .
X 267.
4 Seminiferous tubules of a n 11-day-old r a t ( r a t no. 4, litter no. 52) which
had received ten injections of antigonadotropic serum. X 267.
5 Seminal vesicle of a n untreated 11-day-old r a t ( r a t no. 2, litter no. 5 2 ) .
45.
6 Seminal vesicle of an 11-day-old rat ( r a t no. 4, litter no. 52) r h i c h had
received ten injections of antigonadotropic serum. X 45.
7 Seminiferous tuLiclcs of an untreated 21-dax-old rat ( r a t no. 1, litter
no. 35). X 267.
8 Seminiferous tubules of a 21-day-old r a t ( r a t no. 4, litter no. 3 5 ) which
had received twenty injections of antigonadotropic serum. X 267.
x
190
ANTIHORMONE EFFECTS I N MALE RATS
FLBTE 1
H. N. MA RV I N A N D R. K. MEYER
191
PLATE 2
EXPLANATION OF FIGURES
9 Seminal vesicle of an untreated 21-day-old r a t ( r a t no. 1, litter no. 3 5 ) .
45.
10 Seminal vesicle of a 21-day-old rat ( r a t no. 4, litter no. 3 5 ) which had
received twenty injections of antigonadotropic serum. X 45.
11 Seminiferous tubules of an untreated 21-day-old r a t ( r a t no. 1, litter
no. 42). X 267.
1 2 Seminiferous tubules of a 21-day-old r a t ( r a t no. 2, litter no. 42) which
had received only ten injections of antigonadotropic serum during only the first
1 0 days of life. X 267.
13 Seminal vesicle of an untreated 21-day-old rat ( r a t no. 1, litter no. 42).
x 45.
14 Seminal vesicle of a 21-day-old r a t ( r a t no. 2, litter no. 42) which had
received only ten injections of antigonadotropic serum during only the first
1 0 days of life. X 45.
x
192
ASTIHORMONE EFFECTS IN MALE RATS
PLATE 2
11. h. I\I%lWIN A N D R . X. MEYER
193
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