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Department of Obstetrics and Gynecology (H.W., K.A., L.X., N.K., Y.A., K.Y., R.F.), and First Department of Anatomy (H.H.), Gunma University School of Medicine, Gunma 371-8511; and Department of Obstetrics and Gynecology, Hirosaki University School of Medicine (H.M.), Hirosaki 036-8562, Japan
Address all correspondence and requests for reprints to: Hideki Mizunuma, M.D., Department of Obstetrics and Gynecology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562 Japan. E-mail: mizunuma{at}cc.hirosaki-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4), dihydrotestosterone (DHT), estrone
(E1), and E2 on recombinant human FSH- (rhFSH) resistant type 4
follicles obtained from immature mice. Type 4 follicles of a diameter
of 100–120 µm with one or two granulosa cell layers around the
oocyte and an intact basal lamina with theca cells were isolated from
the ovaries of 11-d-old BDF-1 mice and cultured with medium alone
(control) or with dehydroepiandrosterone, dehydroepiandrosterone
sulfate, T, 4, DHT, E1, or E2 at concentrations ranging
from 1 x 10-11 to 1 x 10-7
M for 4 d. We examined the mean diameters of type 4
follicles, levels of immunoreactive (IR)-inhibin, and E2 and
progesterone in the culture media on day 4. In addition, we evaluated
follicular cell proliferation by immunofluorescence staining with
5-bromo-2'-deoxyuridine. All tested androgens significantly increased
the diameter of type 4 follicles in a dose-dependent manner without the
production of IR-inhibin and E2. The nuclei of granulosa cells in type
4 follicles cultured with all tested androgens exhibited intense
5-bromo-2'-deoxyuridine-positive staining, compared with those of
controls. In contrast, neither E1 nor E2 had any stimulatory effects.
The stimulatory effects of T, 4, or DHT were inhibited
by an AR antagonist in a dose-related fashion but not by an aromatase
inhibitor. Furthermore, all tested androgens had a synergistic effect
with rhFSH on follicular growth and the production of IR-inhibin and
E2. These results demonstrated that neither adrenal nor ovarian
androgens are arteriogenic but that they stimulate type 4 follicles
unresponsive to rhFSH and augment the responsiveness of these follicles
to rhFSH. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (DHT) to normal cycling mice reduces
the number of normal large follicles by 50% (3), ovarian
androgens have long been implicated as an inhibitor of follicular
development (4, 5, 6). Recent in vitro studies,
however, have shown that T and DHT stimulate the production of
progesterone by granulosa cells from hypophysectomized E-treated
immature rats (7, 8, 9) and that ovarian androgens enhance
the cyclic AMP production (10) as well as the aromatase
activity (11, 12) of granulosa cells from normal and
E-primed immature rats. In addition, ovarian androgens were found to
stimulate the growth of small preantral mouse follicles cultured
in vitro (13, 14) and T and DHT to increase the
number of follicles as well as theca and granulosa cell proliferation
in normal cycling rhesus monkeys (15). These results
suggest that ovarian androgens have a stage-specific action on
follicular development and can elicit a stimulatory action on
follicular growth at the preantral stage. However, whether adrenal
androgens have such effects on preantral follicular growth remains
unknown. The fact that congenital adrenal hyperplasia causes ovarian
dysfunction and premature adrenarche promotes the onset of puberty in
women raises the notion that adrenal androgens are involved in ovarian
function (16). Very early follicular growth is gonadotropin independent (17), and the involvement of intra- and extraovarian factors such as activin or GH has been thought important in regulating the first step of folliculogenesis. In immature mice, intra- and extraovarian factors may stimulate small preantral follicles at a level at which they acquire responsiveness to FSH (18, 19). Studies of androgen-resistant testicular feminization mouse (Tfm) female mice have shown impaired reproductive performance, such as premature luteinization and progressive decrease in primordial follicles followed by premature cessation of reproduction (20, 21), although to the best of our knowledge, whether these animals have delayed puberty or a normal response to FSH remains to be clarified. Because ovaries have ARs at birth (22), understanding the role of androgens on follicular growth at very early stages is important for understanding the pathophysiology of prepubertal follicular growth. The present study investigates the physiological role of gonadal and adrenal androgens and estrone (E1) and E2 on follicular growth of immature mouse type 4 follicles that remain unresponsive to FSH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4),
5-dihydrotestosterone (DHT), 1,3,5(10)-estratriene-3-ol-17-one (E1),
and 1,3,5(10)-estratriene-3,17ß-diol (E2) from Sigma
(St. Louis, MO). Recombinant human FSH (rhFSH) (lot no. 19505064) was
supplied by Organon Co. (Oss, The Netherlands). The AR
antagonist, OH-Flutamide, and the aromatase inhibitor, Fadrozole
hydrochloride hydrate, were also supplied by Nippon Kayaku Co. (Tokyo, Japan) and Novartis Pharma Co. (Tokyo,
Japan). All other chemicals were of analytical grade or of the highest
quality commercially available.
Animals
BDF1 hybrid female lactating mice with 7-d-old female
pups were purchased from Japan Charles River Laboratories, Inc. (Tokyo, Japan) and housed in a temperature- (24–26 C) and
light-controlled room with a 14-h light/10-h dark photoperiod in
accordance with the principles of the Animal Care and Experimentation
Committee, Gunma University, Showa Campus. The maternal mice were given
food and water ad libitum, and the pups were nursed for
4 d. Eleven-day-old immature mice were killed by cervical
dislocation.
Follicle isolation and culture
Follicles were mechanically isolated from the ovaries of
11-d-old immature female mice as described (18, 23).
Briefly, ovaries were aseptically removed and placed in 15-mm Falcon
plastic Petri dishes (FALCON 3037, Becton Dickinson and Co., Rutherford, NJ) containing DMEM (Life Technologies, Inc., Tokyo, Japan) at room temperature. After removing the
surrounding tissue, the ovaries were microdissected using two 27-gauge
needles attached to 1-ml syringes under a stereomicroscope
(Olympus Corp., Tokyo, Japan), and approximately 60
preantral follicles were isolated from each ovary. We cultured type 4
follicles of a diameter of 100–120 µm according to the
classification of Pedersen and Peters (24) in a humidified
chamber with 5% CO2 in air at 37 C. Type 4
follicles consisted of one to two layers of granulosa cells around the
oocyte and an intact basal lamina with theca cells. Histological
examination revealed that 86.9% of the sections of these type 4
follicles had at least one thecal cell. This agrees with previous
findings that type 4 follicles have 84.5 ± 12.8 granulosa cells
per section (18).
Ten type 4 follicles were transferred into 30-mm plastic Falcon Petri
dishes containing 1 ml serum-free DMEM supplemented with 6.25 µg/ml
of insulin, 6.25 µg/ml of transferrin, 6.25 ng/ml of selenious acid,
5.35 µg/ml of linoleic acid, 0.15% BSA, 15 mM HEPES, 45
µg/ml of penicillin G, 350 µg/ml of streptomycin, and 1.75 µg/ml
of Amphotericin before being cultured in a humidified
chamber with 5% CO2 in air at 37 C. Type 4
follicles were cultured with medium alone (control) or with
DHEA, DHEAS, T, 4, or DHT at
concentrations of 1 x 10-11 to 1 x
10-7 M, and E1 or E2 at
concentrations of 1 x 10-11 to 1 x
10-7 M for 4 d without media
changes. Next, to ascertain the direct effect of androgens, type 4
follicles were cultured in the presence of T,
4, or DHT at concentrations of 1 x
10-7 M in combination with an AR
antagonist or an aromatization inhibitor at concentrations of 1 x
10-11 to 1 x 10-5
M. Moreover, to clarify the effect of combined rhFSH and
androgens, type 4 follicles were cultured with rhFSH at concentrations
of 0.1–100 mIU/ml in the presence of 1 x
10-7 M DHEA, DHEAS, T,
4, or DHT at for 4 d. Each experiment was
repeated at least three to five times. Less than 10% of follicles
remarkably degraded during 4 d of culture, and these were removed
from the medium.
Measurement of follicular diameter and hormone assay
The mean diameter of two-dimensional maximal and minimal lengths
in each follicle was measured daily using an inverted microscope
(IMT-2, Olympus Corp.). To determine the levels of
immunoreactive (IR)-inhibin, E2, and progesterone, the culture medium
was collected on day 4 and stored at –20 C. All samples were assayed
in duplicate by RIA. IR-inhibin concentrations were measured by
double-antibody RIA using rabbit antiserum against bovine follicular
fluid inhibin as described (25). E2 levels were determined
using an anti-E2 antiserum supplied by Dr. W. F. Crowley, Jr.
(26) and estradiol-6-(O-carboxymethyl)
oximino-(2-[125I]iodohistamine) (Amersham Pharmacia Biotech, Buckinghamshire, Little Chalfont, UK). The
concentrations of progesterone were also measured by double-antibody
RIA using a specific rabbit antiserum as described (27).
Progesterone-11-hemisuccinate-(2-[125I]iodohistamine)
was also purchased from Amersham Pharmacia Biotech. The
sensitivity levels of the assays for IR-inhibin, E2, and progesterone
were 0.04 ng/ml, 1.00 pg/ml, and 0.01 ng/ml, respectively. The intra-
and interassay coefficients of variation for IR-inhibin and E2 were
3.2%, and 4.0% and 2.7% and 2.8%, respectively.
Assays of cell proliferation in follicles
Follicular cell proliferation in type 4 follicles cultured with
DHEA, DHEAS, T, 4, or DHT was
evaluated by immunofluorescence staining with 5-bromo-2'-deoxyuridine
(BrdU) using a cell proliferation kit (Amersham Pharmacia Biotech). Type 4 follicles were cultured with 1 x
10-7 M DHEA, DHEAS, T,
4, or DHT for 4 d and with 1 x
10-5 M BrdU to label proliferative
cells for 60 min in a humidified chamber under 5%
CO2 in air at 37 C on day 4. After BrdU labeling,
cultured follicles were transferred to
poly-L-lysine–coated slide glasses, fixed in 3%
paraformaldehyde in 0.1 M PBS (pH 7.4) for 30 min at room
temperature, solubilized with 1% Triton X-100 in PBS for 10 min,
washed in PBS, and incubated in 2% gelatin in PBS for 15 min at 37 C.
The follicles were then incubated with sufficient reconstituted
nuclease/anti-BrdU in a wet chamber for 120 min at 37 C, washed six
times in 0.1% BSA/PBS, and further incubated with FITC-labeled
antimouse IgG (Cappel/ICN, Aurora, Ohio) diluted to 1:100
by 1% BSA/PBS for 60 min at 37 C. Nuclei were stained with TOPRO-3
iodide (Molecular Probes, Inc., Eugene, OR) at a 1: 800
dilution. After six washes in 0.1% BSA/PBS, the follicles were mounted
as described (28) and examined under a fluorescence
microscope (Axioplan, Carl Zeiss, Jena, Germany) and/or a
confocal laser scanning microscope (MRC-1024ES, Bio-Rad Laboratories, Inc. Hemel Hempstead, UK). Negative control
sections were processed without the labeling reagent
(10-5 M BrdU) or reconstituted
nuclease/anti-BrdU. The BrdU index was calculated by dividing the
number of BrdU-positive cells by the number of BrdU-positive and
TOPRO-3–positive cells.
Statistics
Results are presented as means ± SEM. One-way
ANOVA followed by Scheffé’s multiple comparison was performed to
determine statistical differences among groups. A value of
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows changes in the
diameter of type 4 follicles cultured with DHEA, DHEAS, T,
4, DHT, E1, or E2 at the indicated
concentrations for 4 d. The diameter of type 4 follicles increased
in a significant dose-dependent manner after stimulation by
DHEA, DHEAS, T, 4, and DHT but
not by E1 or E2. These findings suggested that
the stimulatory effect of androgens was direct and not a consequence of
being converted to E. The minimum effective dose of each androgen was
1 x 10-10 M, and potency did
not significantly differ among the tested androgens. Although all
tested androgens increased follicular diameter, IR-inhibin secretion
was not significantly increased (data not shown). Androgen
administration tended to increase E2 secretion, but the difference was
not statistically significant, suggesting that the major role of
androgens in type 4 follicles is to cause morphological change rather
than functional differentiation.
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shows the morphological changes
of type 4 follicles cultured for 4 d with medium alone (control)
and medium containing DHEA, DHEAS, T,
4, or DHT at a concentration of 1 x
10-7 M (Fig. 2, A–F). The diameter
of follicles cultured in the presence of each of the androgens (Fig. 2, B–F) increased remarkably, compared with that of the control (Fig. 2A). Figure 2, A'–F' and Fig. 2, A''–F'' show BrdU incorporation into
granulosa cells. The incorporation of BrdU by follicles cultured in
medium containing an androgen significantly increased (Fig. 2, B'–F'),
as did the number of cells (Fig. 2B'–F'). These findings indicate
that androgens stimulate the proliferation of granulosa cells of type 4
follicles.
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shows the effects of an AR
antagonist (OH-Flutamide) and aromatase inhibitor on follicular
diameter of type 4 follicles cultured with 1 x
10-7 M ovarian or adrenal androgens.
Although the AR antagonist decreased the diameter of type 4 follicles
stimulated by ovarian and adrenal androgens in a dose-related manner,
an aromatase inhibitor did not, suggesting that the stimulatory effect
of androgens on follicular growth is a direct action.
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shows the effect of rhFSH on the
growth of type 4 follicles cultured for 4 d in the presence of
1 x 10-7 M androgens.
Recombinant human FSH alone did not cause any significant change in
follicular diameter. However, rhFSH caused a significant and
dose-related increase in follicular diameter in conjunction with
androgens at a dose of 1 x 10-7
M. Figure 5 shows IR-inhibin
and E2 concentrations in the culture medium. The increase in follicular
diameter was accompanied by an increase in IR-inhibin and E2 secretion,
suggesting that FSH induced the differentiation of granulosa cells.
Although the androgens did not evoke significantly different levels of
IR-inhibin secretion, their effects on E2 secretion was significantly
different. Among all of the tested androgens, the nonaromatizable
androgen DHT was the least potent in evoking E2 secretion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the response of small preantral follicles from 28-d-old mice is intermediate between immature and adult mice, we speculate that the prepubertal gonadotropin surge is important to the differential responses of immature and adult mice to FSH. The prepubertal gonadotropin surge occurs around 11 d of age and ends by 28 d of age (29). Therefore, an unanswered question is how small preantral follicles of immature mice acquire the initial step in the generation of an FSH response. One putative answer is the presence of intra- and extraovarian growth factors. Because small preantral follicles from 11-d-old mice start to grow as the result of activin A and GH stimulation, we infer that these intra- and extraovarian factors control the initial step of small preantral follicular growth (19). The results of the present study suggest that androgens function in this manner. In particular, adrenal androgens directly stimulate follicular growth, suggesting that the adrenal gland plays an important role in the initiation of prepubertal follicular growth. Despite the fact that the expression and regulation of ARs in neonatal or prepubertal mouse ovaries is not apparent in the adult mouse ovary (30, 31, 32), the results of the present study suggest that ovarian and adrenal androgens play important roles in granulosa cells of type 4 follicles derived from 11-d-old mice acquiring a response to FSH.
It is well known that the various androgens have different levels of
affinity for the AR. Nevertheless, as show in Fig. 3
, the minimal
effective dose of androgens was 1 x 10-10
M for both adrenal and ovarian androgens, suggesting that
both ovarian and gonadal androgens were equally potent in inducing the
growth of type 4 follicles from immature mice. The discrepancy between
the different affinity of androgens to their receptor(s) and biological
potency can be partly accounted for by the metabolism of androgens
during the 4-d culture. It is known that 17ß-hydroxysteroid
dehydrogenases and 5 reductase, which can convert low-activity sex
steroids to more potent forms, are expressed in immature mice ovary
(33, 34).
Both ovarian and adrenal androgens stimulated proliferation and
increased the number of granulosa cells, which was followed by an
increase in the size of the follicles. However, the stimulatory effect
of androgens on E2 or IR-inhibin secretion from type 4 follicles of
immature mice was either relatively weak or absent. We showed that type
4 follicles from 11-d-old mice increase in size and that they secrete
more IR-inhibin and E2 upon stimulation by activin A and GH
(19). Thus, the action of androgens as seen in the present
study is unique. However, Fig. 5 shows that androgen increased
IR-inhibin and E2 production from type 4 follicles stimulated by
androgens in the presence of FSH. Such synergistic action of androgen
with FSH on follicular growth is already known. Several in
vitro studies have demonstrated that androgens in the presence of
FSH increase the production of cAMP, progesterone, and aromatase
activity in cultured granulosa cells (8, 9, 10, 11, 12). T also
significantly increases granulosa cell FSH receptor mRNA at all stages
of follicular development in normal cycling rhesus monkeys, whereas FSH
increases granulosa AR mRNA levels only in primary follicles
(35). The results of the present study demonstrate that
synergistic action of androgen with FSH functions even in the type 4
follicles of immature mice, although the exact mechanisms of how such
follicles acquire FSH responsiveness is as yet unknown.
Significantly more E2 was released into the culture medium by
follicles incubated with FSH and aromatizable androgens than with
those incubated with FSH and the nonaromatizable androgen DHT (Fig. 5).
Because DHT stimulated IR-Inhibin secretion to the same extent as
aromatizable androgens did, the decreased effect of DHT on E2 secretion
is accounted for by the absence of substrates. Aromatizable androgens
may therefore be used as an E precursor by the enzyme cytochrome
P450aromatase in the granulosa cells under the influence of FSH
(36). P450aromatase mRNA and its activity is detectable in
the mouse ovary on the day of birth and thereafter increases in a
gonadotropin-dependent manner (22), suggesting that most
of the E detected in the culture medium was synthesized de
novo by converting aromatizable androgens to E. Moreover, FSH
induces P450aromatase mRNA and activity in rat granulosa cells in a
dose-, time-, and stage-dependent manner and T and DHT synergistically
enhance the levels of P450aromatase mRNA and its activity with FSH
(37). Figure 5 shows that type 4 follicles secreted
detectable amounts of E2 as the result of DHT stimulation. Although the
present study did not clearly define whether type 4 follicles actually
produce androgens, we have shown that type 4 follicles have theca cells
and can produce and secrete Es when there is insufficient amounts of
substrate (18, 19). Thus, our results suggest, firstly,
that androgens in the presence of FSH serve as a precursor of E and
enhance E production and, secondly, that type 4 follicles obtained from
11-d-old mice have an extant mechanism of E synthesis.
In conclusion, both adrenal and ovarian androgens directly stimulate FSH-unresponsive type 4 follicles without producing IR-inhibin and E2 and serve to enhance the sensitivity of FSH responsiveness in type 4 follicles from immature mice. In addition, the present results may answer the question as to how type 4 follicles from immature mice can acquire FSH responsiveness, although biochemical mechanisms are yet to be clarified. In this context, the present results suggest that the adrenal gland plays an important role in the initiation of puberty.
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Acknowledgments |
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Footnotes |
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4, Androstenedione; BrdU;
5-bromo-2'-deoxyuridine; DHEA, dehydroepiandrosterone; DHEAS,
dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; E1, estrone;
IR, immunoreactive; rhFSH, recombinant human FSH. Received January 29, 2001.
Accepted for publication July 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase, 17ß-ol-dehydrogenase and
aromatase in immature rat ovary. J Steroid Biochem 14:1369–1377[CrossRef][Medline]
-hydroxysteroid dehydrogenase enzymes in
immature, mature, and pregnant rats. Endocrinology 138:2886–2892This article has been cited by other articles:
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, P < 0.05, 
, P < 0.05,