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The Journal of Clinical Endocrinology & Metabolism 86(9):4206 – 4215
Copyright © 2001 by The Endocrine Society

Dynamics of Inhibin Subunit and Follistatin mRNAduring Development of Normal and Polycystic Ovary
Syndrome Follicles
TOSHIHIRO FUJIWARA*, YISRAEL SIDIS, CORRINE WELT, GERALYN LAMBERT-MESSERLIAN,
JANIS FOX, ANN TAYLOR, AND ALAN SCHNEYER
National Center for Infertility Research and Reproductive Endocrine Unit, Massachusetts General Hospital, Boston,
Massachusetts 02114; Department of Pathology and Laboratory Medicine, Women and Infants Hospital (G.L.M.),
Providence, Rhode Island 02903; and Department of Obstetrics and Gynecology, Brigham and Women’s Hospital (J.F.),
Boston, Massachusetts 02115
To further explore the developmental dynamics and possibleroles of inhibin, activin, and follistatin in the development of
human antral follicles as well as the relationship between
mRNA and protein levels of these hormones within follicles,
quantitative competitive RT-PCR assays were established todetermine mRNA levels for the inhibin/activin subunits andboth follistatin splice variants. Granulosa cell RNA was obtained by transvaginally aspirating follicles (6 –23 mm) fromcarefully characterized normal women at different times ofthe follicular phase. ␣- and ␤A-subunit mRNA levels increased
significantly with follicle diameter (r ⴝ 0.56; P < 0.01 and r ⴝ
0.45; P < 0.05, respectively) and follicle maturity (r ⴝ 0.65; P <0.001 and r ⴝ 0.58; P < 0.01, respectively), but ␤B mRNA levels,
which were at least 10-fold lower than levels of the othersubunits, showed no relationship to size or maturity. Bothfollistatin 315 and 288 transcripts were detected in granulosa
cells, but neither follistatin transcript varied significantly
across the range of follicle sizes analyzed. In addition, granulosa cells contained three follistatin 315 mRNA transcripts
for each follistatin 288 transcript, and the follistatin 315/288
ratio did not vary with follicle size. ␣-Subunit mRNA levels
were positively associated with dimeric inhibin A protein inhuman follicular fluid from the same follicle aspirates (r ⴝ0.71; P < 0.001). Similarly, ␤A-subunit mRNA was associatedO

VARIAN FOLLICLE development entails both gonadotropin-dependent and gonadotropin-independent
processes. Thus, although FSH is required for the development of antral follicles, local ovarian factors appear to be
critical for regulating preantral follicle development and formodulating the effects of gonadotropins (reviewed in Ref. 1).
For example, it is increasingly evident that selection of thedominant follicle in monoovulatory species involves a careful orchestration between endocrine-derived trophic support
and the regulation of that support by steroidal and nonsteroidal factors produced by the follicle itself (2, 3). In fact, theelaboration of inhibins, the follicle-derived members of the
TGF␤ superfamily that act to suppress FSH secretion fromthe pituitary (4), may be a critical event in selecting thedominant follicle (2). Thus, understanding the dynamics ofinhibin biosynthesis across follicle development and the processes that regulate the biosynthesis of this important endo-

Abbreviations: FS, Follistatin; hFF, human follicular fluid; PCOS,
polycystic ovary syndrome.

with inhibin A (r ⴝ 0.59; P < 0.01), and ␤B mRNA was associatedwith inhibin B (r ⴝ 0.67; P < 0.005) in these samples. Thus, theincrease in inhibin subunit transcription and protein synthesis with follicle size suggests that inhibin biosynthesis might
be important for continued development of the dominantfollicle.
To explore this hypothesis further, we compared mRNAlevels for each of these transcripts in follicles obtained fromsix polycystic ovary syndrome patients (eight follicles) andcompared the results to those from a group (n ⴝ 5) of normal
follicles matched for mean diameter. Comparisons were also
performed for a subset of polycystic ovary syndrome follicles(n ⴝ 5) matched for diameter and size range with the normalgroup. ␣-Subunit mRNA levels were 16-fold lower in both polycystic ovary syndrome follicle groups relative to size-matchednormal follicles (P < 0.02), whereas ␤A-subunit mRNA wassignificantly lower only when all polycystic ovary syndrome
follicles were compared. ␤B-Subunit and follistatin mRNA levels and the follistatin 315/288 ratio were not statistically different for any group. These results suggest that insufficient
production of inhibin ␣ and possibly ␤A-subunits, but not follistatin, is associated with follicular arrest in polycystic ovary
syndrome follicles. (J Clin Endocrinol Metab 86: 4206 – 4215,
2001)

crine modulator might shed light on pathophysiological situations in which follicle development has been disrupted.
It has been known for some time that inhibin biosynthesis
as well as the production of activins, inhibin-related members of the TGF␤ superfamily that stimulate FSH biosynthesis and release from pituitary gonadotrophs (4), are regulated
during mammalian follicle development. In one of the earliest studies in rodents, inhibin and activin subunit mRNAlevels increased up to the evening of proestrus, but fell dramatically after the GnRH surge, only to rise again just after
the secondary FSH surge (5). These studies were extended toprimates and humans using in situ hybridization on surgically derived specimens, which collectively suggest that the␤B-subunit (and presumably activin B) is produced in preantral and/or early antral follicles. As small antral follicles
increase in size, biosynthesis of inhibin ␣- and ␤A-subunits
increase, suggesting an orderly shift to the production of
more dimeric inhibin B, followed by inhibin A and activin A
as the preovulatory stage is achieved (6 –10). Similarly, follistatin (FS) expression appears to increase with antral follicle

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The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4206 – 4215 4207

diameter (6). Thus, this highly coordinated pattern of changing inhibin/activin subunit and FS mRNA expression suggests that preantral follicles might represent a primarily activin B-dominant microenvironment that changes to an
inhibin B-, then an inhibin A-dominant environment as
␣-subunit production becomes more prominent in larger
antral follicles.
Support for this concept was obtained by examining concentrations of inhibins and activin in human follicular fluid(hFF) obtained by transvaginally aspirating follicles fromnormal women across antral follicle development (11). In thisstudy inhibin A increased with follicle size and maturity,
whereas activin A was relatively constant, and inhibin B had
no relationship with follicle development. Other studies using hFF from surgically derived ovaries observed similar
relationships (12). However, as factors regulating the transport of inhibin out of the follicle as well as its metabolism
within the follicle are not yet known, it is not clear whether
these changing levels of inhibin and activin protein concentrations in hFF are due to regulated biosynthesis or to alterations in clearance, an understanding of which might delineate the physiological and pathophysiological role(s) ofinhibins and activins in follicular development.
Although inhibin’s endocrine action in regulating FSH
biosynthesis and release from the pituitary has been known
for some time (4), potential paracrine/autocrine actions ofinhibin and activin are not as well defined. Inhibin has beenshown to enhance the development of preovulatory follicles
when applied to the rat ovary (13) and to increase LH-stimulated thecal androgen production (14), an effect predicted
to enhance granulosa cell E2 biosynthesis. Activin induces
granulosa cell proliferation, FSH receptor number, and hormone biosynthesis in immature granulosa cells, but inhibits
luteinization in more mature granulosa cells, as detected by
reduced progesterone biosynthesis (15, 16). This may explain
why activin injection into rat ovaries resulted in the appearance of follicle atresia (13). The in vivo situation is even more
complex, as activin action is regulated by the structurally
unrelated protein, FS (17), which is expressed as two alternatively spliced mRNAs encoding for proteins of 288 and 315
amino acids (18). Both forms of FS bind activin with highaffinity and nearly irreversible kinetics (19, 20), thereby inhibiting activin’s biological activity and making FS a criticalfactor in determining the overall bioactivity of intrafollicular
activin. However, FS288 can bind cell surface proteoglycans(21), a characteristic that affords a differential tissue distribution of the different FS protein forms (22, 23) as well as
additional actions involved with regulating paracrine or
endocrine-derived activin bioactivity (24) and activin internalization (25). Thus, differences in biosynthesis of the different FS forms could lead to alterations in overall FS activity.
Taken together, these studies suggest important local actions
for inhibin, activin, and FS in modulating follicle development.
Polycystic ovary syndrome (PCOS) is a common reproductive disorder characterized by irregular menstrual cycles
and hyperandrogenism, and associated with insulin resistance and obesity (reviewed in Ref. 26). In addition, ovaries
from women with PCOS exhibit a peripheral array of developmentally arrested small, antral follicles (27). Despite
intensive effort, it is still unclear why these follicles remainarrested in their development, as PCOS patients typicallyhave normal levels of FSH (26), their follicles respond robustly to exogenous FSH (28), and in vitro studies have demonstrated clear, even exaggerated, responsiveness of PCOS
granulosa cells to FSH (29). These observations have led tothe suggestion that follicular arrest results from either thepresence of a growth inhibitor, which would be reduced withtreatments such as wedge resection or follicle cauterization,or to the lack of a developmental stimulator. In either case,the action of a local autocrine/paracrine modulator of follicular development has been postulated (30).
To more directly address the question of inhibin, activin,and FS biosynthesis during antral follicle development andexamine the hypothesis that coordinate regulation of theirproduction at the mRNA level is critical for normal follicle
development, we developed quantitative RT-PCR assays,
which were then applied to analysis of mRNA levels in
granulosa cells aspirated from normal women across antral
follicle development. To investigate the hypothesis that defects in inhibin, activin, or FS biosynthesis might be involvedin the follicle developmental arrest associated with PCOS, we
also examined mRNA levels in granulosa cells aspirated
from PCOS patients and compared these results to those forsize-matched normal follicles. Our results indicate that inhibin ␣- and ␤A-subunit biosynthesis increases with size andmaturity in normal follicles, consistent with the previously
identified increase in inhibin A with antral follicle development (11). Moreover, production of ␣- and ␤A-subunit
mRNAs is significantly reduced in PCOS follicles comparedwith that in size-matched normal follicles, suggesting thata defect in inhibin biosynthesis is associated with PCOS
follicular arrest.
Subjects and Methods
Patients
Evaluation and inclusion criteria for normal volunteers was previously described, and the samples studied in this report represent a
subsample of those women (11) from whom sufficient numbers of granulosa cells were recovered for mRNA analysis. A total of 21 follicles,
representing a mean diameter of 13.1 mm (6 –23 mm range), were obtained by transvaginal aspiration of spontaneously maturing follicles
from 18 normal volunteers (Table 1). These subjects had a history of
regular 26- to 32-d menstrual cycles, were taking no medications for 3
months before this study, received no medication during the study, had
normal TSH and PRL levels, and had normal physical examinations.
Daily blood samples were drawn across a complete menstrual cycle,
continuing up to the day of aspiration in the following cycle. Frequent
pelvic ultrasonography was used to document the development of adominant follicle in the first cycle and to monitor follicle growth during
the aspiration cycle. The largest follicle on either ovary was aspirated
transvaginally, and for the purposes of this study is referred to as thedominant follicle.
Four follicles were aspirated transvaginally from four women with
PCOS. To be included in this study, PCOS patients were otherwise
healthy, between the ages of 18 – 40 yr, had fewer than nine menses peryr, had been taking no medication for 3 months, and had biochemical
and/or clinical evidence of hyperandrogenism. Each patient had at least
two pelvic ultrasounds and 10 d of daily blood sampling to confirm theabsence of follicle maturation and menstrual cyclicity. Four additionalfollicles were obtained from two PCOS patients whose ovaries had been
surgically removed for nonovarian disorders.
To compare normal and PCOS follicles, a subset of normal follicles(n ⫽ 5) was selected whose mean diameter was not statistically different
from that of the PCOS follicles (n ⫽ 8). For a more rigorous comparison,

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Fujiwara et al. • Inhibin, Activin, FS mRNA in Human Follicles

TABLE 1. Summary of patient and sample characteristics

Age (yr)a
BMIa
Ferriman-Gallwey scorea
Number of follicles
Follicle diameter range(mm)

All normal
subjects (n ⫽ 18)

All PCOS patients (n ⫽ 6)

Size/range-matchednormal subjects (n ⫽ 5)

Size/range-matched
PCOS patients (n ⫽ 5)

31.2 (1.7)
24.0 (1.1)
5.7 (0.54)
21
6.0 –23.0

32 (2.9)32 (3.8)
9.0 (0.5)
8
0.5– 8.5

27.6 (2.4)
24.7 (1.9)
5.4 (0.8)
5
6.0 – 8.5

28.8 (2.9)
29.8 (4.5)
9.0 (0.7)
5
5.5– 8.5

Samples from all normal subjects were obtained by transvaginal aspiration of the largest follicle in the cycle after a complete normal cycle.
Samples from PCOS patients were obtained by transvaginal follicle aspiration (n ⫽ 4) after documentation of absence of follicle growth or fromsurgically removed ovaries from PCOS cases (n ⫽ 2). Size/range-matched subjects represent the subset of women from whom follicles wereobtained for the size/range-matched follicle groups in Table 3.
aMean (SD).
we also selected a subset of PCOS follicles (n ⫽ 5) whose range and mean
diameter were nearly identical to those of the normal size-matchedgroup, eliminating the smallest PCOS follicles. Characteristics for normal volunteers and PCOS subjects, in total as well as for the subgroups,
are summarized in Table 1.
All subjects gave informed consent, and the entire protocol wasapproved by the human studies institutional review board at the Massachusetts General and Brigham and Women’s Hospitals.

Sample acquisition
The methods for follicle aspiration have been previously described indetail (11). Follicular aspirates were transferred to the laboratory on ice,and the volume of the aspirate was determined. Granulosa cells werethen allowed to settle in a plastic petri dish, after which they weremanually collected, rinsed in three changes of Dulbecco’s PBS (LifeTechnologies, Inc.-BRL, Grand Island, NY), transferred to a 1.5-ml microcentrifuge tube, and centrifuged at 3000 rpm for 5 min. The supernatant was aspirated, 1 ml TRIzol and 5 ␮g Escherichia coli tRNA (bothfrom Life Technologies, Inc.-BRL) were added, and the tube was storedat ⫺80 C until RNA extraction. The follicular fluid aspirate remaining
in the dish after granulosa cell removal was centrifuged, and the supernatant stored at ⫺20 C until assayed as previously described (11). The
average time interval between sample collection and completion ofsample preparation was 1–1.5 h.

Development of competitive RT-PCR assays
The inhibin/activin subunits ␣, ␤A, and ␤B, as well as FS288 and FS315
transcripts were analyzed by competitive, quantitative RT-PCR. Competitors for each target were created by deleting a small portion (⬃10%)in the middle of each target using PCR to introduce novel restriction
sites, followed by excision and ligation of the shortened fragments, sothat except for this small deletion, the competitor and target are identicaland were similarly amplified by the same primer pair. The religatedcompetitors were then amplified by PCR, cloned into pCRII-Topo (Invitrogen, Carlsbad, CA), amplified, and sequenced. For the quantitative
PCR, each target was amplified in a series of four reactions that included
increasing amounts of competitor ranging from a dose that showed nocompetition to a concentration that eliminated amplification of the target, the optimal range being determined experimentally for each target.
At the point where the signal from competitor and target were equal, theconcentrations of cDNA were equal, thereby allowing calculation of the
precise mRNA concentration for each target in the original sample.
Because the RNA was extracted in the presence of carrier RNA toprevent loss of the small amounts of granulosa cell RNA, recoveries
could not be directly determined, so results for each target were normalized to the ␤-actin concentration and are reported as ratios. All target
mRNA concentrations were in the femtomolar range, with the exceptionof ␤B-subunit mRNA, which was in the attamolar range, indicating that
␤B mRNA levels are substantially lower than those of other targets.

RNA extraction/RT-PCR
Granulosa cell RNA was extracted using TRIzol (Life Technologies,
Inc.-BRL) according to the manufacturer’s protocol. RNA was resolvedin 10 –15 ␮l diethylpyrocarbonate H2O. Five microliters of RNA weretreated for 15 min with deoxyribonuclease I (Life Technologies, Inc.BRL) in a 10-␮l reaction and then reverse transcribed using 100 U
Superscript II (Life Technologies, Inc.-BRL) in a 21.5-␮l reaction for 50
min at 42 C. Ribonuclease H (1.5 U; Life Technologies, Inc.-BRL) was
added, and the sample was incubated at 37 C for 20 min, after which the
enzyme was inactivated. This RT reaction was diluted 1:10 with water
before PCR.
PCR was performed in a volume of 25 ␮l containing 2 ␮l diluted RT
product, 0.2 mm deoxy-NTPs, 1.74 mm MgCl2, 0.5 ␮m of each primer,
0.5 U Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA), and 0.25
␮Ci [␣-32P]deoxy-CTP (NEN Life Science Products, Boston MA). Allprimer pairs used for this analysis have been previously described, andtheir specificity demonstrated (31–34). Targets were amplified within
their respective experimentally determined exponential ranges using atemperature profile of 1 min each at 94, 60, and 72 C. PCR products wereresolved on 5% polyacrylamide gels, after which the radioactive bands
corresponding to both target and competitor were individually cut andcounted in a ␤-scintillation counter. After subtraction of backgroundradioactivity and adjustment for differences in product size, the ratio oftarget/competitor was calculated and plotted against the competitor
concentration. The competitor amount equivalent to a ratio of 1 wastaken as the initial target concentration after compensating for RT dilution. The RNA concentration was then normalized to ␤-actin for each
granulosa cell mRNA sample to control for differences in granulosa cellnumbers and for variable RNA recoveries between samples.
To evaluate the precision of the quantitative RT-PCR assays, RNA
was extracted from a large pool of granulosa-lutein cells using methods
described above, and the cDNA from this pool was analyzed with each
PCR assay, providing a measure of interassay coefficient of variation. In
addition, this pool was analyzed four times for each target PCR to
calculate the intraassay coefficient of variation.

Hormone analysis
Hormone concentrations for the normal follicles represent the subset
of previously reported results for which sufficient granulosa cell mRNA
was recovered for analysis in this study (11). Hormone concentrations
for PCOS follicles were not analyzed, because only two PCOS follicles
contained sufficient granulosa cell mRNA and follicular fluid. Follicular
fluid inhibin A and B and activin A were measured using commerciallyavailable immunoassay kits, whereas free FS was measured using a
two-site immunoassay, all as previously reported in detail (11, 35).
Pro-␣C, a measure of inhibin ␣-subunit protein containing the pro regionof the precursor, was quantified using an immunoassay kit from Serotec(Oxford, UK) as previously described (32). To characterize menstrual
cycles, serum samples were analyzed for E2, LH, and progesterone aspreviously described (11).

Data analysis
For normal subjects, follicle maturity (day of the menstrual cycle) was
estimated by determining the number of days before predicted ovulation that each follicle was aspirated using the daily serum hormone
levels in the preceding study cycle as a guide, as previously described

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The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4206 – 4215 4209

in detail (11). Granulosa cell mRNA content, after normalization to␤-actin, was then compared with follicle diameter, follicle maturity, or
hormone concentration in hFF from the same follicle (11), and correlations were determined using least squares regression analysis. Significant relationships are indicated by inclusion of the regression line alongwith the corresponding r and P values on the appropriate figures. As
PCOS patients were anovulatory, results from these follicles were only
analyzed by follicle size. Normal and PCOS follicles were analyzed
separately for these analyses.
Inhibin A, free FS, and activin A protein results were normally distributed and analyzed directly. Inhibin B, pro-␣C, and all mRNA results
deviated significantly from a normal distribution and were log transformed before analysis. Results are therefore presented in log scale inappropriate figures and as medians and ranges in tables.
To compare PCOS follicles with normal follicles, the five smallest
normal follicles were selected because they closely match both in range
and mean follicle diameter with a subset of the five largest PCOS follicles. These size-matched normal follicles were also compared with thewhole group of PCOS follicles (n ⫽ 8) for which sufficient mRNA was
obtained for analysis. t tests were used to compare means. P ⬍ 0.05 wasused to indicate statistical significance.

levels were more than 10-fold lower than ␤A levels. No associations were noted between mRNA levels and follicle sizeor maturity in granulosa cells from PCOS patients.
The FS315 and FS288 mRNA alternatively spliced transcripts were investigated individually so we could determine
whether their ratio varied as a function of follicle maturation.
Steady state FS315 mRNA levels in granulosa cells fromnormal or PCOS subjects did not vary consistently whenanalyzed in relation to either follicle diameter (Fig. 2A) or
maturity (Fig. 2B). Similar results were obtained for the lessabundant FS288 mRNA transcript (Fig. 2, C and D). Interestingly, the expression of these two transcripts were highly
correlated with each other for both normal and PCOS follicles(Fig. 2E; r ⫽ 0.85; P ⬍ 0.001 and r ⫽ 0.99; P ⬍ 0.001 respectively). In addition, the FS315/FS288 ratio was the same fornormal and PCOS follicles (mean, 2.98 ⫾ 1.2 and 2.90 ⫾ 1.2,
respectively) and did not vary with size (Fig. 2F) or maturity(not shown).

Results
Granulosa cell mRNA

Granulosa cells were obtained from 21 normal and 8 PCOS
follicles (Table 1), and steady state mRNA levels for inhibin
␣-subunit, inhibin/activin ␤A- and ␤B-subunits, both FS alternative splice variants, and ␤-actin were assessed by quantitative competitive RT-PCR. Intra- and interassay variations
for each target were determined using a pool of granulosa
cell mRNA derived from in vitro fertilization patients (Table
2). As the assay variability is less than the observed differences between follicles, these results support the utility of the
RT-PCR procedures. ␣-Subunit mRNA levels in granulosa
cells from normal women were positively correlated withboth follicle diameter (Fig. 1A; r ⫽ 0.56; P ⬍ 0.01) and maturity (Fig. 1B; r ⫽ 0.65; P ⬍ 0.001). Similarly, inhibin/activin
␤A-subunit mRNA levels in granulosa cells from normal
women were positively associated with both follicle size (Fig.
1C; r ⫽ 0.45; P ⫽ 0.05) and maturity (Fig. 1D; r ⫽ 0.58; P ⬍0.01), and there was a strong correlation between ␣- and␤A-subunit mRNA levels (r ⫽ 0.91; P ⬍ 0.001). These mRNA
results are consistent with our previously observed positive
association of hFF and serum dimeric inhibin A protein concentration with follicle size and maturity (11, 36). In contrast
to ␣- and ␤A-subunits, ␤B-subunit mRNA levels varied
widely and were not associated with either follicle size or
maturity (Fig. 1, E and F, respectively), in agreement with ourpreviously reported lack of association of inhibin B proteinwith follicle size and maturity (11). However, ␤B mRNATABLE 2. Characterization of intra- and interassay coefficients ofvariation (CV) for semi-quantitative RT-PCR

␣-Subunit␤A-Subunit
␤B-Subunit
FS315
FS288
␤-ActinIntraassay CV (%)

Interassay CV (%)

16.7
11.8
14.0
2.8
11.1
9.5

21.5
25.5
19.1
11.0
24.6
13.1

A pool of cDNA that was reverse transcribed from granulosa cell
mRNA was assayed four times in a single experiment. The interassay
variation was calculated from same pool run in four consecutive experiments.

Comparison of mRNA and hormone concentrations innormal follicles

Hormone and mRNA levels from individual follicle aspirates were compared only for samples obtained from normal
subjects, as there was insufficient follicular fluid for hormonal analysis in most PCOS follicles from which mRNA
was also acquired. Granulosa cell ␣-subunit steady statemRNA levels were positively associated with dimeric inhibin A concentrations in hFF from the same follicles (Fig. 3A;r ⫽ 0.71; P ⬍ 0.001). However, no association was observed
between ␣-subunit mRNA levels and inhibin B (Fig. 3B).
Moreover, there was no association between ␣-subunit
mRNA and pro-␣C protein, a measure of ␣-subunit monomer or dimer containing the pro region of the ␣-inhibinprecursor molecule (Fig. 3C).
As expected, ␤A-subunit mRNA levels were positively
associated with dimeric inhibin A (Fig. 4A; r ⫽ 0.59; P ⬍ 0.01).
Interestingly, ␤B mRNA levels were positively associatedwith dimeric inhibin B concentrations in hFF (Fig. 4B; r ⫽0.67; P ⬍ 0.005) despite neither parameter being associatedwith follicle growth or maturation, indicating that ␤B mRNAand inhibin B protein biosynthesis are regulated independently of follicle development. Neither FS288 nor FS315 levels were associated with free FS hormone levels in hFF (Fig. 5,
A and B), and activin A was not associated with ␤A-subunit
mRNA.
Comparison of normal and PCOS follicle meanmRNA levels

A subset of five normal follicles was selected to match themean follicle diameter for all PCOS follicles. As shown in
Table 3, ␣-subunit mRNA levels were 10-fold (P ⬍ 0.03)
lower in PCOS follicles relative to normal follicles, while␤A-subunit was 5-fold lower in PCOS follicles (P ⬍ 0.02).
Although ␤B-subunit mRNA levels were 2.5-fold lower in
PCOS follicles, this difference did not reach statistical significance. As the PCOS follicle group contains three verysmall (0.5–2 mm) follicles that were not directly matched in
our normal follicle subset, we also examined a subset of
PCOS follicles (n ⫽ 5) that overlapped the normal follicle

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Fujiwara et al. • Inhibin, Activin, FS mRNA in Human Follicles

FIG. 1. Inhibin/activin subunit mRNA levels in granulosa cells from aspirated follicles. Quantitative RT-PCR assays were used on RNA
extracted from granulosa cells aspirated from normal (F) or PCOS (e) subjects to determine the concentrations of inhibin ␣-, ␤A-, and ␤B-subunits
relative to that of ␤-actin. Significant relationships of mRNA level with follicle size or maturity [days before predicted ovulation (11)] areindicated. Note the change of scale for ␤B-subunit, because its concentration was at least 10-fold lower than those of the other subunits. No
significant relationships were observed for PCOS follicles.

group in both mean follicle diameter and range of follicle
sizes. As shown in Table 3, even in this restricted subset of
PCOS follicles, ␣-subunit mRNA levels were 16-fold lower
than those in normal follicles (P ⬍ 0.02), whereas the ␤A
mRNA levels were no longer significantly different (P ⫽0.059). Thus, at least the reduced ␣-subunit mRNA level in
PCOS follicles appears to be a robust characteristic of thesedevelopmentally arrested follicles. Neither the FS mRNAvariants nor their ratio were different between normal and
PCOS follicles, suggesting that defects in FS biosynthesis are
not associated with PCOS.
Discussion

To determine whether biosynthesis of inhibin, activin, andFS is modulated during antral follicle development in normal

women, we examined mRNA levels for these hormones in
granulosa cells transvaginally aspirated from 6- to 23-mm
spontaneously growing normal human follicles. Competitive RT-PCR assays were established for each mRNA target
to allow quantitative assessment of each target in the minutequantities of recovered RNA. Consistent with suggestions
from earlier in situ hybridization and immunocytochemical
studies on surgical specimens (6, 9) as well as with measurements of dimeric inhibin A in hFF from follicle aspirates(11), we observed that both ␣- and ␤A-subunit mRNA levels
increase as antral follicles grow and mature. In contrast,
␤B-subunit RNA levels had no relationship to follicle maturity or size, which also agrees with our previous assessmentof inhibin B protein in hFF (11). Thus, relative to inhibin B andpossibly activin B, which cannot be quantitated at present,

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The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4206 – 4215 4211

FIG. 2. Analysis of FS315 and FS288 mRNA levels in granulosa cells from aspirated follicles. Quantitative RT-PCR assays were developed foreach alternative splice variant, and concentrations were expressed as a ratio with the ␤-activin concentration. Results are displayed as a functionof follicle size (A and C) or maturity (B and D). E, FS315 and FS288 variants are highly correlated for both normal and PCOS follicles. F, The
FS315/FS288 ratio does not change across follicle development, and the ratio for both normal and PCOS follicles is about three FS315 transcripts
for each FS288 transcript. Significant relationships are indicated by lines.

biosynthesis of ␣- and ␤A-subunit mRNA and production of
dimeric inhibin A protein is greater in larger follicles, suggesting the hypothesis that increasing inhibin A concentrations are important for maintenance of dominant follicle
development.
To further examine this hypothesis, mRNA levels in PCOS
follicles, a model for follicular developmental arrest, werecompared with those in a group of size-matched normal
follicles. Consistent with the hypothesis, ␣- and ␤A-subunit
mRNA levels were 10- and 5-fold lower, respectively, in thewhole PCOS follicle group (P ⬍ 0.03 and P ⬍ 0.02, respectively), and a trend toward reduced ␤B-subunit mRNA levels

was observed as well. To eliminate the possibility that verysmall follicles could bias the PCOS follicle group mean analysis, we also examined a subset of five PCOS follicles whose
size and diameter range were nearly identical to those in thenormal size-matched group. Even with this limited subset offollicles, ␣-subunit mRNA levels were significantly (16-fold)lower than those in normal follicles (P ⬍ 0.02), and the ␤A
mRNA levels were nearly so (5-fold; P ⫽ 0.059). These results
suggest that ␣-subunit biosynthesis and perhaps ␤A- and␤B-subunit production are defective in PCOS follicles and,thus, that PCOS follicular arrest might be associated with
inhibin insufficiency.

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Fujiwara et al. • Inhibin, Activin, FS mRNA in Human Follicles

FIG. 4. Relationship of inhibin ␤-subunits to their respective
dimeric hormone in hFF. ␤A-Subunit mRNA levels were positively
associated with dimeric inhibin A protein concentrations (A), and␤B-subunit mRNA levels were correlated with dimeric inhibin B
concentrations (B) in the same follicles. Significant relationships
are indicated by lines.

FIG. 3. Relationship of ␣-subunit mRNA concentration to ␣-subunit-containing proteins in hFF of the same normal follicle. ␣-Subunit mRNA levels were correlated with dimeric inhibin A levels in
the hFF aspirated from the same follicle (A), but not with inhibin
B (B) or pro-␣C (C). Significant relationships are indicated by lines.

It is unclear at this point, however, whether the deficientinhibin subunit biosynthesis in PCOS follicles is secondary toa deficiency of some as yet unidentified factor or if the inhibin
deficiency is a primary cause of follicular growth arrest.
Inhibin has been previously proposed to function as an autocrine or paracrine regulator of follicular development ei-

ther by direct action on granulosa cells or by enhancing
LH-induced androgen production (14, 37). In addition, treatment of female rats with inhibin A injected under the ovarianbursa resulted in enhanced antral follicle development (13).
It is also becoming clear from animal studies that productionof inhibin sufficient to reduce circulating FSH is a criticalevent in selection of the dominant follicle (1, 2). Moreover, ina previous study of hFF inhibin concentrations in women
undergoing ovarian hyperstimulation for in vitro fertilization, we found that inhibin A concentrations increased withfollicle size in non-PCOS patients, but did not vary significantly in women with PCOS (38). Taken together, these results suggest that insufficient production of dimeric inhibin
is one potential explanation for follicular arrest typicallyobserved in PCOS patients, indicating that further study into
the role of inhibin in follicular development is warranted.
Our examination of both mRNA levels and the corresponding protein hormone concentrations in hFF from thesame follicles demonstrates for the first time that inhibin

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Fujiwara et al. • Inhibin, Activin, FS mRNA in Human Follicles

The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4206 – 4215 4213

FIG. 5. Relationship of FS mRNA levels to free FS protein concentrations in hFF. Neither FS315 mRNA (A) nor FS288 mRNA (B) wereassociated with free FS levels in hFF from the same follicles. No
significant relationships were noted.

subunit mRNA biosynthesis in granulosa cells is tightly
linked to protein hormone production in normally developing follicles in vivo. Interestingly, no relationships between
granulosa cell mRNA and hFF hormone concentrations wereobserved when follicles aspirated from gonadotropin-hyperstimulated women were examined (32). These results indicate that administration of exogenous gonadotropins to
women stimulates inhibin biosynthesis along with folliculardevelopment, but the usual relationship between mRNA andhormone levels is lost. This may be due to hormone biosynthesis in excess of its diffusion rate from the follicle, resulting
in an accumulation of inhibin in hFF, or to the production ofexcess mRNAs that are not directly translated into protein.
However, the physiological implication of this loss of association is unclear, as we were unable to identify any relationship between hFF dimeric inhibin concentration and invitro fertilization outcome (32). Nevertheless, the present
study indicates that inhibin biosynthesis is strongly regulated at both the mRNA and protein levels during normal
antral follicle development.

Although biosynthesis of inhibin ␣- and ␤A-subunits andinhibin A protein appear to be gonadotropin regulated, recent evidence suggests that inhibin B protein and ␤B message
are not (39 – 42). In the present study ␤B-subunit biosynthesis
did not vary with follicle size or maturity, and in our previous study inhibin B protein concentrations in hFF weresimilarly not associated with follicle development (11). Previous studies have also been unable to detect any association
between hFF inhibin B concentrations and follicle size (12,43). Taken together, these results suggest that the normal
increase in serum inhibin B observed during the early follicular phase of the human menstrual cycle (44, 45) may be
due to an increased number of developing small antral follicles actively secreting inhibin B, rather than to a direct effectof FSH on inhibin B secretion. However, in the present study,
␤B-subunit biosynthesis and inhibin B hormone concentrations were tightly correlated. These results suggest that inhibin B biosynthesis is tightly regulated at the mRNA andprotein levels, but that this regulation does not vary withfollicle size or maturity. Recent data suggest that, rather than
gonadotropins, intrafollicular growth factors such as IGFs
and/or other members of the TGF␤ superfamily might regulate ␤B mRNA transcription and/or inhibin B protein levels
in antral follicles (39 – 42). Interestingly, mean ␤B message
levels were less than 10% of the ␤A levels, consistent with alower rate of ␤B message biosynthesis at this stage of development. In addition, although not significant in this study,
␤B mRNA levels were lower in PCOS follicles, suggestingthat further analysis of ␤B-subunit biosynthesis regulation
might yield important clues to the developmental arrest in
PCOS follicles.
By designing RT-PCR assays that could differentiallyquantitate FS315 and FS288 transcripts, we could examine,for the first time, the quantitative relationship between these
two transcripts and whether this relationship changes duringfollicular development. There was no consistent variation in
expression for either transcript as follicles matured or grewin size. Based on all normal follicles, the mean FS315:FS288
ratio was 2.98. These observations indicate that the alternative splicing event that produces FS288 is not regulated during follicular development and produces approximately
three full-length FS315 transcripts for every one FS288 transcript. It has been previously demonstrated that the major FS
protein species in follicular fluid is the 300/303 intermediatesize that must be derived by proteolytic processing of theFS315 protein, and that full-length FS315 and FS288 isoforms
are relatively rare (23, 46). Moreover, it is currently unknown
to what degree the FS288 transcript is actually translated in
granulosa cells under normal physiological conditions. Thus,
our results do not support the concept that the alternative
splicing event itself is important to FS’s role in the ovary.
Based on candidate gene analysis of affected sibling pairs,
it was previously suggested that a mutation in or near the FS
gene was statistically associated with PCOS (47), although amore recent analysis of additional PCOS families casts doubt
on this association (48). Consistent with this latter finding,our results fail to support the presence of a defect in FS
biosynthesis in PCOS follicles relative to that in size-matchednormal follicles, nor was there any difference in the FS315/
FS288 transcript ratio. In fact, we recently reported that freeDownloaded from jcem.endojournals.org at Jichi Med Univ on December 25, 2009

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Fujiwara et al. • Inhibin, Activin, FS mRNA in Human Follicles

TABLE 3. Comparison of inhibin/activin subunit and follistatin mRNA levels in granulosa cells from size- and range-matched PCOS
follicles compared to size-matched normal follicles
Size/range-matched normal
follicles (n ⫽ 5)a

Mean follicle diameter (mm)Follicle diameter range (mm)␣-Subunitb
␤A-Subunitb
␤B-Subunitb
Follistatin-315b
Follistatin-288b
Follistatin 315:288 ratioa

7.3 (0.46)
6.0 – 8.5
4.8 (0.2– 6.41)c,d
8.3 (1.7–39.3)c
0.149 (0.07– 0.49)
5.6 (1.5–11.8)
2.1 (0.7–3.6)
2.98 (1.2)

Size/range-matched PCOS
follicles (n ⫽ 5)

All PCOS follicles (n ⫽ 8)

7.0 (0.57)
5.5– 8.5
0.3 (0.2– 0.5)c
1.6 (0.6 –5.6)
0.03 (0.01– 0.2)
9.2 (1.5– 63.9)
3.2 (0.8 –11.2)
2.90 (1.2)

5.0 (1.1)
0.5– 8.5
0.5 (0.2–2.0)d
1.5 (0.5–5.6)c
0.06 (0.01– 0.4)
12.8 (1.5– 64.0)
4.5 (0.8 –12.2)
2.80 (1.2)

Also shown are results for all PCOS follicles analyzed. Quantitative, competitive RT-PCR assays were used to determine mRNA levels in
granulosa cells obtained from follicle aspirates. mRNA results are expressed as ratios (⫻10⫺2) with ␤-actin.
aMean (SEM).
bGeometric mean (min–max).
c
P ⬍ 0.02.
d
P ⬍ 0.03.

FS levels in hFF from developing human follicles were notassociated with follicle growth or maturity, but were in large
excess over activin in nearly all antral follicles examined (11).
Taken together, these studies strongly indicate that free FS
levels in hFF are not a critical parameter for antral follicle
development, nor are they important for PCOS folliculardevelopmental arrest.
By aspirating follicles at precise times from women withwell characterized normal menstrual cycles and development of quantitative RT-PCR assays, we were able to determine the developmental pattern of inhibin and activin biosynthesis from a predominantly ␤B microenvironment to an
␣/␤A microenvironment as normal antral follicles mature. In
addition, we have clearly demonstrated that FS mRNA levels
do not vary with follicle development, that the FS315/FS288
ratio does not change, and that approximately three FS315
transcripts are synthesized for every FS288 mRNA. Moreover, by aspirating developmentally arrested follicles fromwomen with PCOS and comparing them to size-matchednormal follicles, we have determined that these PCOS follicles contain vastly reduced ␣- and ␤A-subunit mRNA levels,
suggesting that defective inhibin biosynthesis might be related to the aberrant follicle development seen in womenwith PCOS. Further analysis of relative inhibin protein concentrations between PCOS and size-matched normal follicles
should help elucidate the prevalence and importance of this
distinction.
Acknowledgments
We are grateful to Drs. Ann Delbaere and Miyuki Sadatsuki for theirtechnical assistance, and to Brooke Wexler for her excellent patient
recruitment skills. We also appreciate the efforts of the REU Immunoassay Laboratory, including Patty Smith, Sheila Mallette, and Patrick
Sluss, for their timely attention to assay requests. We are indebted to Drs.
Janet Hall and Drew Tortoriello for critical review of the manuscript.
Received January 24, 2001. Accepted May 17, 2001.
Address all correspondence and requests for reprints to: Dr. Alan
Schneyer, Reproductive Endocrine Unit, BHX-5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: schneyer.alan@
mgh.harvard.edu.
This work was supported by Grant U5429164 from the National
Center for Infertility Research, Massachusetts General Hospital, andNIH Grant R01-DK-55838 (to A.L.S.).

* Present address: Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan.

References
1. McGee EA, Hsueh A. 2000 Initial and cyclic recruitment of ovarian follicles.
Endocr Rev 21:200 –214
2. Ginther OJ, Beg MA, Bergfelt DR, Donadeu FX, Kot K, Follicle selection inmonoovular species. Biol Reprod, in press
3. Zeleznik AJ, Follicle selection in primates: Many are called but few are chosen.
Biol Reprod, in press
4. Vale W, Rivier C, Hsueh A, et al. 1988 Chemical and biological characterization of the inhibin family of protein hormones. Recent Prog Horm Res
44:1–34
5. Woodruff TK, D’Agostino J, Schwartz NB, Mayo K 1988 Dynamic changes
in inhibin messenger RNAs in rat ovarian follicles during the reproductive
cycle. Science 239:1296 –1299
6. Roberts VJ, Barth S, El-Roeiy A, Yen SS 1993 Expression of inhibin/activin
subunits and follistatin messenger ribonucleic acids and proteins in ovarianfollicles and the corpus luteum during the human menstrual cycle. J Clin
Endocrinol Metab 77:1402–1410
7. Roberts VJ, Barth S, El-Roeiy A, Yen SSC 1994 Expression of inhibin/activin
system messenger ribonucleic acids and proteins in ovarian follicles fromwomen with polycystic ovarian syndrome. J Clin Endocrinol Metab 79:1434 –1439
8. Schwall RH, Mason AJ, Wilcox JN, Bassett SG, Zeleznik AJ 1990 Localizationof inhibin/activin subunit mRNAs within the primate ovary. Mol Endocrinol
4:75–79
9. Yamoto M, Minami S, Nakano R, Kobayashi M 1992 Immunohistochemical
localization of inhibin/activin subunits in human ovarian follicles during themenstrual cycle. J Clin Endocrinol Metab 74:989 –993
10. Yamoto M, Minami S, Nakano R 1993 Immunohistochemical localization of
inhibin subunits in polycystic ovary. J Clin Endocrinol Metab 77:859 – 862
11. Schneyer A, Fujiwara T, Fox J, et al. 2000 Dynamic changes in the intrafollicular inhibin/activin/follistatin axis during human follicular development:
relationship to circulating hormone concentrations. J Clin Endocrinol Metab
85:3319 –3330
12. Magoffin DA, Jakimiuk AJ 1997 Inhibin A, inhibin-B and activin-A in thefollicular fluid of regularly cycling women. Hum Reprod 12:1714 –1719
13. Woodruff TK, Krummen LA, Lyon RJ, Stocks DL, Mather JP 1993 Recombinant human inhibin A and recombinant human activin A regulate pituitaryand ovarian function in the adult female rat. Endocrinology 132:2332–2341
14. Hillier SG 1991 Regulatory functions for inhibin and activin in human ovaries.
J Endocrinol 131:171–175
15. Findlay JK 1993 An update on the roles of inhibin, activin, and follistatin as
local regulators of folliculogenesis. Biol Reprod 48:15–23
16. Cataldo NA, Rabinovici J, Fujimoto VY, Jaffe RB 1994 Follistatin antagonizes
effects of activin-A on steroidogenesis in human luteinizing granulosa cells.
J Clin Endocrinol Metab 79:272–277
17. DePaulo LV, Bicsak TA, Erickson GF, Shimasaki S, Ling N 1991 Follistatin
and activin: a potential intrinsic regulatory system within diverse tissues. Soc
Exp Med Biol 200:500 –512
18. Shimasaki S, Koga M, Esch F, et al. 1988 Primary structure of the humanfollistatin precursor and its genomic organization. Proc Natl Acad Sci USA
85:4218 – 4222
19. Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H 1990 Activinbinding protein from rat ovary is follistatin. Science 247:836 – 838

Downloaded from jcem.endojournals.org at Jichi Med Univ on December 25, 2009

Fujiwara et al. • Inhibin, Activin, FS mRNA in Human Follicles

The Journal of Clinical Endocrinology & Metabolism, September 2001, 86(9):4206 –4215 4215

20. Schneyer AL, Rzucidlo DA, Sluss PM, Crowley Jr WF 1994 Characterizationof unique binding kinetics of follistatin and activin or inhibin in serum. Endocrinology 135:667– 674
21. Nakamura T, Sugino S, Titani K, Sugino H 1991 Follistatin, an activin binding
protein, associates with heparan sulfate chains of proteoglycans on follicular
granulosa cells. J Biol Chem 266:19432–19437
22. Schneyer AL, Hall HA, Lambert-Messerlian GM, Wang QF, Sluss PM, Crowley WF 1996 Follistatin-activin complexes in human serum and follicular fluid
differ immunologically and biochemically. Endocrinology 137:240 –247
23. Sugino K, Kurosawa N, Nakamura T, et al. 1993 Molecular heterogeneity offollistatin, an activin-binding protein. J Biol Chem 68:15579 –15587
24. Delbaere A, Sidis Y, Schneyer AL 1999 Differential response to exogenous andendogenous activin in a human ovarian teratocarcinoma-derived cell line
(PA-1): regulation by cell surface follistatin. Endocrinology 140:2463–2470
25. Hashimoto O, Nakamura T, Shoji H, Shimasaki S, Hayashi Y, Sugino S 1997
A novel role of follistatin, an activin-binding protein, in the inhibition of activin
action in rat pituitary cells. J Biol Chem 272:13835–13842
26. Taylor AE 1998 Polycystic ovary syndrome. Endocrinol Metab Clin North Am
27:877–902
27. Adams J, Polson DW, Franks S 1986 Prevalence of polycystic ovaries inwomen with anovulation and idiopathic hirsutism. Br Med J 293:355–359
28. Franks S 1995 Polycystic ovary syndrome. N Engl J Med. 333:853– 861
29. Mason HD, Willis DS, Beard RW, Winston RML, Margar R, Franks S 1994
Estradiol production by granulosa cells of normal and polycystic ovaries:
relationship to menstrual cycle history and concentrations of gonadotropins
and sex steroids in follicular fluid. J Clin Endocrinol Metab 79:1355–1360
30. Erickson GF, Magoffin DA, Gabriel Gazzo V, Cheung AP, Chang RJ 1992
Granulosa cells of polycystic ovaries: are they normal or abnormal. Hum
Reprod 7:293–299
31. Sidis Y, Fujiwara T, Leykin L, Isaacson K, Toth T, Schneyer AL 1998 Characterization of inhibin/activin subunit, activin receptor, and follistatin messenger ribonucleic acid in human and mouse oocytes: evidence for activin’s
paracrine signaling from granulosa cells to oocytes. Biol Reprod 59:807– 812
32. Fujiwara T, Lambert-Messerlian G, Sidis Y, et al. 2000 Analysis of follicular
fluid hormone concentrations and granulosa cell mRNA levels for the inhibinactivin-follistatin system: relation to oocyte and embryo characteristics. Fertil
Steril 74:348 –355
33. Di Simone N, Hall HA, Welt C, Schneyer AL 1998 Activin regulates ␤-A
subunit and activin receptor messenger ribonucleic acid and cellular proliferation in activin-responsive testicular tumor cells. Endocrinology 139:1147–
1155
34. Welt CK, Lambert-Messerlian G, Zheng W, Crowley WF, Schneyer AL 1997
The presence of activin, inhibin and follistatin in epithelial ovarian carcinoma.
J Clin Endocrinol Metab 82:3720 –3727

35. McConnell D, Wang QF, Sluss P, et al. 1998 A two site chemiluminescentassay for activin-free follistatin reveals that most follistatin circulating in men
and normal cycling women is in an activin-bound state. J Clin Endocrinol
Metab 83:851– 858
36. Welt CK, McNicholl DJ, Taylor AE, Hall JE 1999 Female reproductive aging
is marked by decreased secretion of dimeric inhibin. J Clin Endocrinol Metab
84:105–111
37. Mather JP, Woodruff TK, Krummen LA 1992 Paracrine regulation of reproductive function by inhibin and activin. Proc Soc Exp Biol Med 201:1–15
38. Lambert-Messerlian G, Taylor A, Leykin L, et al. 1997 Characterization of
intrafollicular steroid hormones, inhibin, and follistatin in women with andwithout polycystic ovarian syndrome following gonadotropin hyperstimulation. Biol Reprod 57:1211–1216
39. Welt C, Schneyer A 2001 Differential regulation of inhibin B and inhibin A by
FSH and local growth factors in human granulosa cells from small antral
follicles. J Clin Endocrinol Metab 86:330 –336
40. Eramaa MJ, Tuuri T, Hilden K, Ritvos O 1994 Regulation of inhibin ␣ and ␤-Asubunit messenger ribonucleic acid levels by chorionic gonadotropin andrecombinant follicle-stimulating hormone in cultured human granulosa-luteal
cells. J Clin Endocrinol Metab 79:1670 –1677
41. Eramaa M, Ritvos O 1996 Transforming growth factor-␤1 and -␤2 induce
inhibin and activin ␤B subunit messenger ribonucleic acid levels in cultured
human granulosa-luteal cells. Fertil Steril 65:954 –960
42. Lanuza GM, Groome N, Baranao JL, Campo S 1999 Dimeric inhibin A and
B production are differentially regulated by hormones and local factors in rat
granulosa cells. Endocrinology 140:2549 –2554
43. Welt CK, McNicholl DJ, Taylor AE, Hall JE 1999 Female reproductive aging
is marked by decreased secretion of dimeric inhibin. J Clin Endocrinol Metab
84:105–111
44. Welt C, Martin KA, Taylor A, et al. 1997 Frequency modulation of folliclestimulating hormone (FSH) during the luteal-follicular transition: evidence for
FSH control of inhibin B in normal women. J Clin Endocrinol Metab 82:2645–
2652
45. Groome NP, Illingworth PJ, O’Brien M, et al. 1996 Measurement of dimericinhibin-B throughout the human menstrual cycle. J Clin Endocrinol Metab
81:1401–1405
46. Esch FS, Shimasaki S, Mercado M, et al. 1987 Structural characterization offollistatin: a novel follicle stimulating hormone release inhibiting polypeptide
from the gonad. Mol Cell Endocrinol 1:849 – 855
47. Urbanek M, Legro RS, Driscoll DA, et al. 1999 Thirty-seven candidate genes
for polycystic ovary syndrome: strongest evidence for linkage is with follistatin. Proc Natl Acad Sci USA 96:8573– 8578
48. Urbanek M, Wu X, Vickery KR, et al. 2000 Allelic variants of the follistatin
gene in polycystic ovary syndrome. J Clin Endocrinol Metab 85:4455– 4461

Downloaded from jcem.endojournals.org at Jichi Med Univ on December 25, 2009