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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
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{at}mgh.harvard.edu
Abstract
To further explore the developmental dynamics and possible roles 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 to determine mRNA levels for the
inhibin/activin subunits and both follistatin splice variants.
Granulosa cell RNA was obtained by transvaginally aspirating follicles
(6–23 mm) from carefully characterized normal women at different times
of the follicular phase. 
- and ßA-subunit mRNA levels
increased significantly with follicle diameter (r = 0.56;
P < 0.01 and r = 0.45; P
< 0.05, respectively) and follicle maturity (r = 0.65;
P < 0.001 and r = 0.58; P
< 0.01, respectively), but ßB mRNA levels, which were at
least 10-fold lower than levels of the other subunits, showed no
relationship to size or maturity. Both follistatin 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
in human follicular fluid from the same follicle aspirates (r =
0.71; P < 0.001). Similarly,
ßA-subunit mRNA was associated with inhibin A (r =
0.59; P < 0.01), and ßB mRNA was
associated with inhibin B (r = 0.67; P <
0.005) in these samples. Thus, the increase in inhibin subunit
transcription and protein synthesis with follicle size suggests that
inhibin biosynthesis might be important for continued development of
the dominant follicle.
To explore this hypothesis further, we compared mRNA levels for each of
these transcripts in follicles obtained from six polycystic ovary
syndrome patients (eight follicles) and compared the results to those
from a group (n = 5) of normal follicles matched for mean
diameter. Comparisons were also performed for a subset of polycystic
ovary syndrome follicles (n = 5) matched for diameter and size
range with the normal group. -Subunit mRNA levels were 16-fold lower
in both polycystic ovary syndrome follicle groups relative to
size-matched normal follicles (P < 0.02), whereas
ßA-subunit mRNA was significantly lower only when all
polycystic ovary syndrome follicles were compared.
ßB-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 ßA-subunits, but not follistatin, is
associated with follicular arrest in polycystic ovary syndrome
follicles.
OVARIAN 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 for modulating the effects of gonadotropins (reviewed in Ref. 1). For example, it is increasingly evident that selection of the dominant 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, the elaboration of inhibins, the follicle-derived members of the TGFß superfamily that act to suppress FSH secretion from the pituitary (4), may be a critical event in selecting the dominant follicle (2). Thus, understanding the dynamics of inhibin biosynthesis across follicle development and the processes that regulate the biosynthesis of this important endocrine 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 TGFß
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 mRNA levels 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 to primates and humans using in situ hybridization
on surgically derived specimens, which collectively suggest that the
ßB-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
ßA-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, 7, 8, 9, 10). Similarly, follistatin (FS) expression appears to
increase with antral follicle 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 from normal women across antral follicle development (11). In this study 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) of inhibins 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 of inhibin and activin are not as well defined. Inhibin has been shown 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 high affinity and nearly irreversible kinetics (19, 20), thereby inhibiting activin’s biological activity and making FS a critical factor 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 remain arrested in their development, as PCOS patients typically have 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 to the suggestion that follicular arrest results from either the presence of a growth inhibitor, which would be reduced with treatments 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 and examine the
hypothesis that coordinate regulation of their production 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 involved in 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 for size-matched normal follicles. Our results
indicate that inhibin - and ßA-subunit
biosynthesis increases with size and maturity in normal follicles,
consistent with the previously identified increase in inhibin A with
antral follicle development (11). Moreover, production of
- and ßA-subunit mRNAs is significantly
reduced in PCOS follicles compared with that in size-matched normal
follicles, suggesting that a 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 a dominant
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
the dominant follicle.
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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, we also selected a subset of PCOS follicles
(n = 5) whose range and mean diameter were nearly identical to
those of the normal size-matched group, 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 was approved 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 in detail (11). Follicular aspirates were transferred to the laboratory on ice, and the volume of the aspirate was determined. Granulosa cells were then allowed to settle in a plastic petri dish, after which they were manually collected, rinsed in three changes of Dulbecco’s PBS (Life Technologies, 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 µg Escherichia coli tRNA (both from Life Technologies, Inc.-BRL) were added, and the tube was stored at -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 of sample preparation was 1–1.5 h.
Development of competitive RT-PCR assays
The inhibin/activin subunits , ßA,
and ßB, 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, so that except for
this small deletion, the competitor and target are identical and were
similarly amplified by the same primer pair. The religated competitors
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 no competition 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, the concentrations 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 to prevent loss of the small amounts of
granulosa cell RNA, recoveries could not be directly determined, so
results for each target were normalized to the ß-actin concentration
and are reported as ratios. All target mRNA concentrations were in the
femtomolar range, with the exception of
ßB-subunit mRNA, which was in the attamolar
range, indicating that ßB 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 resolved in 10–15 µl diethylpyrocarbonate H2O. Five microliters of RNA were treated for 15 min with deoxyribonuclease I (Life Technologies, Inc.-BRL) in a 10-µl reaction and then reverse transcribed using 100 U Superscript II (Life Technologies, Inc.-BRL) in a 21.5-µl 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 µl containing 2 µl diluted RT
product, 0.2 mM deoxy-NTPs, 1.74 mM
MgCl2, 0.5 µM of each primer, 0.5 U
Taq DNA polymerase (Fisher Scientific,
Pittsburgh, PA), and 0.25 µCi
[-32P]deoxy-CTP (NEN Life Science Products, Boston MA). All primer pairs used for this analysis
have been previously described, and their specificity demonstrated
(31, 32, 33, 34). Targets were amplified within their respective
experimentally determined exponential ranges using a temperature
profile of 1 min each at 94, 60, and 72 C. PCR products were resolved
on 5% polyacrylamide gels, after which the radioactive bands
corresponding to both target and competitor were individually cut and
counted in a ß-scintillation counter. After subtraction of background
radioactivity and adjustment for differences in product size, the ratio
of target/competitor was calculated and plotted against the competitor
concentration. The competitor amount equivalent to a ratio of 1 was
taken as the initial target concentration after compensating for RT
dilution. The RNA concentration was then normalized to ß-actin for
each granulosa cell mRNA sample to control for differences in granulosa
cell numbers 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 commercially available 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 region of 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 as previously 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 in detail (11). Granulosa cell mRNA content, after normalization to ß-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 along with 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 in appropriate 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 the whole group of PCOS follicles (n = 8) for which sufficient mRNA was obtained for analysis. t tests were used to compare means. P < 0.05 was used to indicate statistical significance.
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 ßA- and
ßB-subunits, both FS alternative splice
variants, and ß-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 with both follicle
diameter (Fig. 1A; r = 0.56;
P < 0.01) and maturity (Fig. 1B; r = 0.65;
P < 0.001). Similarly, inhibin/activin
ßA-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 ßA-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
ßA-subunits, ßB-subunit
mRNA levels varied widely and were not associated with either follicle
size or maturity (Fig. 1, E and F, respectively), in agreement with our
previously reported lack of association of inhibin B protein with
follicle size and maturity (11). However,
ßB mRNA levels were more than 10-fold lower
than ßA levels. No associations were noted
between mRNA levels and follicle size or maturity in granulosa cells
from PCOS patients.
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) or maturity (Fig. 2B). Similar
results were obtained for the less abundant 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 for normal 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).
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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 state mRNA 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 -inhibin precursor
molecule (Fig. 3C).
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; r = 0.59; P <
0.01). Interestingly, ßB mRNA levels were
positively associated with dimeric inhibin B concentrations in hFF
(Fig. 4B; r = 0.67; P < 0.005) despite neither
parameter being associated with follicle growth or maturation,
indicating that ßB mRNA and 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 ßA-subunit
mRNA.
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A subset of five normal follicles was selected to match the mean
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 ßA-subunit was 5-fold lower in
PCOS follicles (P < 0.02). Although
ßB-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 very small (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 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
ßA 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 these developmentally arrested follicles. Neither the
FS mRNA variants nor their ratio were different between normal and PCOS
follicles, suggesting that defects in FS biosynthesis are not
associated with PCOS.
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To determine whether biosynthesis of inhibin, activin, and FS 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
minute quantities 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 ßA-subunit mRNA
levels increase as antral follicles grow and mature. In contrast,
ßB-subunit RNA levels had no relationship to
follicle maturity or size, which also agrees with our previous
assessment of inhibin B protein in hFF (11). Thus,
relative to inhibin B and possibly activin B, which cannot be
quantitated at present, biosynthesis of - and
ßA-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, were compared with those in
a group of size-matched normal follicles. Consistent with the
hypothesis, - and ßA-subunit mRNA levels
were 10- and 5-fold lower, respectively, in the whole PCOS follicle
group (P < 0.03 and P < 0.02,
respectively), and a trend toward reduced
ßB-subunit mRNA levels was observed as well. To
eliminate the possibility that very small 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 the normal size-matched group. Even with this limited subset of
follicles, -subunit mRNA levels were significantly (16-fold) lower
than those in normal follicles (P < 0.02), and the
ßA mRNA levels were nearly so (5-fold;
P = 0.059). These results suggest that -subunit
biosynthesis and perhaps ßA- and
ßB-subunit production are defective in PCOS
follicles and, thus, that PCOS follicular arrest might be associated
with inhibin insufficiency.
It is unclear at this point, however, whether the deficient inhibin subunit biosynthesis in PCOS follicles is secondary to a 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 either 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 ovarian bursa resulted in enhanced antral follicle development (13). It is also becoming clear from animal studies that production of inhibin sufficient to reduce circulating FSH is a critical event in selection of the dominant follicle (1, 2). Moreover, in a previous study of hFF inhibin concentrations in women undergoing ovarian hyperstimulation for in vitro fertilization, we found that inhibin A concentrations increased with follicle 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 typically observed 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 the same follicles demonstrates for the first time that inhibin 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 were observed 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 follicular development, but the usual relationship between mRNA and hormone 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 of excess 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 in vitro 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
ßA-subunits and inhibin A protein appear to be
gonadotropin regulated, recent evidence suggests that inhibin B protein
and ßB message are not (39, 40, 41, 42).
In the present study ßB-subunit biosynthesis
did not vary with follicle size or maturity, and in our previous study
inhibin B protein concentrations in hFF were similarly 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 effect of FSH on inhibin B secretion. However, in the present
study, ßB-subunit biosynthesis and inhibin B
hormone concentrations were tightly correlated. These results suggest
that inhibin B biosynthesis is tightly regulated at the mRNA and
protein levels, but that this regulation does not vary with follicle
size or maturity. Recent data suggest that, rather than gonadotropins,
intrafollicular growth factors such as IGFs and/or other members of the
TGFß superfamily might regulate ßB mRNA
transcription and/or inhibin B protein levels in antral follicles
(39, 40, 41, 42). Interestingly, mean ßB
message levels were less than 10% of the ßA
levels, consistent with a lower rate of ßB
message biosynthesis at this stage of development. In addition,
although not significant in this study, ßB mRNA
levels were lower in PCOS follicles, suggesting that further analysis
of ßB-subunit biosynthesis regulation might
yield important clues to the developmental arrest in PCOS
follicles.
By designing RT-PCR assays that could differentially quantitate FS315 and FS288 transcripts, we could examine, for the first time, the quantitative relationship between these two transcripts and whether this relationship changes during follicular development. There was no consistent variation in expression for either transcript as follicles matured or grew in 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 intermediate size that must be derived by proteolytic processing of the FS315 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 a more 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-matched normal follicles, nor was there any difference in the FS315/FS288 transcript ratio. In fact, we recently reported that free FS levels in hFF from developing human follicles were not associated 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 follicular developmental arrest.
By aspirating follicles at precise times from women with well
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
ßB microenvironment to an
/ßA 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 from women with PCOS and comparing
them to size-matched normal follicles, we have determined that these
PCOS follicles contain vastly reduced - and
ßA-subunit mRNA levels, suggesting that
defective inhibin biosynthesis might be related to the aberrant
follicle development seen in women with 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.
|
We are grateful to Drs. Ann Delbaere and Miyuki Sadatsuki for their technical 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.
Footnotes
This work was supported by Grant U5429164 from the National Center for Infertility Research, Massachusetts General Hospital, and NIH Grant R01-DK-55838 (to A.L.S.).
1 Present address: Department of Obstetrics and Gynecology, University
of Tokyo, Tokyo 113-8655, Japan. 
Abbreviations: FS, Follistatin; hFF, human follicular fluid; PCOS, polycystic ovary syndrome.
Received January 24, 2001.
Accepted May 17, 2001.
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) subjects to determine the concentrations of inhibin 
