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Instituto de Biología Experimental (G.M.L., J.L.B.), CONICET and Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Argentina; School of Biological and Molecular Sciences (N.P.G.), Oxford Brookes University, Oxford OX3 OBP, United Kingdom; and Centro de Investigaciones Endocrinológicas (S.C.), Hospital de Niños "Ricardo Gutierrez", Buenos Aires, 1425 Argentina
Address all correspondence and requests for reprints to: J. Lino Barañao, Ph.D., Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina. E-mail: lbaranao{at}dna.uba.ar
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß disulfide-linked subunits. Heterodimerization
of -subunit with either of the two forms of the ß-subunit, ßA
and ßB, generate dimeric inhibin A and inhibin B, respectively.
Whereas inhibin is an heterodimer, consisting of an -chain and one
of the two highly homologous ß-chains, another gonadal peptide,
activin, is formed by dimerization of the ß-chains. Combinational
assembly of the ßA and ßB can therefore generate activin A
(ßA-ßA), activin B (ßB-ßB), and activin AB (ßA-ßB).
Although gonads have been shown to produce an excess of free
-subunit (4), both free and ß-subunits have no biological
activity, at least in terms of the inhibition of FSH secretion (5, 6).
On the other hand, no differential biological effects have been
described for inhibins A and B so far (5, 6).
Since the development of immunoassays for measuring inhibins in sera, a
potential application in the physiology of the reproductive system, as
well as in the diagnosis of its disorders, has opened (7). Previous
assays, using a heterologous RIA (8), had a significant
cross-reactivity with biologically inactive free inhibin -subunit,
which limited its utility (7, 9). Recently, this problem seemed to be
solved with the development of ultrasensitive two-site enzyme-linked
immunosorbent assays (ELISAs) specific for dimeric inhibins, as well as
for their precursors (10, 11, 12, 13).
Inhibins A and B are the relevant forms of circulating dimeric inhibins in women, and changes in their plasma concentration in the menstrual cycle have been described. Inhibin A remains low during the early- and midfollicular phases and predominates in the luteal phase of the cycle, whereas inhibin B levels are maximal in the early follicular phase and fall in the late follicular phase before ovulation, remaining low during the luteal phase (13, 14).
In the adult female rat, differing patterns of circulating inhibin A and B levels have been also observed during the estrous cycle. Inhibin A levels peak on proestrus, whereas high inhibin B levels are observed on metestrus, diestrus, and proestrus (15). These differences may be ascribed to either different sources within the follicle or to a differential regulation of the secretion of each dimer.
This study was aimed at assessing the effect of FSH and putative intraovarian regulators, such as steroids and peptide growth factors, on inhibin A and B production in cultured rat granulosa cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Granulosa cell preparation and culture
Ovaries were obtained from 24- to 25-day-old female Sprague
Dawley rats after 3 days of DES treatment [sc SILASTIC implants
(Dow Corning Corp., Midland, MI] containing 5 mg DES).
Granulosa cells were prepared and cultured as previously described
(16). Briefly, the ovaries were punctured with a 30-gauge needle and
incubated in DMEM (4.5 g glucose/liter)-Ham’s F12 (F12; 1:1,
Gibco BRL, Gaithersburg, MD), EGTA (6.8 mM),
and HEPES (10 mM; 15 min at 37 C), and then washed and
incubated in DMEM-F12 (1:1), sucrose (0.5 M), and HEPES (10
mM; 5 min at 37 C). After incubation, the medium was
diluted with 2 vol DMEM-F12 and HEPES (10 mM), and ovaries
were allowed to sediment. Granulosa cells were obtained by pressing
ovaries within two pieces of nylon mesh (Nytex 50, Nytex,
Geneva, Switzerland). To eliminate contaminating theca/interstitial
cells, the crude granulosa cell suspension was layered over a 40%
Percoll solution in saline and centrifuged at 400 x g
for 20 min. The purified granulosa cell layer was aspirated from the
top of the Percoll solution and resuspended in DMEM-F12 (1:1)
containing bicarbonate (2.2 g/liter; pH: 7.4). Cells were seeded on
plastic 96-well plates (Nunc, Roskilde, Denmark) precoated with rat
tail collagen. The initial plating density was 3 x
105 viable cells/cm2. Cells were maintained at
37 C with 5% CO2. After 2 h, media were changed to
remove nonattached cells and were replaced by fresh media containing
the different factors to be tested.
Isolation of oocytes
Bovine oocytes, used in rat granulosa cell cocultures, were
isolated as previously described (17). Briefly, bovine ovaries were
collected, at a local slaughterhouse, from beef cows and heifers just
after slaughter. Cumulus-oocyte complexes were manually aspirated from
2- to 8-mm follicles using an 18-gauge needle fitted to a 5-ml syringe.
Cumulus cells were removed by vortexing cumulus-oocyte complexes in
TC199-HEPES, containing 0.1% hyaluronidase, for 2 min, followed by
gentle pipetting and were carefully washed 3 times in CR1aa medium.
Dimeric inhibin ELISA
Granulosa cells were cultured for 72 h in the presence of
different stimulus. Inhibins A and B in the culture media were measured
using a two-site ELISA specific for each peptide, as previously
described (13, 14). Briefly, conditioned media was diluted in FCS
according to the amount of inhibin present. Before assay, 0.5 vol 6%
aqueous SDS was added to all samples and standards and were heated for
3 min at 100 C. Samples were treated with freshly prepared 1% hydrogen
peroxide solution for 30 min at room temperature. A sensitive
amplified-enzyme assay (Ampak, DAKO Corp.,
Cambridgeshire, UK) was used to amplify the alkaline phosphatase
activity. Recombinant human inhibins A and B were used as standards.
Activin A, activin B, and follistatin had less than 0.1%
cross-reaction in both assays. Inhibin A had less than 0.5%
cross-reaction in the inhibin B assay; whereas inhibin B had less than
0.1% cross-reaction in the inhibin A assay (11, 13). The assay
sensitivity was 7 pg/ml for inhibin A and 15 pg/ml for inhibin B.
Intra- and interassay coefficients of variation were less than 10% for
both assays. The human inhibin A and B assays had been successfully
used in rat serum (15). We have validated these assays for use on rat
granulosa cell-conditioned media. Comparison of the slopes of the
regression lines of transformed data for assay standards and
conditioned media or serum samples indicated no significant departure
from parallelism.
Statistical analysis
Results are expressed as mean ± SEM of
triplicate cultures. Statistical comparisons of the results were made
using ANOVA and Tukey-Kramer’s test for multiple comparisons after
logarithmic transformation of data (18). Experiments were carried out
at least three times, with similar results. Analysis of the dose
response curves were performed using a computer program (ALLFIT) based
on a four-parameter logistic equation (19).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, addition
of increasing doses of FSH elicited dose-dependent increases in both
inhibins, with a more pronounced stimulation of inhibin A (9.9- and
4.7-fold increase over basal production of inhibin A and B,
respectively, at a dose of 20 ng/ml). This preferential stimulation of
inhibin A production increased the apparent ratio of inhibin A/inhibin
B from 14 ± 4 to 34 ± 2.
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). In the
presence of this steroid, a further stimulation was elicited by FSH in
both inhibins. The effects of estradiol and FSH were not
supra-additive (two-way ANOVA).
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, although the
dose-response curve for FSH was bell-shaped for both inhibins, maximal
stimulation of inhibin B was observed at comparatively lower
concentrations of the gonadotropin.
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). This seemingly specific stimulation
of inhibin B by TGF-ß was dramatically amplified in the presence of
FSH (Fig. 4). As shown in Fig. 4, a
combination of 60 ng/ml FSH plus TGF-ß produced a 75-fold increment
in inhibin B levels, whereas those of inhibin A were increased only
14-fold. In this latter condition, both inhibins were produced at
roughly the same levels (inhibin A/inhibin B apparent ratio, 0.9–1.8,
Table 1).
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and Table 1
. This action was quantitatively more pronounced than that
elicited by TGF-ß and was also amplified, although to a lower extent,
by the addition of FSH.
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and Table 1). A slight amplification
of this effect by FSH was found in some experiments, but it was not
consistent. Inhibin levels in control media containing only oocytes
were undetectable (data not shown).
Serum levels
In contrast with the observations made in culture media from
purified granulosa cells, when serum inhibin A and B levels were
measured in estrogen-primed rats, inhibin B was found to be the
predominant dimer (inhibin A, 0.74 ± 0.36; inhibin B, 5.50
± 1.45 ng/ml). The proportion between inhibin A and B (A/B apparent
ratio, 0.13 ± 0.02) was markedly different from that observed in
granulosa cells cultured under basal conditions (A/B apparent ratio,
12 ± 3, legend to Fig. 1 and Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Considerable evidence has accumulated indicating that ovarian inhibin
production is controlled by FSH and local regulators (2, 3). These
results were obtained by using an heterologous RIA that did not
discriminate between the dimers and the free -subunit (7, 20, 21).
More recent studies have measured the messenger RNA (mRNA) levels of
the different subunits (, ßA, and ßB) under various experimental
conditions (22, 23, 24, 25, 26, 27). However, this measurement does not necessarily
reflect the actual inhibin protein production, because ß chains can
also dimerize to produce activin.
Because recombinant rat inhibin standards are at present unavailable, the absolute levels of both dimers could not be determined, and values were expressed in terms of the respective human standards. The pattern of dimeric inhibin production by granulosa cells studied here is presented as fold increase, as a more reliable expression of the selective stimulation of each inhibin by the factors tested, because the changes in the ratios are likely to be maintained when measured against rat inhibin standards.
We have found that production of both inhibin A and inhibin B was stimulated by FSH, with a more pronounced effect on inhibin A. These results are in agreement with previous reports showing that inhibin subunit expression is regulated by this gonadotropin, acting through a cAMP-dependent pathway (23, 24, 25). We have shown that FSH stimulated inhibin secretion in a biphasic fashion. Similar biphasic effects had been observed for the interaction of this gonadotropin with IGF-I, sex steroids, TGF-ß, or activin on granulosa cell proliferation (16, 28, 29). In addition. a biphasic modulation of inhibin mRNA levels by PMSG had been previously reported (30). These types of biphasic responses to FSH have been attributed to activation of different G protein by the FSH receptor, a mechanism that seems to be involved in the transduction of pleiotropic actions of FSH isoforms (31). Data presented herein would suggest that not only the total levels of inhibin, but also the ratio between the dimers, may change markedly within the range of physiological concentrations of FSH. It remains to be established whether the different FSH isoforms exert a selective regulation on the inhibin A and B production.
Estradiol was also able to stimulate inhibin A production with a minor
effect on inhibin B. Stimulatory effects of estrogens had previously
been demonstrated on mRNA levels for - and ß-subunits (26).
The relative sensitivity of inhibin A and inhibin B to FSH stimulation
was markedly affected by IGF-I. The addition of this growth factor, in
the presence of low doses of the gonadotropin, produced a marked
decrease in the inhibin A/B ratio. Li et al. (32) have
postulated that IGF-I signaling is obligatory for FSH stimulation of
inhibin -subunit expression in rat granulosa cells. Our results
would further suggest that IGF-I may regulate the relative expression
of both inhibins by modulating FSH action on the ß-subunit genes.
TGF-ß induced the secretion of both inhibin A and B with a
clear preferential stimulation of the B dimer. Previous reports had
shown that TGF-ß enhanced basal and FSH-stimulated total inhibin
production (20), as well as - and ßA-subunit mRNA content (24) in
cultured immature rat granulosa cells. More recently, Erämaa and
Ritvos (27) reported that TGF-ß induces ßB-subunit expression in
human granulosa-luteal cells without affecting or ßA mRNA levels.
Our results further suggest that, in rat granulosa cells, the
stimulation elicited by TGF-ß can be amplified by FSH.
Activin A was even more potent that TGF-ß in stimulating inhibin B
production. Xiao et al. (21) have shown that recombinant
activin A causes a dose-related increase in inhibin production in rat
granulosa cells. Activin A was also able to stimulate -subunit
expression in rat granulosa cells (33), whereas in human
luteal/granulosa cells, activin may selectively stimulate ßB mRNA
transcripts (34).
Coculture with meiotically immature oocytes was also associated with a selective stimulation of inhibin B, resembling the effect observed with TGF-ß or activin. This is consistent with data from different laboratories, including our own, indicating that a still unidentified factor, probably belonging to the TGF-ß superfamily, produced by the oocyte, regulates granulosa cell function (17, 35, 36, 37). Recently a member of the TGF-ß superfamily, the growth/differentiation factor-9 (GDF-9) was identified and found to be specifically expressed in the oocyte (38). Ovarian follicles in GDF-9 knock-out mice form only one layer of granulosa cells, indicating that this factor may be required for normal folliculogenesis (39). Further studies will be required to assess the role of GDF-9 in the regulation of granulosa cell inhibin production.
The role of the germ cell in regulating inhibin production in the testis is well documented. Inhibin secretion from Sertoli cells is stimulated by the presence of germ cells (40, 41), and ß-subunit levels fluctuate with the stage of spermatogenesis (42). However, to our knowledge, the present results are the first demonstration of a specific effect of the oocyte on granulosa inhibin secretion, indicating that a similar paracrine regulation does exist in the ovary.
In immature, estrogen-treated rats (from which the granulosa cells have been isolated for the in vitro studies presented herein), inhibin B was found to be the predominant dimer, in contrast with the high inhibin A/B ratio observed in nontreated granulosa cell cultures. Serum values are consistent with previous reports describing relative amounts of circulating inhibin A and B (15). The discrepancy between the proportion of inhibin dimers in serum and those produced by isolated granulosa cells may suggest that intraovarian factors, such as those identified in the present study, are able to shift the ratio toward the predominance of inhibin B. It is noteworthy that stimulation with TGF-ß, activin A, or oocyte-derived factor(s) were the only experimental conditions where the relative proportion of inhibin A and B could be diminished. These results might suggest that the inhibin/activin ßB-subunit gene is a specific target for this superfamily of peptides.
Using cultures of rat granulosa cell at early stages of differentiation, we found that production of inhibin A is predominant. It had been previously postulated that inhibin B was characteristic of early preantral follicles, whereas inhibin A is preferentially secreted by more differentiated cells from antral follicles (21, 15, 43, 44). Our results seem to indicate that the change in the ratio of the inhibin dimers may be a reflection of the change in the concentrations of intrafollicular regulators.
Inhibins A and B might exert differential endocrine or autocrine/paracrine actions. However, at present, no distinct biological effects of both dimers have been demonstrated. Inhibin A and inhibin B are equally potent in attenuating FSH secretion in the rat pituitary cells’ in vitro bioassay, although ovine pituitary cells are relatively insensitive to human inhibin B (6). On the other hand, at the ovarian level, although a preliminary report indicates that human theca cells show similar responses to both dimers in the regulation of androgen production (45), further studies are required to determine whether inhibin A and inhibin B have the same paracrine/autocrine effects.
Then the question arises as to what could be the physiological meaning of the marked changes in the ratios of the two forms with seemingly identical action. One possibility is that inhibins A and inhibin B may be mediators responsible for synchronizing different events during follicular development. Accordingly, each inhibin may be responding to different environmental signals acting on granulosa cells. Inhibin A would be more sensitive to FSH stimulation during the later stages of follicular growth, whereas inhibin B would reflect the action of the members of the TGF-ß superfamily in preantral follicles.
In this regard, it is worth noticing that we have found a marked simulation of inhibin production by oocytes. We have previously shown that, under the same experimental conditions, oocytes can stimulate granulosa cell growth and that this effect decreases after meiotic maturation. The effect of meiotic maturation of the oocytes on the regulation of inhibin production is, at present, being investigated.
On the other hand, it has been shown that inhibin can modulate meiotic maturation of bovine oocytes (46) and that inhibin levels in follicular fluid are related to the meiotic stage of the oocyte in the pig (47). Taken together, these data would support the notion that inhibin serves not only as a peripheral marker of the progression of gametogenesis, regulating FSH levels, but also as a paracrine effector, mediating the interaction between the oocyte and somatic cells during meiotic maturation.
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Acknowledgments |
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Footnotes |
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2 An established investigator from the CONICET.
Received September 16, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit in human serum:
implications for radioimmunoassay. J Clin Endocrinol Metab 70:1208–1212[Abstract]
C-containing
forms in human serum by a new ultrasensitive two-site enzyme-linked
immunosorbent assay. J Clin Endocrinol Metab 80:2926–2932-ßA dimer) and total a
inhibin in the peripheral circulation and ovaries of rats after
gonadotrophin-induced follicular development and during the normal
oestrous cycle. J Endocrinol 147:271–283[Abstract]
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