The effects of cyproterone acetate, a potent antiandrogen, on preovulatory follicles were studied both in vivo and in vitro. In vivo administration of cyproterone.
BIOLOGY
OF
REPRODUCTION
21,
Effects
929-936
(1979)
of Cyproterone
Acetate,
a Potent
Antiandrogen,
on the Preovulatory Follicle J. J. PELUSO,
I. BROWN Department
Loyola
and of
Biology,
University Chicago,
R. W. STEGER’
of
Chicago,
Illinois
60626
and Department
of
Michigan East
State
Lansing
Physiology,1 University,
Michigan
48824
ABSTRACT The effects of cyproterone acetate, a potent antiandrogen, on preovulatory follicles were studied both in vivo and in vitro. In vivo administration of cyproterone acetate induced acid phosphatase activity and reduced mitosis within the granulosa cell layer, accelerated the rate of atresia and subsequently transformed the preovulatory follicle into an ovarian cyst. Serum LII levels were not altered, but serum FSU levels were initially suppressed by cyproterone acetate treatment. In vitro, cyproterone acetate reduced the number of granulosa cells incorporating lIIl -thymidine preovulatory
by follicle
These observations that their action
50%.
and
suggest is mediated
INTRODUCTION Ovarian
follicles
granulosa
cell
divisions
are
with
FSII
(Goldenberg
be
of
FSH
and
within
the
1975).
However,
promotes
in and
a!.,
role
both
an
in
et
a!.,
intraovarian an
rat
cycling of
1975);
rate
and
mechanism
are
the
In
immature
treatment
preovulatory induce
induces
a!.,
follicles an
ovulatory
within LH
et
River
48
h but (Peluso
pyknosis
necessary the
to
which
Experiment between
a!.,
occurs
for
present
follicular
investigations
determine
the
a potent
antiandrogen,
preovulatory
effects
follicles
of on degen-
AND
METHODS
1. Serum
does et
Temporal LII
and
Relationship Ovarian
Content
Sixty-four
female Charles River rats, 21 days of housed in a controlled environment (24#{176}C; 14L:1OD). On Day 24 at 0930 h, 32 rats received an i.p. injection of 5 IU PMSG and the remaining 32 rats received an i.p. injection of saline. Eight PMSG primed and 8 saline treated rats were decapitated at 48, 60, 72
age,
of
not a!.,
or
Accepted Received
are
MATERIALS
rats,
development
surge
of the
involved
is required
Charles
and
capacity
decreased,
et fol-
(Mon
the
phosphatase
The then
in
capacity
erate.
Androgen
24-day-old
the
acid
is
not This
1978).
ovulation,
at
do
sequence
lose
1977).
is reduced
acetate,
rate
time
first
FSll
undertaken
follicular
1977).
PMSG
bind
androgens
cyproterone
et
a!.,
Steger,
and
were
follicles
develop
et
to
and
follicular
(Kumari
which
ovulation
a!.,
and
mitoses
Since
preantral 3)
of
growth
play
(Beyer rat
atresia
hCG-induced
to
stimulate
cells
(Peluso
et
appear
immature
induce
may
synthesis
regulating 1)
these
estrogen
(Dorrington
LH
cells
(Peluso
the
degenerate. a precise
granulosa
bind
activity
aromatizing
also
mature
2)
(Louvet
for
the
the
1978);
licles
cells
Androgens
1974)
division
estrogen
androgens
important
growth
cell enhanced
preovulatory
subsequently follows
the
to
stimulatory
induces
granulosa
development.
in
FSH
estrogen formation
The
an
which
involved in maintaining intraovarian mechanism.
the
but
degeneration
These
and
Thus,
ovulate
repeated
antrum
granulosa
since
enzymes
FSH
1972).
through
synthesis,
to 1972).
for
a!., on
mediated
an
by
necessary et
effects
due
(Pederson,
stimulated
being
1977).
grow
divisions
that androgens are in part by a direct
were
96
h after
injection.
Serum
was
collected
from
trunk blood and assayed for LH content by a double antibody radioimmunoassay. LI-I values were cxpressed as ng NIAMDD LH-RP-1/mI serum. At autopsy, the ovaries were removed, trimmed of
August 15, 1979. April 10, 1979.
929
PELUSO
930
fat, weighed, homogenized saline and extracted with
in 1 ml phosphate 3 ml ethyl ether.
buffered l’he ether
extract was evaporated to dryness, resuspended in I ml phosphate buffered saline and assayed for androgen content by a double antibody radioimmunoassay. The materials for the androgen assay were purchased from Bio-Endocrinology, Inc. (Montreal, Canada). The antiandrogen serum not only bound testosterone but also showed a 3% cross reactivity with dihydrotestosterone. however, the antiandrogen serum did not cross react with cholesterol, C21 steroids or C15 steroids. Androgen content was expressed as ng/mg ovarian weight.
Experiment Acetate on
of
2. Effect Cyproterone the Preovulatory l”ollicle
Sixty female Charles River rats, 24 days old, received 5 IU PMSG, i.p. Groups of rats were then injected s.c. with either 5 mg cyproterone acetate (Schening AG Berlin/Berkmen 45013314) suspended in 0.2 ml corn oil, or 0.2 ml corn oil at 48, 60, 72 or 96 h after PMSG treatment. One hour after each injection, a group of 5 control and 5 treated rats were decapitated. The ovaries were removed and either fixed in Bouin’s fluid, or placed in embedding medium (OCT compound: Ames Co., Elkart, IN), frozen in a dry ice-ethanol bath and stored at -20#{176} C. Serum was prepared from the trunk blood and stored at -20#{176}C until assayed for FSII and Lii content. FSII and LII were measured with radioimmunoassay. LII and FSH values were expressed as ng NIAMDD LH-RP-1 or FSH-RP-1/ml serum, respectively. The Bouin fixed ovaries were embedded in paraffin wax, sectioned at 10 m and stained with hematoxylin and eosin. Histological examinations were made on follicles 500 m in diameter or greater. Largest diameters were determined by measuring sections containing the were
oocyte with a germinal classified as atretic if
nuclei persed thinned. A
were and/or mitotic
present, the index
the granulosa was
vesicle. 3 or
cumulus cell
calculated
The more
for
was both
were disseverely
the
48 and
60 h control and treated groups. The mitotic index represents the percent of granulosa cells with mitotic figures in the section of the follicle containing the oocyte with a germinal vesicle. Histochemical
Experiment on
3. Effect
Incorporation
of
Preovulatory
of Cyproterone
Acetate
-thymidine
[3jjJ
by
I”ollicles
Five female Charles River rats, 24 days old, were injected with 5 IU PMSG, i.p. Forty-eight hours after PMSG treatment, the rats were decapitated; the ovaries were removed and sliced into cubes of 1-3 mm3. The ovarian fragments were randomly placed on a small piece of lens paper covering a stainless steel mesh platform which was then set in an organ culture dish (Falcon Plastics, 3010). The dishes contained 0.8 ml Eagles minimum essential medium with Earles salts and glutamine to which either 0.1 ml ethanol or 0.1 ml 2.5 mM solution of cyproterone acetate in ethanol was added. The dishes were placed in airtight chambers, gassed with 5% CO2 in air and incubated for 1 h at 37#{176}C. After the first hour of incubation, the grids containing the tissue fragments were immediately transferred to a second set of culture dishes containing medium which was supplemented with 0.1 MCi [53 ill -thymidine. The chambers were regassed and further incubated for 1 h. After the second incubation, the ovarian fragments were fixed in Bouin’s fluid, embedded in paraffin wax and sectioned at 10 m. The sections were then dipped in Kodak NTB-3 emulsion, exposed for 21 days, developed and then stained with hematoxylin and eosin. The labeling index was determined for all follicles greater than 500 tim in diameter. The labeling index was determined by calculating the percentage of labeled nuclei within the largest diameter of the follicle. A nucleus was considered labeled when an average of 3 grains were present over its area. The probability that a cell was considered labeled as a result of background was less than 2% for each group of follicles. The data from all 3 experiments were analyzed by Student’s t test.
follicles pyknotic
cells layer
ET AL.
RESULTS
Experiment
I.
between
Serum
Ovarian
Androgen
Serum PMSG Although
LU treated there
Temporal LII
Relationship
and Content
levels rats was
were than no
higher in
the
difference
at all controls
times (Fig.
in
serum
in 1). LH
localization of acid phosphatse was at 48 and 60 h (P>0.05), serum LH levels were carried out Ofl frozen sections which were mounted on reduced at 72 and 96 h after PMSG treatment slides and air dried. The sections were covered with an (P
