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Responses of the reproductive system of hypophysectomized rats to injections of pregnancy-urine extracts. II. The female

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RESPONSES O F THE REPRODUCTIVE SYSTEAI O F
HYPOPHYSECTOMIZED RATS TO INJECTIONS
O F PREGNANCY-URINE EXTRACTS
11. T H E F E M A L E '
SAMUEL L. LEONARD2 AND PHILIP E. SMITH
Department of A n a t o m y , College of Physicians and Surgeons, Columbia University
TWO PLATES (ELEVEN FIGURES)
Investigations on the action of the gonadotropic principle
in pregnancy urine have been largely carried on with normal
aiiimals, particularly immature rats and mice. It is recognized, however, that the effects of the injections of the gonadotropic urinary substance in normal animals may not be due
to the urinary principle alone but to a combined action with
the A.P. hormone liberated by the animal's own gland. That
the pituitary hormones and the urinary gonadotropic hormones have a synergistic action has been demonstrated in
immature females by Evans, hleyer and Simpson ('32), Leonard ( '32 b) and Fevold et al. ( ' 3 3 ) . To secure a true picture
of the action of the urinary principle, only, on the gonads it
seems necessary to use hypophysectomized animals. By instituting treatment at varying intervals after the ablation of
the pituitary, animals can be secured in which not only has
the A.P. hormone been eliminated but also in which varying
degrees of gonad atrophy have taken place. It is thus possible to determine the capacity of the urinary principle to
replace the A.P. gonadotropic hormones and t o determine its
action per se.
Aided by a grant from the National Research Council, Committee
of Sex.
* National Research Fellow.
175
011
Problems
176
S A M U E L L. LEONARD A X D PHILIP E. S M I T H
Several i11\7estigators have reported results obtained from
tlle injection of extracts of pregnancy urine (P.U.) in hypophysectomized rabbits and rats, most of the reports on
rats being given in preliminary papers only. ’CVhite and
Leonard ( ’33) drew the conclusion that P.U. extracts induced
ovulatioii in rabbits without a synergistic action with the
intact anterior pituitary gland. Iii a female rat which had
been hypophpsectomized for 33 months, Reichert et al. (’32)
report no effect with P.U. injections on either the ovaries
or the reproductive tract, a failure of response which also
obtained in pituitaryless dogs. In a footnote, however, which
was apparenbly added later, they state that a few r a t s showed
some response, the nature of which is not stated. Nognchi
( ’31) in adult hypophysectornized rats foiiiid that ‘short’
periods of treatment cansed a proliferation of the theca, a
disappearance of the granulosa and no formation of corpora
lutea. With long treatments, there was shrinkage of the
ovary and a destruction of the follicles. The time betiveen
operation and the beginning of treatment was not given but
apparently little or no interval intervened. There was ail
oestrous response in the vagina and uterus. !,Trade, Katzman and Jorgensen (’33) in mature female rats also secured
oestrus, in treatments of 3 to 15 days’ duration, instituted
2 to 47 days after liypopliysectomy. Tliey report a granulosa
aiid theca luteinizatioii. Collip, Selye and Thompson (’33a ,
b ) report the effect of an extract of the placenta (A.P.L.)
( d i i c h is stated to be physiologically similar to the urinary
gonadotropic hormone) on hypophpsectomizecl mature aiid
immature female rats. Apparently the injections were begun
soon after the pituitary ablation. Oestrus did not result iii
the immature but was continuous in the mature animal. Lnteiiiizatioii of the them cells only was reported, with the formation of tliecal corpora lutea. Size changes in the o r a r p
a r e not definitely described though a ‘shrinking ovary-’ is
mentioned in connection with the oestrons smears.
Smith and Leonard ( ’33 a, b ) , in a series of studies 011
hypophysectornized mature r a t s in which injections f o r vary-
EFFECT OF PREGNANCY U R I N E O N OVARIES
177
ing periods were begun immediately after operation o r after
an interval of 16 to 81 days, report an enlargement of the
ovaries. This enlargement did not continue indefinitely with
prolonged injections. The enlargement was due to an hypertrophy of the existing interstitial tissue and of the theca cells,
and the transformation of some.of the follicles into bodies apparently structurally similar to corpora Iutea. Oestrin was
secreted by the ovaries of the animals injected immediately
after hypophysectomy, as revealed by the response of the
uteri and an oestrous type of vaginal smear. Occasionally,
this reaction was noted in the postponed injections as well.
The only investigator who claims that the injection of P.U.
extract gives an effect after A.P. ablation similar to that of
A.P. treatments is Freud ('32) who used only adult rats. His
injections begun 3 days after operation gave an oestrous
effect except in pregnant animals-an
exception described
only by this investigator.
The results reported have not thus f a r been entirely harmonious-a fact which is not surprising, since the condition
of the ovaries at the beginning of treatment may cause a
variation in the response as has been suggested by Collip.
It seems essential that the age of the animals and the period
elapsing between the pituitary ablation and beginning of
treatment be definitely stated. It is further essential that the
weight and structure of the ovaries be approximately known
when injections are begun. This necessitates the autopsy of
reference controls (littermates whose treatment has been
identical up to time when injections are started) or unilateral
ovariectomies. With this information, with careful autopsy
data and structural studies, and with data on the daily vaginal
smears it should be possible to determine the effect of the
injection of the gonadotropic urinary principle and to contrast it with the effects given by A.P. treatments. This we
have attempted to do and present the results obtained in the
female, in this paper. It is recognized that with different
dosages or periods between hypophysectomy and treatment
a variation from the findings given here might be obtained.
THE ANATOMICAL RECORD, VOL. 5 8 , NO. 2, AND SUPPLEMENT
178
SAMUEL L. L E O N A R D A N D PHILIP E. SMITH
The hypophysectomized female rat has not responded with
as great uniformity to P.U. treatment as has the male.
CONDITIONS O F THE EXPERIMENT
Piebald rats of the Long-Evans strain were used. As evidence upon the completeness af the pituitary ablation we have
daily weighings, lengths taken a t the time of operation and
at autopsy, weights of the thyroids and the adrenals and
serial sections of the pituitary capsules. The latter were
fixed in situ, then carefully stripped from the bone and serially sectioned in every case. I n many of the ‘postponed’“
injections we have the additional evidence of the atrophy of
the gonads f o r in most of these cases a unilateral ovariectomy
was done before treatment was instituted.
I n all, except some of the postponed injections, litters composed of several (four to seven) animals were used. This
enabled the series to include reference and untreated hypophysectomized controls and in the experiments requiring
it, as in determining the rate of regression of the gonads after
cessation of treatment, treated operated controls. The data
upon the effects obtained by the various experimental procedures are thus based upon accurately inferred weights and
structure of the gonads at the beginning of the procedure.
Besides this, the uteri were weighed a t autopsy, the vaginal
smears examined daily and often sections of the vaginae were
made.
Both mature and immature females have been used. In
the latter, it has been possible to make some especially instructive structural studies for any corpora lutea found after
treatment were definitely known to have been formed as a
result of the injections, since these structures are never found
in untreated immature hypophysectomized females.
With both the mature and immature animal ‘immediate’
and ‘postponed’ treatments were given, i.e., injections were
begun at the time of hypophysectomy or after a period (16
a Treatments begun a f t e r a n interval following hypophysectomy a r e desigllated
as ‘postpoiled,’ those begun at the time of operation as ‘immediate.’
EFFECT O F PREGNANCY URINE O N OVARIES
179
to 135 days) had elapsed. Certain animals in each of these
series were autopsied at the termination of the treatment ;
others were not sacrificed for some time in order to secure
data upon the rate of regression of the gonads when treatment
was stopped.
Three pregnancy-urine preparations have been injected,
antuitrin S (Parke, Davis & Co.), follutein (E. R. Squibb &
Sons) and one prepared by o u r ~ e l v e s . ~Antuitrin S was used
in a majority of the experiments. We have used in most of
the experiments a constant dosage of 25 R.U. per day, injected subcutaneously. Neither the follutein nor the antuitrin S contained effective quantities of the female sex hormone as shown by a complete failure of response when doses
up to 500 R.U. were injected in castrated sensitized female
rats. I n several cases, pregnancy blood serum was injected,
which contained the female sex hormone as well as the gonadotropic p r i n ~ i p l e . ~
Over sixty hypophysectomized females have been used in
the study.
EXPERIMENTAL FINDINGS
Hypophysectomixed females
W e i g h t changes in the ovaries. The injection of P.U. extract, both in the series in which treatment was begun at the
time of hypophysectomy and in those in which an interval
elapsed, has almost invariably induced an enlargement of the
ovaries (tables 1, 2). This enlargement was of such magnitude as to make it unquestionable. The increase in size of
the gonads was rapid but, as will be pointed out below, was
not maintained with prolonged treatment. I n the experiments
with immediate treatments, injections up to 15 days gave an
increase over the reference control ovaries up to 315 per cent
in the immature animals and up to 69 per cent in the adult
'We are indebted t o Dr. 0. P. Kamm for the antuitrin S and to Dr. J. A.
Morrell for the follutein. The dosage was based upon their determinations of the
potency of the extracts.
The pregnancy-blood serum was furnished through the kindness of Dr. R.
Kurzrok, of the Department of Obstetrics and Gynecology.
~
W 9571 Ref. contr.
GH 9568 Hypsect.
W 9570 Hypsect.
W 9569 Hypsect.
GH9567 Hypsect.
G H 9565 Hypsect.
G 9528 HypsectGH 9531 Hypsect.
G 9530 Hypsect.
Hypsect.
G 9483
G 9456 Ref. contr.
W 9457 Hypsect.
G 9455 _ -Hypsect.
-- _ GH 9453-~Hypsect.
- W 9488 Ref. contr.
B 9486 Hypsect.
G 9485 Hypsect.
BH 9487 Hypsect.
BH 9490 Hypsect.
W 9489 Hypsect.
B - 9484 _ _Hypsect.
GH 8326 Ref. contr.
GH 8329 Hypsect.
GH 8325 Hypsect.
GH 8324 F T v n w c +
Immature
DESIGNATION
.4ND TYPE
'
I
-
-
~~~
1
43
47
23
25
54
54
22
22
Ant. S
Ant. S
-
~
1
1
25
25
25
25
25
13
10
10
20
20
25
25
25
12
12
10
25
11
-
25
25
- .
VAQINA
.
- .
,
~
-
~
~-
-
-
Vagina closed
Opened 3rd day; corn.
Closed throughout
Opened 4th day; mixed, le, corn.
Opened 4th day; corn.
Closed throughout
Opened 11th day; le
Opened 4th day; corn.
-
-
Opened 12th day; corn., and mixed
Opened 5th day; corn. Closed throughout
- -.
-
Opened 4th day; corn.
Closed
_-- - Opened 10th day- - .~- _ - _ _ _ -
Opened 10th day; corn.
'
I
~
j
1
I
'
1
I
1
i
-_ I
Opened 4th day; corn., dioestrus
Closed
Closed
----
, Opened Gth day; corn. and mixed
~
I
--_--___
25
25
25
-
5
5
10
10
20
TREATMENT
Extract
- -
None
Ant. S
Follut.
I1
Wt.,
gm. ,
49
I
I
,
22
-
days
Age,
EXPERIMhNT
BEQUN
-
--
0
62
G4
55
64
67
44
47
46
47
~
noic
0.043
0.032
0.0269
0.036
0.019
0.035
0.161
0.024
0.034
0.181
_ -
0.029
0.116
0.142
0.094
-
'
~
nnnn
0.0123
0.0083
0.0154
0.0082
0.0135
0.0245
0.0080
0.0120
0.0240
0.0090
0.0148
0.0182
0.0200
0.0185
0.0072
0.0245
0.0210
- .
0.022
0.134
0.053
46
50
58
~
0.0140
0.0070
0.0056
0.0495
0.0240
0.0190
0.027
0.025
0.022
0.245
0.195
0.202
55
70
GO
59
55
2o0 1
10 I 75
in
76
0
10
10
10
20
20
14
13
12
-
5
J
)
0
10
20
10
10
20
- --
AUTOPSY
E f f e c t on reproductive organs of injections of P.U. extract beginning a t the time of hypophysectomy
TABLE 1
I
" * F
-33
+
+
-47
- 1 2 ,
59
-- 48
-22
56
+35
+ 22
(+32)
+
+
+254
71
36
(-49)
(+75)
(+50)
- 50
- 60
+
-
-
-
-
+
-
-
' +
i -
-
I -
1
,
+
-
+
+
.~
~
+
+
+
+
+
-
-
-
IIypsect.
Hypsect.
Hypsect.
Control -
~
187
181
173
181
187
i73
161
159
' N O I ~ ~
iNoiic
1Slit.S
iPrcg. bl.
hit. S
1
181
176 Ant. S
185 IAnt. S
172 lAIlt. s
164
/Ant. S
I
97 Aut. 8
112 Preg. bl.
107 Aiit. S
90 (None
i
1-
10
10
6
6
_--
10
10
I U
~
~
!
~
'
1
--
~
45
25
25
cc.
25
ZD
'
CYI...
~
Dioestrus
Corn.
Mixed and rorii.
Diocstrus
Corn.
Corn.
Corn., dioestrus
~
-
~
Corn., mixed
Mixed, corn.
Corn.
Corn., last 5 days tlioestrus
~
,
Opened 5th day; corn., mixed
Opened 4th day; corn., dioestrus
Opeucd 8th day; coru.,
diocstrus
.
-~
U p ~ l I L " 1 ) L U U,,J
1
~
1
I
10
10
10
_-
10
10
30
10
155
142
136
'
I
,
1
+ 55
+ 69
+ 1
~
(1
18)
+
+
0.0273
- 35
0.0357 1
0.0236
- 34
0.0603
69
0.0530
48
0.01174 - 34
0.0417
0.0646
0.0705
0.0420
+
+
-
+215
0.0475
0.007631
1
0.0262
-76 1
0.153 0.0293
0.388 10.0535
1
0.384
I - 0.0910
1 0.221 I 0.0343
I
129
0.081 0.0217
1 0.237
.
0.0485
-
~
'
1
0.177
0.373
0.393
0.338
0.244
0.045
0.137
0.135
1 0.231
179
0.141
136 1 0.320
167
0.286
163
0.100
-
132
1
-
30
I
162
178
146
-
' 106
97
, 121
0
6
6
15
_
10
30
10
+
-
-
--
-
-
-
-
~
+
+
+
+
+
+
+
+
+
+
+
--
i +
i
I
+
j +
I
,
B 8115 Hypsect.
R 8116
_ _ Hypsert. 1
W 8004 Ref. coutr.
GH8005 Hypsect.
W
8002_ _Hypsect.
[
-.
-___
i t:i
~
j
~
'
+
10
20
110
'Follut.
(+ 49)
110
Foll.+A.P.j , ~_10 1 _ 20
(+155)
_
-~
j -~
119
119
:None
Dioestrus
I
20
- 34 1 1
8 ' 40
Mixed, corn.
8
119_ _1 149 IAnt. S
.
41 _ - --I -+ -~
-____-~
No injection first day.
Right ovary only. Auimal was iiijccted for 10 days. Theu after a lo-day no-iujectioii period the l r f t ovary was removctl, 0.0070 gm. Autopsy
10 days later.
3 R i g I ~ovary
t
only. Animal n a s injected for 10 days. Tlien a f t e r a lo-day no-injeetion period the left ovaly was l e ~ l l o ~ e i 0.0080
l,
gal. Autopsy
10 days later.
Right ovary only. Aiiimal A':LS iujectcd f o r 10 days. Tlieii after a 10-day n o - i u ~ c e t i o ~
period
i
the left ovary a a s 1c111o~cd,0.0148 gm. $utopsy
10 days later.
'Pyiidine extract of slicep A . P . was mixed with the follutein.
101
101
101
G H 8094 Ref. coiitr.
W 8091 Hypsect.
GH8093 Hypsect.
1
84
84
84
84
84
46
46
46
46
--
Ref. coutr.
Ilypsect.
Hypsect.
Hypsect.
Hypsect.. -
B 8451
G H 8452
GH8455
G H 84.54
GH8453
Xature
G H 8312
BH8313
B H 8311
G H 8316
-
W 6.509
RH 737
G H 6427
GH 6196
B
8111
8112
-w 1 4 i 7
GH 6527
B
6H-8355
G H 8356
- -__
B 8079
B 8080
_B
_ 8078
Mature
B H 345
m
B 7972
B 7974
B 7973
- - -G H 6967
GHSt.
RH 6910
BH 6912
-.BH 6909
B 8710
Immature
G H 8805
G H 8802
RAT
~~
-
110
110_ - 138
150
219
235
238
333
__-
~
_
-
60
72
72
72
73
75 _
91
91
91
-110-
- -
46
46
46
31
31
I
1
1
1
II -
1
-
22
4848
48
8157 78
78
78
57-
54
54
54
35
41
39
40
~-
57
57
16
135
_
16
-
'
1
I
I
1
I
_I
,
Extract
0.0074
0.0127
u.uiui
-
0.0051
0.0020
0.0048
0.0048
0.0077
0.0054
0.0032
'
1
P.U. cxt.
P.U. ext._
r . u . exz.
Folluteiii
-FoGuteiiiFolluteiii
Folluteiii
__ _
Ant. S.
None?
Ant. S
Ant. -S -
P.U. ext.
Ant. S
Ant. S
Ant. S
Ant. S
- ~__ -~
Ant. S
0.0022
0.0022
Ant. S
0.0021
Ant. S
1 0.0038
I
P R E - T R h A T M h N T DhTA
_
8
-
10
10
26
15
11
lo
10-
13
+ 6
9
8
_
10
+
10
19
6
20
+15
30
30
15
Number
of days
TRhATXhNT
1
-
,
1-
1
i
'
11
25
25
18
12
19
10
0.25 cc.
1cc.
0.5 cc.
1
50
,
20
1
10
10
10
25
I
I
I
I
25
1 -.-25 I
25
25
25
46
46
38
I
1
1
'
'
~
I
'
1
l
I
I
i
1
I
0.0102
0.0081
0.0110
0.0095
0.0065
0.0026
0.0035
0.0106
0.0100
-0.0232
0.0340
0.0255
_.
_0.0077
0.0105
0.0140
0.0146
0.0128
0.0215
0.0095
0.0230
0.0216
0.01830.0153
0.0178
0.0122
0.0149
0.0095
0.0247
-
'
1-
ar:.7..A
le
le
le
le
- __-I__
le
I
-.-I
I
Vagina
Ciosca
Closed
le
le
1
I
I
I
~
1
-
+
+
+
18
+++40459
+168
+113
+700
+533
+333
____
Per cent
change
in ovary
-_,.,.x'
I ah
+++
0.0550
Closed
1
4-212.
.0.115
le
Coriiified
0.160
smear
0.172 .. dixcd-~
__
Closed
+285
0.0580
Cornified
+I18
0.2750
le
+191
0.0710
le
0.0850
$1;;
lc
0.0585
-~~
-3%
le
0.106
86
Cornificd
+350
0.216
le
+321
0.238
40
1 Coriiified
0.2210
Cornificd
Coriiifienl 1
16
I
47
'
le
28
0.0790
0.0340
0.0580
0.1220
_ .
0.041-
0.105
~-
0.0220
Jterus, gm.
TABLE 2
Postponed anjectzuna of pregnancy wane extracts an Iiypopkyseetomzzed female rats
-
-
-
EFFECT O F PREGNANCY URINE O N OVARIES
183
animals. I n the untreated hypophysectomized littermates, on
the other hand, the characteristic pronounced decrease in size
of the ovaries occurred. Thus the injections not only overcame the regression but also usually increased the initial
weights of the gonads. I n the postponed treatments an even
greater percentage increase in the weights of the gonads is
shown, 168 to 700 per cent in the immature and 16 to 350 per
cent in the adult. I n prolonged treatments, regression of
the ovaries took place in spite of the continued injections.
The postponed-treated animals show a greater percentage
increase than do those given immediate treatments. The
actual weight increase of the gonads, however, is the reverse
of this, being greater in the immediate than in the postponed
treatments. The greater percentage increase in the postponed treatments is due to the fact that the ovaries have
regressed to a minute size so that the increase which follows
treatments, though actually small, gives a larger percentage
value.
Our data, though not extensive, indicate that the enlargement of the ovaries is not maintained. If animals were autopsied at closer intervals during prolonged treatments, we feel
the increase and regression would be better manifested.
If after a short period of treatment the injections are
stopped, a very rapid regression in the size of the ovaries
takes place. For example, rat GH8093, autopsied at the end
of a 10-day treatment period (45 R.U./day), had ovaries
which weighed 0.0603 gm. (table 1). A littermate, TV8090,
received the same treatment for 10 days. Injections were
then stopped for 10 days at the end of which period one
ovary was removed and weighed (0.0148 gm.). There had
thus been a loss of some 50 per cent in weight in the 10-day
period following cessation of treatment. Further regression
was very slow, for the remaining ovary removed after another
10-day period weighed 0.0117 gm. This rapid decrease is
largely due to the changes in the interstitial tissue and theca
cells which will be discussed in the next section.
184
SAMUEL L. LEOXARD A N D PHILIP E. SMITH
Strzlctural changes i n the ovaries. Interstitial tissue. One
of the most characteristic morphological effects of the pregnancy-urine extract on the hypophyscctomized rat oraries is
the hypertrophy of the interstitial cells. This can best be
demonstrated in the immature rat with postponed treatment
(figs. 1, 2 ; see also fig. 9), f o r tliere are not persistent corpora
lutea and no follicles with aiitra a t the time that treatment
is begun to confuse the picture.
The interstitial cells of the ovary of the untreated hypophysectomized rat are quite characteristic. They are usually arranged in radiating cords or may be in circular rows
when in the process of being formed from atretic follicles.
Tbere is little cytoplasm and the crowded nuclei appear
deeply stained (figs. 1, 6, 7 ) . With P.U. injections, the
changes that occur in these cells are very marked. The cytoplasm increases and becomes eosinophilic and laden with fat,
as seen by appropriate stains. The nuclei become large,
vesicular, lighter in their staining reaction and well separated (fig. 9). The hypertrophied cells resemble lutein cells
and indeed are very difficult to distinguish from them. The
ovary becomes almost homogeneous in structure throughout,
being composed largely of hypertrophied interstitial cells
(figs. 3, 5). In some cases, the organization, as well as the
structure of the cords of interstitial cells, resembles small
corpora. I n contrast to true corpora, however, these bodies
are not sharply separated from each other. This tppe of
reaction was observed in all the ovaries of hypophysectomized treated animals regardess of age or time of treatment.
It is the increase in cell size in this type of tissue that largely
imparts the heavier weight to the ovaries of the postponedtreated animals and is especially prominent in them. I t also
contributes to the %eight increase in the immediate treatments though t o a lesser degree, f o r in them the responses
of other tissues also contribute to the weight increase.
The fresh ovary of the P.U. treated animal has a yellowpinkish tinge in marked contrast to the white color of the
ovaries of the untreated animals which have been hypophys-
EFFECT O F PIIEGNANCY UBIXE O N O V 1 R I E S
185
ectomized f o r some time. This color change is largely due to
the interstitial cell changes.
Regression of the interstitial cells is characteristic of the
cessation of treatment or of prolonged treatment. The large
fat-containing cells that formerly had abundant eosinophilic
cytoplasm become shrunken and do iiot stain with the acid
dyes. The nuclei which were large, lightly staining and
vesicular become shrunken and deeply staining (fig. 10).
Follicles. With P.TJ. iiijectioiis there is no evidence of
follicular maturation and growth o r of cyst formation which
is the characteristic occurrence with hypophyseal implants
in the hypophysectomized rat. Primordial follicles without
a formed cavity continue to persist during P.U. treatment.
Whethcr these are reduced in number we cannot definitely
state, but wc feel certain that they are not increased.
There is also quite a striking contrast between the ovaries
of liypopliysectomized and normal rats treated with P.U.
extracts. I n the former, follicular cysts and blood follicles
are almost completely absent, whereas with the normal female
they are characteristically present.
Theca cell hypertrophy. W e have also observed the Ihecal
luteinization mentioned by Collip and his associates, particnlarly in the immature female operated on at 22 days of age
and then immediately treated. The cells of the theca interna
of the young follicles undergo hypertrophy characteristic of
the interstitial cells, as was noted above. I n some of the
degenerating follicles, the theca so increased in size that it
obliterated the degenerating ovum and surrounding granulosa
cells and presented the appearance of a small lutein body.
The theca luteiiiization is undoubtedly better depicted in the
younger animals than in those slightly older, although such
reactions may be seen in hypophysectomized females of any
age group. The hypertrophy of the theca cells likewise regresses with continued treatment or with the cessation of
treatment.
Corpora lntea. The formation of corpora lutea, in which
the granulosa seemed definitely to be involved, usaally though
186
SAMUEL L. LEONARD AND PHILIP E. SMITH
not invariably, occurred in the females with immedate treatment (figs. 9, 10, 11). I n four young animals (age 22 to 29
days at time of operation) there was a complete failure to
form corpora. Of these four, one (25 days old) failed to
show even theca luteinization (G9453), the interstitial tissue
only being slightly influenced.
I n the other 22- and 29-day-old rats in which treatment was
begun at the time of operation corpora were formed. Noguchi
states that the immature hypophysectomized rat will not form
corpora lutea with pregnancy urine and Collip failed to produce corpora lutea also. The fact that corpora lutea definitely
failed to form with P.U. injections in several of our younger
operated animals, whereas there was no failure in the older
immature rats, suggests that if still younger animals were
used, corpora would invariably fail to form.
911 females 29 and 46 days of age (except one animal mentioned above), though immature at the time of hppophpsectomy, showed newly formed corpora lutea when treatment
was begun immediately. With the cessation of treatment o r
long-continued treatment, these newly formed corpora persisted as discrete bodies in the atrophied interstitial cells
(figs. 10, 11). These bodies were unquestionably formed as
a result of the treatment, for the reference control ovaries,
removed at the time treatment was begun, contained no corpora. It is impossible to state definitely that new corpora are
formed in the adults in either the postponed or the immediate
treated animals because of the presence of persistent lutein
bodies from previous ovulations.
Whether adual corpora lutea were formed in the postponed-treated animals is difficult to determine. That the untreated rats operated upon between the ages of 31 and 46
days, in which the vaginae were closed, had no corpora is
certain, for none were present in the control ovaries removed
16 to 70 days after hypophysectomy (fig. 1). I n some of
these (GHS355, BS079, BSOSO) the remaining ovaries, following a course of treatment, presented bodies structurally similar to corpora (fig. 3). Furthermore, these bodies persisted
E F F E C T O F PREGNANCY U R I N E O N OVARIES
187
in the midst of regressing interstitial cells as do the corpora
of mature animals after hypophysectomy. These newly
formed bodies, however, are not as large as the corpora of
normal animals and have the appearance shown by these
bodies when regressing. That they were formed as a result
of the treatment is certain and we hesitate to call them typical
corpora only because they were not as robust as those which
are recently formed in normal animals.
Physiological effects of P.U. extract
So far we have considered only morphological changes produced in the gonads by P.U. injections. Interesting information was also obtained on their functional stimulation by
studying the changes in the accessory organs.
The production of oestrin was the characteristic physiological effect of the injection of pregnancy-urine extract in
the hypophysectomized females. This reaction was more
clearly shown in the immediate than in the postponed treatments. The secretion of oestrin was detected by three reactions, an increase in the size of the uterus, the opening of
the vagina and the production of cornified cells in the vagina.
The increase in size and vascularity of the uterus was noted
in both the postponed- and immediate-treated animals. The
effect was much greater in the immediate-treated animals.
The time of opening of the vagina in the immature female
with immediate treatment varied considerably (from the
fourth to the twelfth day). One immature animal did not
open after a 14-day treatment (69453). This was an exceptional case for not only did the vagina fail to open but also
the uterus did not enlarge. The ovary contained no corpora
lutea, the interstitial cells were only moderately hypertrophied and the theca cells were not enlarged. The weight also
failed to increase as in the others. We are a t a loss to explain the behavior of this animal.
The production of cornified cells in the vagina occurred
most constantly in the animals in which treatment was begun
immediately. The cornification characteristically persisted in
188
SAMUEL L. LEONARD A N D P H I L I P E. SMITH
most cases throughout the course of treatment, although leucocytes occasionally appeared with the cornified cells. This
continuous cornification in treated hypophysectomized females is in marlied contrast to the normal female treated with
similar extracts in which the cornified cells nearly all disappear when treatments a r e continued. I n r a t GH8453 (table 1)
a complete regression of the ovary took place with prolonged
treatment and a t this time a dioestrous smear occurred.
W e have made no tests to determine whether the corpora
lutea formed iii the hypophysectomized r a t s were fnnctional
or iiot. I n the normal immature female, Shelesnyali ('33)
has demonstrated by producing deciduomata that the corpora
formed with P.U. injections a r e functional. The constant
prodnction of oestrin in the treated hypophysectomized r a t
might prevent the action of the lutein hormone, even though
it was formed.
In several cases, in both the postponed and immediate treatments, blood serum of pregnant women was injected instead
of pregnancy-urine extract. The results obtained mere similar to those with the urinary product. However, the oestrous
eft'ect produced cannot be definitely ascribed to the action of
the gonadotrophic hormone became of contamination with the
fernale sex hormone.
In a few cases, partially hypopliysectomized females were
treated with the extracts. Tlie reactions mere quite similar
to those of the completely hypophysectomized animals, the
ovaries and uteri, however, showed a greater response and
the follicles appeared to be larger. This is ell illustrated
by r a t W9569 (table l), in which there was found a fragment
of definite A.P. tissue. I n this animal, a s in some of the other
partial hypophpsectomies, the vaginal smear quickly became
a dioestrons type which is characteristic of the injected normal immature female (see below). If, as our data show, a
continuous oestrous smear is characteristic in the immediate
treatments with P.U. extract in the hypophysectomized rat,
then some of the other animals which a r e designated a s partial were in reality fuiictioiially complete hppophysectomies.
EFFECT O F P R E G N A N C Y U R I N E O N OVARIES
189
We have been severely critical of our hypophysectomies and
have considered all epithelial-like cells in the capsules as
anterior pituitary tissue. I n reality, these fragments may be
pars intermedia o r other epithelioid tissue in some of the
cases.
An observation which interested us was made on the 22-dayold operated females given immediate treatment. I n three
cases, even though a definite fragment of the gland was found
in the capsule, the reaction obtained with P.U. treatment could
not be distinguished from those of the animals which, as
shown b:~ cxamination of the capsule, were completely hypophysectomized. It is possible that the circulation of the
S
with due to the manipularemaining fragment T ~ interfered
tion at operation, and that the pituitary hormone could not be
supplied in sufficient quantities to be effective. If a longer
period had elapsed after the operation, the effects of the remaining hypophyseal tissue probably would have become
manifested when the circulation through it was re-established.
The addition of small amounts of a pituitary sex hormone
likewise magnified the action of the P.U. extract (table 1,
B8116). Not only were the corpora lutea greatly increased
in size, but also follicles with cavities were formed.
NORMAL F E M A L E S
The effect of pregnancy-urine extract on the normal female
has been studied a t length by several workers. Zondek ('31)
gives a good rCsum6 of his work on this problem. I n the immature female rat, there is a limit to the size of the ovary
that can be produced with pregnancy-urine extract in 5 days
(Evans et al., '31; Collip et al., '31). However, ovaries of
600 mg. o r more have been reported from longer injections.
I n our experiments, injections with 10 units of follutein per
day for 30 days produced ovaries Lip to 335 mg. In these
prolonged injections, the ovaries sometimes did not remain
large but regressed to the size of the controls in spite of
the continued injections, as has also been reported by Collip
et al. ('33 c).
190
SAMUEL L. LEONARD AND PEILIP E. SMITE
The ovaries of these long-treated animals contained large
corpora lutea, hemorrhagic follicles and cysts. The heavier
ovaries produced in these treatments were due in part to the
large lutein and follicular cysts.
The effects of short periods of treatment are well known.
Follicular maturation and ovulation in rats and mice injected
with pregnancy-urine extract have been reported (Borst e t
al., '31). It appears that the proper dosage is needed to
produce this result, for corpora lutea atretica are usually
formed and prevent ovulation. Blood points are common with
the short periods of treatment.
The oestrous cycle was followed for a period covering several cycles in adult females treated with P.U. extract. There
was a tendency toward a rhythmical vaginal change in spite
of the continued injections. I n some cases, however, the
smear was continuously dioestrous. It has been noted in the
experiments in this laboratory that the oestrous cycles of
adult females have a tendency to be normal in a healthy animal even when treated with fairly large doses of growth
hormone containing the sex principle, though the ovary will
be increased markedly in size. The uteri of such animals
became very large, weighing in some cases over 600 mg.
We have pointed out above that injected normal immature
females have a different vaginal smear from those which we
have described in treated hypophysectomized females. Five
normal littermates beginning on the twenty-second day of life
received cc. of antuitrin S daily for 15 days. All opened
on the fourth day with only cornified cells in the vagina but
within a few days the smear changed to that typical of the
dioestrous type which persisted for the duration of the injections. This type of smear is in marked contrast to that of
the hypophysectomized immature females treated in the same
manner with antuitrin S in which smears of cornified cells
predominate.
+
EFFECT O F PREGNANCY URINE ON OVARIES
191
DISCUSSION
There are many similarities in the action of pregnancyurine extracts on the ovaries of normal and hypophysectomized females. I n the normal females there is an increase
in the weight of the ovaries ; in the hypophysectomized females
there is either an increase for a time or a slowing of the
regression which invariably takes place in untreated hypophysectomized animals. I n both types there is an hypertrophy of the theca interna of many of the follicles. I n both
also there is a formation of corpora lutea. This formation
of corpora lutea, though not occurring in all the younger (22-,
29-day-old) immature operated animals, constantly took place
with immediate treatment in the older immature animals. I n
them, it could be shown that these bodies were formed postoperatively as the result of the treatment, f o r there were no
pre-operatively formed corpora present to be confused with
recently formed ones. I n the operated adult rats it seems
probable that corpora were also formed by the treatments,
though the presence of pre-operatively formed bodies made
the absolute determination of this impossible. Occasionally,
small bodies resembling corpora lutea formed in the postponed-treated operated animals.6
There are also differences in the action of P.U. extract in
the normal and hypophysectomized female. It has not been
Irradiation of the ovaries of mice, particularly adults (Rrambell and Parkes,
'27), induces physiological and structural effects which in several respects are
strikingly similar to the effects of hypophyseotomy followed by P.U. injections
i n rats. In both types there is 110 follicular growth. There is a secretion of
oestrin, however, which i n our aniinals was usually coiitiuuous, but in the irradiated
mice was usually rhythmic, though sometimes prolonged. The oestriu must be
secreted in our animals by the same type of tissue t o which Brambell and Parkes
attributed oestriii production, namely, iiiterfollicular or interstitial tissue. In
both types of animals, the interstitial tissue and theca cells hypertrophy and
become lutein-like, the ovaries appearing structurally homogeneous. In our
animals this hypertrophy does not persist even with coiitinued treatment, whereas
i n the irradiated mice of Brambell and Parkes the hypertrophy persisted f o r the
duration of the experiment (73 days). I n the ovaries of both types of animals,
persisting bodies, structurally similar to corpora lutea, are formed. Some of
these Brambell and Parkes believed were true corpora, whereas others they designated as corpora lutea atretica, these being formed both from graiiulosa and theca.
192
SBMI'EL L. LEOTTARD A S D PHILIP X. SMITH
possible to produce a n ovary in an hypophysectomized r a t
a s large a s in the normal, treated with the same amount of
extract. It appears that the animal's own gland is furnishing
an element necessary for tlie production of these large
ovaiies. This increase in size of tlie ovaries in the h y p o p h ~ s ectomized r a t s is due to an interstitial and theca cell l i g e r trophy and to a n extent, particularly in the immediate-treated
animal, to the formation of new corpora ltitea. I n the treated
normal rats there is, in addition, follicular growth and cyst
formation. I n the postponed injections, the actual weight
increases of the ovaries a r e not as great as usually occiirred
in the immediate treatment, although the percentage increase
was much greater. The relative weight increase is greater
in the postponed than in the immediate treatments because
a n intense atrophy had taken place in the ovaries before
treatment. Although the actual increase was small compared
to the immediate-treated animals, in relation to the control
ovary, the percentage increase was higher.
The regression of the weight of the ovaries with continned
treatment is of much interest. A rapid loss would be expected
with cessation of treatment, but the regression with continued
injections, tlioiigh unexpected, is not disharmonious with the
findings obtained in prolonged injections in normal females.
Zondek ('31) and Collip et al. ('31) report that with continned
treatments with pituitary-like hormones from pregnant
womcii, the enlarged gonads in normal females return to the
size of the normal control. The ovaries of the controls (10
monlhs old) of the latter investigator, however, showed a
great variation in weight (0.016 to 0.098 gm.). I n o m experiments on some thirty females injected for periods up to 35
days, we found quite a variation in the weights of the oraries
a t antops>-. I n some of the ovaries the weights were the same
a s in the littermate controls (about 0.035 gm.), while others
weighed up to 0.335 gm. The control ovaries were quite uniform in animals of the same age. Since in the shorter treatments the ovaries were invariably enlarged, it seems that in
the longer treatments some of them must have regressed,
although others continued to enlarge.
EFFECT O F PREGNANCY URINE ON OVARIES
193
A possible reason why the gonads of the normal female
return to normal size is based on the fact that there is a
reduction in the potency of the A.P., as evidenced by implants,
when P.U. extract is injected (Kuschinsky, '31 ; Leonard, ' 3 3 ) .
If it is assumed that there is an accompanying reduction in
the liberation of the gonadotropic hormone into the circulation, then there would be a decreasing amount of the A.P.
factor to interact with the P.U. gonadotrophic principle. An
A.Y. factor seems to be essential f o r a pronounced hypertrophy of the ovaries with P.U. injections for we have failed
to secure the marked enlargement after hypophysectomy that
is seen in the normal animal.
This explanation, however, does not suffice for the hypophysectomized female (or male), for regressioii of the interstitial tissue does not begin for 20 days after injections have
been instituted. I t is hardly conceivable that circulating A.P.
hormone persists for such an extended period. Furthermore,
this explanation that a circulating A.P. hormone is essential
f o r the interstitial cell hypertrophy fails absolutely in both
sexes with postponed treatment f o r the hypertrophy takes
place even after the lapse of a 2;-month post-operative
period, aiid it is not conceivable that the A.P. sex hormone
continues t o be present f o r this extended period. A similar
regression also occurs with continued injection in the postponed treatments.
With regard to the formation of corpora lutea, the data
presented show that they can form with P.U. treatment in
the absence of the hypophysis. Collip et al. ('33 b) have
stated that corpus luteum formation can occur with P.U.
treatment only in the presence of some circulating pituitary
factor. I t is evident from the control (uninjected) operated
animals that the pituitary hormones are lost very rapidly
from the blood and consequently the later changes in the
ovaries in the postponed injections, a t least, must come from
the action of this substance, only, on the end organ. It is
quite probable that some previous action (sensitization) of
the A.P. on this organ is necessary in order for the P.U. to
194
SAMUEL L. LEONARD A N D PHILIP E. SMITH
elicit a reaction, but that corpora will form without the presence of circulating A.P. hormone is certain. However, our
results show that many more normal corpora are formed in
the immediate than in the postponed treatments so that we
do not deny that circulating A.P. hormone greatly facilitates
the formation of these bodies. This would be expected for the
A.P. hormone maintains the development of follicles and it
seems certain that corpora formed from large normal follicles
would be more typical than those formed from the relatively
undeveloped follicles characteristically present after hypophy seet omy.
The reason why corpora lutea failed to form in several of
the operated 22-day-old immature females and in one 29-dayold animal, whereas they formed in other animals of these
ages, cannot be definitely determined. It is known that very
young female rats will not respond to A.P. treatment (Smith
and Engle, '27 ; Corey, '28), nor will they respond characteristically to the injection of A.P.L. (Selye and Collip, '33). I t
seems probable that if female rats much younger than 22 days
were hypophysectomized, they would invariably fail to produce corpora lutea with P.U. treatment. That the stage of
the pre-ovulatory cycles (Hargitt, '30) at which the operation
was performed and the injections begun might also influence
the response seems not improbable.
The corpora lutea that formed in the immature treated
hypophysectomized females seem to be like those in normal
animals. Structurally, they are similar and with the cessation of treatment they persist as discrete bodies in the midst
of atrophied interstitial cells, as do typical pre-operatively
formed corpora in mature animals after hypophysectomy. As
it has been demonstrated that corpora lutea formation in the
rat involves the granulosa, it seems probable that the granulosa cells are involved in the formation of these bodies in the
hypophysectomized animals. It will only be by careful cptological studies that the participation of the granulosa can be
excluded.
E F F E C T O F P R E G N A N C Y U R I N E ON OVARIES
195
As shown by the response of the accessory reproductive
organs, there was a secretion of oestrin in almost all of the
operated animals with immediate treatment whether they
were mature or immature. The smears usually contained
many cornified cells, though leucocytes and epithelial cells
were also sometimes present. I n the normal adult female the
injections of similar quantities of extract usually do not completely prevent the rhythmical vaginal changes, though some
animals will show continuous dioestrum. The normal immature treated female will usually open with cornified cells in
the vagina but invariably changes t o a continuous dioestrous
smear in a few days. Collip, Selye and Thomson ('33a)
found that oestrous smears did not occur in immature hypophysectomized rats with A.P.L. injections unless the
animals had been previously treated with A.P.L. hormone for
5 days before the operation. We have found in hypophysectomized females that the injection of a gonadotropic A.P.
hormone in addition to the P.U. extract and that the injection
of P.U. extract, only, in incomplete hypophysectomies gives
smears resembling those of treated normal animals. The
fact that the vaginal smears of hypophysectomized treated
females are characteristic, supplies evidence that in certain
cases the ablations were functionally complete though from
examination of the capsule we had considered them to be incomplete. It is possible that if a small fragment of A.P. did
remain, the circulation may have been so interrupted that, for
a time a t least, effects identical with a complete operation
would result. White ('33) obtained a functional failure of
the pituitary by blocking its circulation in the rabbit.
The results that are presented in this paper were obtained
with extracts that appear to be free of any pituitary factor.
It has been shown by a number of investigators (Riddle, '28;
Schockaert, '33) that pregnancy-urine extracts are without
effect on the testes of birds. The extracts used by us elicited
no response in this form. Other tests have also shown that
our extracts do not contain pituitary substance, namely, limitation of size of the ovary produced by P.U. extracts in imma-
196
S A M U E L L. L E O N A R D A N D PHILIP E. SMITH
ture rats (Evans et al., '31), the rabbit ovulation test
(Leonard, '32), and finally the hypophysectomized rat itself.
Indeed, one of the most characteristic qualitative differences
between P.U. extracts and the pituitary gonadotropic hormone (particularly when given as implants) is the failure of
the former to produce follicles with cavities or follicular cysts
iii the hypophysectomized rat, whereas these are characteristically formed when pituitary implants are given.
Pregnancy urine has a definite capacity to stimulate the
ovaries of hypophysectomized rats. It, however, does not
cause follicular maturation and does not maintain normal
ovarian function after hypophysectomy. I n this, its action
differs from that in males (Smith and Leonard, '33 b ) , in
which its injection, as we have reported, maintains: within the
limits of time and dosage of our experiments, the reproductive capacity.
SUMMARY
Immature and mature hypophysectomized rats were injected for varying periods with pregnancy-urine (P.U.)
extracts (antuitrin S, follutein). Some of the treatments
were begun a t the time of pituitary ablation; others after a
period of 16 to 78 days had elapsed. The experiments were
fully controlled by littermates autopsied a t the time treatments were begun, by untreated operated littermates and by
unilateral ovariectomies. Moderate doses, only, of P.U. estract were given.
Treatments begun a t the time of operation increased or
maintained the weights of the ovaries for a time, though
regression took place with prolonged injections. Postponed
treatment caused an invariable increase too in the weights of
the ovaries.
Follicular growth was not induced by the P.U. treatments.
The interstitial and theca cells hypertrophied and became
much like lutein cells, causing a structural homogeneity of
the ovary. The hypertrophy did not persist when the treatments were prolonged or were stopped. It persisted, however, in certain newly formed bodies which appear to be
E F F E C T O F PREGNANCY U R I N E O N OVARIES
197
structurally identical with corpora. These regressed but little
if at all, within the time limits of the experiment, thus behaving as do typically formed corpora present at the time of
hypophysectomy. I n the younger prepubertal animals corpora were not invariably formed in the immediate injections
nor were they invariably formed in the postponed treatments.
Those formed in the latter type of treatment were small.
Oestrin was secreted by the ovaries in both the mature and
immature hypophysectomized animals injected immediately
after the operation. I n them a cornified smear was usually
continuous, in contrast to P.U. injected normal rats in which
cornification was discontinuous. There was a secretion of
oestrin also in some of the postponed treatments.
The effects induced by P.U. injections in hypophysectomized
rats are dissimilar to those produced by A.P. implants, the
latter producing larger ovaries which contain normal and
cystic follicles and larger corpora. The effects of P.U. injection in hypophysectomized rats are also not the same as in
normal rats for in the latter there is a development of follicles, cysts and blood follicles.
The pituitary sex hormone facilitates the action of P.U.
on the ovaries as shown by results obtained in partial hypophysectomies and in concurrent injections of A.P. and P.U.
in hypophysectomized and in normal immature animals.
The action of pregnancy-blood serum is similar to that of
the P.U. extracts.
Hypophysectomy followed by P.U. injections induces
ovarian changes in rats which are strikingly similar, in many
respects, to those produced by irradiation of the ovaries of
adult mice.
LITERATURE CITED
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SAMUEL L. LEONARD AND P H I L I P E. SMITH
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1932 Relative ineffectiveness of prolan i n hypophysectomized animals.
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J. A. 1933 Differences between anterior pituitary sex-stimulating
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H., A N D J. B. COLLIP 1933 Production of exclusively thecal luteinization
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M. C. 1933 The production of deciduomata i n immature rats by
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EFFECT OF PREGNANCY U R I N E ON OVARIES
199
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____
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J . Springer, Berlin.
PLATE 1
EXPLANATION OF FIGURES
1 L e f t (control) ovary of a n immature rat, B8078, removed 54 days a f t e r
hypophysectomy. No treatment. X 40.
2 Right ovary of B8078 removed 15 days later, the animal having been
injected during this period with 38 R.U./day of antuitrin S. X 40.
3 Right ovary of B8080, a littermate of the above, same treatment except
that it was injected for 30 days following the 54-day no-treatment period. The
interstitial cells have regressed and a discrete body (corpus luteum) has persisted. X 40.
4 L e f t ovary of a mature rat, BH6910, removed $8 days after hypophysectomy. No treatment. Note persistent corpora. X 40.
5 Right ovary of BH6910 removed 1 0 days later, the animal having been
injected during this period with 10 R.U./day of follutein. Note hypertrophy of
the interstitial tissue, which has obscured the persisting corpora. x 40.
200
EFPE(’1’ O F PKEGNAX‘CY URTNE ON O V B l i l E S
S A M U E L TI.
J Z O N A R D A N D I’IIILIP E. S X I T H
201
T H E ANATOMICAL IlECOllD, VOT..
58, N O . 2,
AND SUPPLEMENT
PLATE 2
EXPLANATION OF FIGURES
6 Right ovary of aii immature rat, G H 8329, removed 1 0 days after hypophyseetomy. No treatment. X 2G.
7 A p a r t of the ovary shown i n figure G . X 104.
littermate of the above,
8 Right ovary of aii immature liy~~ophysectoniized
GH8325, treated for 10 days with 25 R.U./d;iy of antuitrin S. Iiijectioii begull
at time of operation. x 26.
9 A part of the ovary shown in figure 8. The regioii photographed is
iiidieated by the arrow. Note forming corpora (below) aiid hypertrophied interstitial cells (above). X 104.
10 Right ovary of mi immature hgpophyseetomized littermate, GH8324, of the
above animals. It was also injected with 25 R.U./day of antuitriil S f o r 1 0 days,
treatment begiiiiiiiig a t the time of operation. Following this a 10-day iio-treatmeiit period elapsed before autopsy. Note persisting corpora aiid regressed
iiiterstitial tissue. x 2G.
11 A part of the ovary sliowii in figure 10. x 104.
202
PLATE 2
EFFECT O F P R E G K A K C Y C I t l N E ON OVARIES
SAMUEL L. LEONARD A X D PHTT.11’ F,. SMITII
203
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