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Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Ph.D., Stanford University School of Medicine, Department of Obstetrics and Gynecology, 300 Pasteur Drive, Stanford, California 94305-5317.
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Abstract |
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content of
the cultured follicles was increased by treatment with the combination
of FSH and 8-bromo-cGMP, demonstrating the induction of follicle cell
differentiation during culture. Therefore, we demonstrated that
activation of the cGMP pathway promotes the survival of cultured
preantral follicles and that in the presence of a cGMP analog, FSH is a
growth and differentiation factor for preantral follicles. The present
serum-free follicle culture model system will be useful in further
evaluation of the regulation of growth and differentiation of preantral
follicles. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, apoptosis has been shown to be the underlying mechanism of follicle atresia (14). Although gonadotropins and several local ovarian factors suppress apoptosis in cultured preovulatory and early antral follicles (15, 16, 17), few studies have involved preantral follicles. Because increases in follicle cell number can occur by either increased cell division or decreased cell death, analysis of apoptosis in cultured preantral follicles is also important.
In this study, we have evaluated apoptosis in preantral follicles in vivo in rats as well as investigated the effects of activators of the cAMP and cGMP pathways in the regulation of preantral follicle cell apoptosis in vitro. Furthermore, we used the antiapoptotic properties of cGMP analogs to develop a serum-free culture system for intact, mechanically dissected preantral follicles. Using this system, we demonstrated that FSH induces both growth and differentiation of preantral follicles when apoptosis is prevented.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and ovarian dissection
All animal experiments were performed in accordance with NIH and
institutional guidelines. Sprague-Dawley rats were obtained from
Simonsen (Gilroy, CA) and housed under standard conditions. Animals
were anesthetized with CO2 and killed by cervical
dislocation before dissection of ovaries. Apoptotic DNA fragmentation
in ovarian cells was assessed in rats at 12, 15, 16, 18, and 21 days of
age. Ovaries were snap-frozen or formalin-fixed for DNA fragmentation
analysis. Ovaries for follicle cultures were obtained from 12- or
14-day-old rats, immediately placed in warmed culture medium, and
dissected.
Preantral follicle culture
The effects of hormones or cyclic nucleotide analogs on
apoptosis of preantral follicles were first examined in a 24-h in
vitro culture system, as described previously (15). Ovaries were
collected from 12-day-old rats, and preantral follicles (160–200 µm
in diameter) were dissected microscopically using fine needles. Twenty
follicles per experimental group were cultured in 500 µl Eagle’s MEM
(Life Technologies, Grand Island, NY) supplemented with penicillin,
streptomycin sulfate, L-glutamine, and 0.1% BSA (Sigma
Chemical Co., St. Louis, MO) in the absence or presence of different
hormones or agents. The cultures were performed at 37 C under 95%
O2 and 5% CO2. After 24 h, follicles were
collected and stored frozen until further analysis.
To assess the growth and survival of individual follicles, a more
prolonged culture system was also developed. Preantral follicles of
160- to 170-µm diameter were dissected from the ovaries of 14-day-old
rats under a dissecting microscope using fine needles. Follicles were
cultured individually in 96-well dishes lined with polycarbonate
membranes in 150 µl medium overlaid with 75 µl sterile mineral oil
at 37 C in a moist atmosphere of 5% CO2 and 95% air.
Basal (control) medium consisted of MEM supplemented with 1% ITS
(insulin, 10 ng/ml; transferrin, 5.5 ng/ml; selenium, 5 ng/ml) and
Pen/Strep (penicillin, 100 U/ml; streptomycin, 100 µg/ml), all from
Sigma, as previously described (5). Serum (5%) from hypophysectomized
rats was included in some groups. Follicle diameter was measured daily
as the average distance between the outer edges of the basement
membrane in two perpendicular planes. At the end of the 48-h
incubation, follicles were collected for further analysis.
Apoptotic DNA fragmentation analysis
Genomic DNA was extracted from ovaries and follicles as
previously described (15). Briefly, tissues were digested with 200 µl
buffer containing 0.5% SDS, 0.1 M NaCl, 0.05 M
Tris, 4 mM EDTA, and 100 µg/ml proteinase K for 4 h
at 57 C. DNA was isolated by chloroform extraction and ethanol
precipitation before resuspension in sterile water. After digestion of
RNA with 10 µg/ml ribonuclease at 37 C for 1 h, DNA was isolated
after phenol-chloroform extraction and ethanol precipitation. The DNA
concentration was estimated by absorbance at 260 nm. A 200-ng aliquot
of DNA from each sample was labeled at the 3'-ends with
[32P]dideoxy-ATP (3,000 Ci/mmol; Amersham, Arlington
Heights, IL) using terminal deoxynucleotidyl transferase (Boehringer
Mannheim, Indianapolis, IN). After 1-h incubation at 37 C, labeled DNA
was isolated by phenol-chloroform extraction and precipitated in
ethanol with transfer RNA as a carrier. Labeled samples were
fractionated through 2% agarose gels by electrophoresis before
exposure using Fuji x-ray films (Fuji Photo Film, Minami-Ashigarashi,
Japan) at -70 C. After autoradiography, gels of each lane
corresponding to DNA less than 15 kilobases (kb) were cut out and
counted in a scintillation counter for quantification of the degree of
apoptotic DNA fragmentation.
In situ DNA fragmentation analysis
DNA fragmentation in ovarian or cultured follicle sections
was evaluated by nonradioactive labeling of DNA using Apop Tag Kit
(Oncor, Gaithersburg, MD). Ovaries or follicles were fixed with 4%
buffered formaldehyde for 48 h at room temperature. The tissues
were embedded in paraffin and sectioned. Three-micron sections were
deparaffinized and incubated with 10 µg/ml proteinase K for 30 min at
37 C. After washing, sections were treated with 2%
H2O2 for 5 min to inactivate endogenous
peroxidase. The 3'-ends of DNA fragments were labeled with
digoxigenin-deoxy-UTP by incubation with terminal deoxynucleotidyl
transferase for 1 h at 37 C. The sections were further incubated
with antidigoxigenin antibody conjugated to peroxidase for 30 min at
room temperature. DNA fragmentation was visualized by staining with
3',5' diaminobenzidine. Negative control sections were processed
without terminal transferase.
Quantification of viable follicle cells
Cell proliferation was verified using the tetrazolium salt-based
Cell Proliferation Kit I from Boehringer Mannheim (18). This assay is
based on the ability of living cells to metabolize yellow tetrazolium
salt to blue formazan crystals that can be solubilized and quantified
spectrophotometrically. At the completion of the experiments,
replicates of four follicles from each treatment group were moved to
single wells containing 100 µl basal medium.
3-\[4,5-Dimethylthiazol-2-yl\]-2,5-diphenyl tetrazolium bromide
labeling reagent was added to the wells and incubated for 4 h at
37 C under 6.5% CO2. Solubilization solution was then
added for 18 h before determination of absorbance at 560 nm.
Absorbance levels were standardized to known quantities of granulosa
cells.
Immunoblot analysis of inhibin- content of cultured
follicles
Twenty follicles from each treatment group were collected in
Eppendorf tubes at the end of the experiment and kept frozen. Follicles
were later thawed in lysis buffer (50 mM Tris-HCl, 150
mM NaCl, 1% SDS, 5 mM EGTA, 0.5 mM
MgCl2, 0.5 mM MnCl2, and 0.2
mM phenylmethylsulfonylfluoride) and homogenized with a
glass rod before storage at -70 C. Inhibin- antigen levels in
homogenates were determined using immunoblot analysis as previously
described (19). Briefly, electrophoresis was performed in a 10%
polyacrylamide gel. Proteins were transferred to nitrocellulose
membranes, and immunoblotting was performed with a mouse monoclonal
antibody to inhibin- (Serotec, Oxford, UK), followed by incubation
with a biotinylated antimouse second antibody and immunofluorescent
imaging with the ECL Western System (Amersham). Quantification of
autoradiographs was performed using densitometric analysis.
Data analysis
All experiments were repeated at least three times, and a
representative autoradiogram is presented where appropriate.
Quantitative results obtained from 
-counting of radiolabeled low mol
wt DNA fragments represent the mean ± SEM of combined
data from replicate experiments. Statistical significance between mean
values was determined by ANOVA followed by t test analysis.
Significance was accepted at the 0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ovarian
internucleosomal DNA fragmentation was very low until day 18 of life
and continued to be elevated on day 21. Quantification of low mol wt
DNA fragments (<15 kb) after scintillation counting indicated 55% and
180% increases on days 18 and 21 of age, respectively, compared to the
low mol wt DNA labeling at 15 days of age (Fig. 1B). In situ
analysis of ovarian sections was performed to identify the types of
follicles in which DNA fragmentation occurred. As shown in Fig. 2A, at 12 days of age, many follicles had reached the
secondary stage, although small antra were beginning to form in a few
follicles. There was negligible incorporation of digoxygenin-deoxy-UTP
in any of these immature follicles. By 16 days of age (Fig. 2B),
slightly larger antra were present within the most advanced follicles,
with minimal DNA fragmentation in any of the follicles. By 18 days of
age (Fig. 2C), there were medium-sized antral follicles in advanced
atresia with heavy staining. By 21 days of age (Fig. 2D), many antral
follicles were present in different stages of atresia. At all ages
studied, there was minimal staining of preantral follicles of any
size.
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, A and B,
negligible DNA fragmentation was found in preantral follicles at the
beginning of culture (0 h), but a substantial increase in
internucleosomal DNA fragmentation was found after 24 h in
serum-free culture. The onset of apoptosis could be suppressed by 65%
with the addition of 10% FCS to the culture medium. However, treatment
with either FSH (100 ng/ml) or 8-br-cAMP analog (5 mM)
failed to suppress apoptosis in preantral follicles (Fig. 3).
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, A and B,
treatment with 8-br-cGMP (5 mM) suppressed apoptosis of
preantral follicles in culture by 76%. In contrast, IL-1ß (250
ng/ml) and SNP (0.5 mM) were less effective, suppressing
apoptosis in these follicles by 19% and 29%, respectively.
Histological analysis of sections from cultured preantral follicles
(Fig. 5) demonstrated that the anatomical integrity of
the follicles was maintained over 24 h in culture. Pyknotic nuclei
can be seen in granulosa cells after 24 h of culture (Fig. 5B),
but not in follicles before culture (Fig. 5A). In situ
staining of cultured follicles for DNA fragmentation indicated that
apoptosis occurred in the granulosa cells of follicles cultured for
24 h without serum (Fig. 5C), and treatment with 8-br-cGMP
markedly reduced apoptosis (Fig. 5D). No staining was seen in the
negative control group when terminal transferase was deleted from the
reaction (Fig. 5E).
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We also used the 48-h culture system to evaluate follicle growth.
Follicles cultured in control medium became slightly larger (3.6%
increase in diameter; from 164.7 ± 0.9 to 170.7 ± 1.85
µm) over the 48-h incubation (Fig. 6A). Although
treatment with FSH or 8-br-cGMP did not significantly alter this growth
pattern, combined treatment with FSH and 8-br-cGMP increased follicle
diameter by 13% to 188.5 ± 1.76 µm (P <
0.01). Because earlier studies had determined that optimal follicle
growth in mice was obtained by treatment with 5% serum and FSH (5), we
also included a group treated with 5% serum from hypophysectomized
rats plus FSH in which a 23% increase in follicle diameter
(P < 0.01) was observed. Compared to the control,
treatment with serum alone resulted in only a modest increase in
follicle growth that was not statistically significant
(P > 0.05; data not shown).
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, treatment with FSH together with the cGMP
analog or 5% serum resulted in more than a 2-fold increase
(P < 0.01) in viable cells per follicle, whereas
treatment with FSH or the cGMP analog alone had no effect on viable
cell number compared with the control value.
FSH promotes preantral follicle differentiation
Earlier studies have demonstrated increases in inhibin-
messenger RNA and protein content with increasing follicle development
(20). To monitor the progression of differentiation of follicles in
culture, we evaluated the inhibin- protein content of cultured
follicles by immunoblot analysis. As shown in Fig. 7, treatment with FSH in combination with either 5% serum or 8-br-cGMP
resulted in an increase in the follicular inhibin- content over that
in control follicles. In contrast, treatment with the cGMP analog or
FSH alone did not increase the inhibin- content (data not shown).
Overall, the relative quantity of inhibin- was consistent with the
increase in size of the follicles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous histological investigations revealed minimal atresia or apoptosis of granulosa cells within preantral follicles in vivo (21, 22, 23), although the large preantral follicles that occur after diethylstilbestrol stimulation of immature rats do undergo apoptosis after estrogen withdrawal (24). However, biochemical studies of preantral follicles are difficult because most analyses use preparations of whole ovaries containing larger follicles and corpora lutea that mask changes in smaller follicles. Our use of juvenile rat ovaries overcomes this problem because at given ages, follicles have reached a predictable maximal stage of development. Additionally, these animals have not received exogenous hormone treatment. Using this model, we have demonstrated that based on fractionation of total ovarian DNA and on in situ analysis of individual follicles, apoptosis occurs widely in antral follicles and is minimal in preantral follicles. This is consistent with earlier studies in which the major wave of atresia was observed in early antral follicles 300–350 µm in diameter (21).
In vitro, however, apoptosis of preantral follicles can be demonstrated when follicles are grown in the absence of serum. In contrast to its effect on larger rat follicles (16) or in hamster preantral follicles (22), FSH or cAMP was ineffective in preventing rat preantral follicle atresia in vitro. We have demonstrated that activation of the cGMP pathway prevents preantral follicle cell death in vitro based on both DNA fragmentation and in situ analysis. In contrast, treatment with IL-1ß or SNP is less effective in preventing preantral follicle apoptosis compared with their potent antiapoptotic effect in preovulatory follicles (17). These data suggest that IL-1ß and SNP may be less effective in activating the cGMP pathway in preantral follicles or that they may also activate pathways independent of cGMP that increase apoptosis, as has been demonstrated in smooth muscle cells in culture (25). IL-1ß has also been reported to have both cytotoxic and morphogenic effects on whole ovarian dispersates in culture (26). It seems likely that the cGMP pathway is regulated by multiple mechanisms within small follicles, possibly with both endogenous ovarian factors as well as serum factors serving to prevent the wide scale follicle loss seen with larger follicles. The physiological stimulators of guanyl cyclase and the in vivo implications of these findings are currently unknown.
The regulation of preantral follicle survival is distinctly different from that in antral and preovulatory follicles. Kinetic studies have shown that follicles grow more slowly in the early phases of development than in later stages (3). As follicles within a cohort enlarge, the time required for granulosa cells to double in number decreases. During this rapid growth, the enlarging follicles are highly dependent on gonadotropin activation of the protein kinase A pathway. The withdrawal of gonadotropins in vivo or in vitro results in atresia of larger follicles (14, 27, 28). In contrast, smaller, slow growing follicles, although responsive to gonadotropins under the appropriate conditions, do not depend upon gonadotropins to prevent granulosa cell death in vitro, suggesting that the apoptosis of preantral follicles is regulated differently from that of larger follicles, and this difference may be related to their different growth rates.
Preantral follicles have been cultured after mechanical dissection or enzymatic dispersion (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 22). Although serum-free cultures of cumulus-enclosed oocytes has been described in the mouse (12), these systems were primarily designed to evaluate oocyte maturation, and 50–90% of complexes are not recovered at the end of the initial incubation. Serum-free culture of preantral follicles with intact basement membrane and theca has not been described in the rat or mouse. The present serum-free culture system allows the evaluation of endocrine or paracrine/autocrine factors that may affect the growth and differentiation of preantral follicles. Culturing intact follicles further allows analysis of the entire follicular unit, with interaction of oocyte, granulosa, basement membrane, and thecal components. Once loss of granulosa cells by apoptosis is prevented, a more accurate assessment of mitogenic factors can be performed.
We have demonstrated that the addition of FSH to medium containing the
cGMP analog results in increased follicular diameter and cell number of
preantral follicles as well as enhances the progression of
differentiation, as demonstrated by increased inhibin- protein
production. This is consistent with earlier studies that suggested a
role for gonadotropins in preantral follicle development (29, 30, 31), and
our recent in vivo studies that demonstrate increased growth
of preantral follicles in juvenile rats treated with FSH (32).
FSH may also be a growth and differentiation factor for human preantral follicles. The present serum-free culture system may be useful in evaluating human follicle growth and differentiation. If FSH affects human preantral follicles as in the rat, there could be important implications for both fertility management and infertility treatment.
Small ovarian follicles have not been studied to the extent of larger antral and preovulatory follicles. We have described a system in which rat preantral follicles can be cultured under serum-free conditions, thereby providing an in vitro model to evaluate these small follicles. Clearly, the regulation of preantral follicle survival and growth differs from that of more mature follicles, and further studies are needed to clarify the regulation of early folliculogenesis.
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Footnotes |
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2 American Society for Reproductive Medicine-NICHHD Fellow of the
Reproductive Scientist Development Program.
Received December 19, 1996.
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References |
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-, ßA-, and ßB-subunits in hormonally induced immature
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J. Gutkowska, M. Jankowski, M. Ram Sairam, N. Fujio, A. M. Reis, S. Mukaddam-Daher, and J. Tremblay Hormonal Regulation of Natriuretic Peptide System during Induced Ovarian Follicular Development in the Rat Biol Reprod, July 1, 1999; 61(1): 162 - 170. [Abstract] [Full Text]
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C.S. Wright, O. Hovatta, R. Margara, G. Trew, R.M.L. Winston, S. Franks, and K. Hardy Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles Hum. Reprod., June 1, 1999; 14(6): 1555 - 1562. [Abstract] [Full Text] [PDF]
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M. Hayashi, E. A. McGee, G. Min, C. Klein, U. M. Rose, M. v. Duin, and A. J. W. Hsueh Recombinant Growth Differentiation Factor-9 (GDF-9) Enhances Growth and Differentiation of Cultured Early Ovarian Follicles Endocrinology, March 1, 1999; 140(3): 1236 - 1244. [Abstract] [Full Text]
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S.-Y. Chun, E. A. McGee, S. Y. Hsu, S. Minami, P. S. LaPolt, H. H.-C. Yao, J. M. Bahr, A. Gougeon, D. W. Schomberg, and A. J.W. Hsueh Restricted Expression of WT1 Messenger Ribonucleic Acid in Immature Ovarian Follicles: Uniformity in Mammalian and Avian Species and Maintenance during Reproductive Senescence Biol Reprod, February 1, 1999; 60(2): 365 - 373. [Abstract] [Full Text] [PDF]
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A. Kaipia, S. Y. Hsu, and A. J. W. Hsueh Expression and Function of a Proapoptotic Bcl-2 Family Member Bcl-XL/Bcl-2-Associated Death Promoter (BAD) in Rat Ovary Endocrinology, December 1, 1997; 138(12): 5497 - 5504. [Abstract] [Full Text] [PDF]
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