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Endocrinology Vol. 144, No. 5 1876-1886
Copyright © 2003 by The Endocrine Society

Evidence for a Negative Intrafollicular Role for Inhibin in Regulation of Estradiol Production by Granulosa Cells

F. Jimenez-Krassel, M. E. Winn, D. Burns, J. L. H. Ireland and J. J. Ireland

Molecular Reproductive Endocrinology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: J. J. Ireland, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824-1225. E-mail: ireland{at}msu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5–1000 µg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the antiinhibin antibodies with bINH precursors or bovine pro-{alpha}C suppressed the capacity of antiinhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both antiinhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INHIBIN IS A DIMERIC glycoprotein comprised of an {alpha}- and one of two different ß-subunits ({alpha}ßA, inhibin A; {alpha}ßB, inhibin B), which is produced primarily by granulosa cells (1). Inhibin binding sites and receptors are located in the pituitary, thecal, and granulosa cells (2, 3, 4, 5, 6, 7, 8, 9). The presence of inhibin receptors in these tissues implies that inhibin has potentially complex interdependent endocrine, autocrine, and paracrine actions important for regulation of dominant follicle development. In support of a key antagonistic role for inhibin in follicle growth, deletion of the {alpha}-subunit gene of inhibin markedly elevates serum concentrations of FSH and activin [inhibin antagonist comprised of two inhibin ß-subunits (1)] and causes gonadal tumors in immature mice (10, 11, 12). In contrast, overexpression of inhibin’s {alpha}-subunit in mice reduces serum concentrations of FSH and estradiol and results in formation of follicular cysts (13, 14). Results of several studies demonstrate that a classical endocrine negative feedback pattern exists for secretion for FSH and inhibin A during the follicular phases of the menstrual cycle in humans (15) and estrous cycles in rats (16) and cattle (17, 18). These data, coupled with the positive effect of immunoneutralization of inhibin on FSH secretion and ovulation rate in single- and multiple-ovulating species (19, 20, 21, 22, 23), strongly support a physiologically important endocrine role for inhibin in regulation of number of follicles that ovulate during reproductive cycles. Nevertheless, despite the remarkably high concentrations of inhibin in follicular fluid of antral follicles in single-ovulating species like cattle (24, 25) or humans (26) and the likelihood that antral follicles contain high-affinity inhibin receptors (2, 3, 4, 5, 6, 7, 8, 9), little is known about the intrafollicular role of inhibin on growth, differentiation, and function of dominant follicles.

In cattle and perhaps humans (27), antral follicles grow in waves that begin several months before puberty and continue unabated every 7–10 d throughout most of reproductive life (28, 29). Each follicle wave in cattle is preceded by a transient 2- to 4-fold increase in serum FSH concentrations, which stimulates growth of several dozen 2- to 4-mm antral follicles. Because serum concentrations of FSH decline during the initial stages of a follicle wave, a single dominant follicle continues to grow to ovulatory size (12–20 mm) and produces markedly enhanced amounts of estradiol, but all other subordinate follicles lose their capacity to produce estradiol and undergo atresia. There are usually two or three waves of follicular development during 21-d estrous cycles in cattle (28, 29). The dominant follicles that develop during the follicular phase ovulate. In contrast, the dominant follicle that develops in follicle waves during the luteal phase loses its capacity to produce estradiol and undergoes atresia. Our laboratory has taken advantage of the dominant follicle growth model in cattle to examine the intrafollicular factors that regulate estradiol production. We became keenly interested in the potential negative autocrine or paracrine role of inhibin in regulation of estradiol production by granulosa cells because intrafollicular concentrations of inhibin A and estradiol vary inversely during dominant follicle development, but intrafollicular concentrations of activin A remain relatively unchanged (24, 30, 31, 32). Collectively, these observations led us to hypothesize that inhibin suppresses the capacity of granulosa cells from dominant follicles to produce estradiol.

To begin to test our hypothesis, the objective of the present study was to use a homologous model system to examine the potential autocrine or paracrine effects of inhibin on the capacity of bovine granulosa cells to produce estradiol. An immunoneutralization approach was chosen to evaluate the effects of inhibin because granulosa cells produce relatively high quantities of inhibin during culture (33, 34, 35, 36), which could mask or alter effects of inhibin treatments. In addition, the direct effects of purified bovine inhibin and a synthetic bovine inhibin peptide fragment on granulosa cell estradiol production were examined. The results of our studies demonstrate that inhibin has a profoundly negative autocrine-paracrine role in regulation of estradiol production by granulosa cells from healthy estrogen-active dominant follicles, which have a high in vivo and in vitro capacity to produce estradiol, or from early atretic estrogen-inactive dominant or subordinate follicles, which have a very low in vivo and in vitro capacity to produce estradiol.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
To develop a homologous approach to examine inhibin’s action on bovine granulosa cells, only reagents isolated from, or generated in, cattle were used for our in vitro studies unless specified otherwise. All procedures involving live cattle were sanctioned by the All University Committee on Animal Use and Care at Michigan State University.

Development of bovine antibovine inhibin antisera
Bovine inhibin {alpha}C1–26gly.tyr (bINH, 40 mg) was conjugated to 20 mg human {alpha}-globulins (HAG; Sigma, St. Louis, MO) using bisdiazotized benzidine, as previously described (37). At 180 d of age, each Angus-Hereford cross-bred steer calf (293 ± 4 kg, n = 9) was given a primary immunization (sc, four sites on the neck) of 1000 µg bINH:500 µg HAG dissolved in 0.5 ml sterile deionized water and emulsified in 2 ml Freund’s complete adjuvant. Booster injections of 500 µg bINH:250 µg HAG emulsified in incomplete Freund’s adjuvant were given at 84, 112, 140, and 168 d after primary immunization. Jugular blood (20 ml) was collected at time of primary injection and 10 d after each injection to determine bINH antibody titer (38). The three steers with the highest bINH antibody titer were exsanguinated under general anesthesia. After clotting, anti-bINH antiserum was harvested by centrifugation and stored at -20 C.

To avoid potential confounding effects of serum on differentiation of granulosa cells (39, 40, 41, 42), the bovine anti-bINH antibodies used to immunoneutralize inhibin in our study were purified by the following two procedures.

Partial purification of bovine anti-bINH antiserum
The bovine IgG (bIgG) fraction of anti-bINH antiserum was isolated with use of the MAbTrap protein G kit (Amersham Pharmacia Biotech, Piscataway, NJ), according to the manufacturer’s instructions.

Purification of bovine anti-bINH antiserum
To purify bovine anti-bINH antiserum, aliquots (20 ml) of anti-bINH antiserum were subjected to caprylic acid precipitation to isolate IgGs (43). The mixture was then centrifuged at 10,000 x g for 60 min, and the bIgG-enriched supernatant was concentrated and desalted with use of Centriprep-30 concentrator (Millipore Corp., Bedford, MA) by following the manufacturer’s instructions. Anti-bINH antibodies were then purified from the acetic-caprylic acid-isolated bIgGs by using Affi-Gel 10:bINH peptide affinity chromatography (44). The Affi-Gel 10:bINH-anti-bINH antibody complex was washed extensively with 50 mM sodium HEPES containing 1 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (pH 7.5) followed by three 50-ml washes with 5 M NaCl to remove nonspecifically adsorbed proteins and low-affinity anti-bINH antibodies. After completion of washing, the anti-bINH antibodies were eluted from the Affi-Gel 10:bINH-anti-bINH antibody complex with 1 N acetic acid. The eluate was neutralized with 5 M Tris-base (pH 11), concentrated, and washed with and exchanged into PBS buffer by using Centriprep-30 concentrators. The purified anti-bINH antibodies were desiccated and stored frozen at -80 C. From approximately 400 mg acetic-caprylic acid isolated bIgGs, 1–5 mg purified anti-bINH antibodies were routinely isolated.

Isolation of inhibin from bovine follicular fluid
Bovine ovaries were obtained from a local abattoir, and bovine follicular fluid (bFF) was removed from antral follicles and pooled as previously explained (45). Purified bovine anti-bINH antibodies were isolated as explained above and linked to an Affi-Gel 10 column according to the manufacturer’s instructions. The immunoaffinity column was then used to isolate inhibin from bFF by procedures previously validated in our laboratory (45). The immunoaffinity-purified preparation of inhibin was subjected to preparative SDS-PAGE coupled with electroelution to isolate inhibin precursors or 29-kDa pro-{alpha}C, which are predominant in bFF (45, 46).

Sources and procedures for isolation of bovine granulosa cells from dominant or subordinate follicles
Granulosa cells were obtained either at a local abattoir from first-wave dominant or subordinate follicles or by ultrasound-guided needle aspiration of dominant ovulatory or first-wave dominant nonovulatory follicles from conscious dairy cows.

Ovaries containing dominant follicles from the first wave of follicle growth were obtained at a local abattoir, and granulosa cells were isolated using a previously published procedure (47). In brief, stage of the estrous cycle for each pair of bovine ovaries obtained on 23 separate visits to a local abattoir was estimated based on the external appearance of the corpus luteum (48). Only ovaries from d 4–10 of the estrous cycle, which coincides with development of the first-wave dominant nonovulatory follicle (49), were obtained. Within 20–30 min of slaughter, each cow’s ovaries were placed separately into a bottle containing ice-cold PBS solution and transported to the laboratory within 3–4 h of slaughter. In the laboratory, the largest (presumed dominant) and sometimes the second largest follicle (subordinate) per cow were dissected. Stroma was removed from each follicle, and the diameter of each dissected follicle was determined with a caliper. Each follicle was punctured with a 22-gauge needle attached to a 3-ml syringe, and bFF was aspirated. Samples of bFF were stored at -20 C for subsequent determination of concentrations of estradiol and progesterone by RIA. In some studies, the ratio of concentration of estradiol to progesterone in bFF was used to separate first-wave dominant nonovulatory and subordinate follicles in hindsight into two categories: estrogen-active (estradiol greater than progesterone in bFF) or estrogen-inactive (progesterone greater than estradiol in bFF). Estrogen-active follicles have biochemical characteristics of healthy growing dominant follicles, but estrogen-inactive follicles are destined for atresia (50, 51). In addition, granulosa cells from estrogen-active dominant follicles have a high basal capacity to produce estradiol in vitro, whereas granulosa cells from estrogen-inactive dominant or subordinate follicles have a low in vitro capacity to produce estradiol (47).

Granulosa cells isolated from individual follicles were prepared for culture as follows: After follicle dissection and removal of bFF, each follicle shell was bisected under sterile conditions, and granulosa cells were scraped gently from the basement membrane with a spatula into Ham’s F12 media (Invitrogen Corp., Carlsbad, CA) supplemented with sodium bicarbonate (0.01 M) and antibiotics (10,000 IU penicillin and 10 mg streptomycin/ml; Invitrogen Corp.). For each experiment, granulosa cells from each follicle were centrifuged (400 x g for 5 min at 4 C) and washed three times with Ham’s F12-supplemented media. Number of granulosa cells was estimated with a Coulter Counter Particle Z1 (Beckman Coulter, Inc., Fullerton, CA), and cells were initially diluted to a concentration of 2 x 106 cells/ml. Cell viability was assessed with Trypan blue exclusion dye (Invitrogen Corp.). Total elapsed time from slaughter until initiation of cell culture was approximately 6 h.

Granulosa cells were also obtained from dominant ovulatory or first-wave dominant nonovulatory follicles from conscious, nonlactating, multiparous Holstein cows. Cows were 4–7 yr of age and housed in facilities at Michigan State University. Estrous cycles were synchronized with two injections of 25 mg prostaglandin F2{alpha} (PGF2{alpha}, Lutalyse; Upjohn and Pharmacia, Kalamazoo, MI) 11 d apart. Follicle growth was monitored by daily ultrasound scanning (7.5-MHz transducer; Aloka Ultrasound, Wallingford, CT) with follicle mapping beginning 7 d after the first PGF2{alpha} injection. Thirty-six hours after the second PGF2{alpha} injection, granulosa cells were aspirated from the ovulatory follicle (largest follicle) of each cow using an ultrasound (5-MHz transvaginal transducer; Pie Medical Ultrasound, Indianapolis, IN) guided needle. After cell recovery, each cow was injected with 100 µg GnRH (Cystorelin; Merial Ltd., Duluth, GA) to induce an LH surge, ovulation, and/or luteinization of the aspirated dominant follicle and growth of the first-wave nonovulatory dominant follicle. Growth of first-wave follicles was monitored by daily ultrasound and follicle mapping. On d 6 after GnRH, granulosa cells were aspirated from the first-wave dominant nonovulatory follicle (largest follicle) of each cow.

Granulosa cells were recovered from conscious cows as follows: Each cow was partially anesthetized with an epidural injection (5 ml) of 2% lidocaine (Xylocaine; AstraZeneca Pharmaceuticals LP, Wayne, PA) before cell collection. The cow’s vulva was washed with a 1% betadine solution (American Livestock Supply, Inc., Madison, WI) before the ultrasound probe was inserted into the vagina. Rectal palpation was used to guide the ovary with the dominant follicle to the ultrasound probe. The cell collection apparatus consisted of a stainless steel cannula (3 mm diameter) that traversed the ultrasound probe. The posterior end of the cannula was attached to an 18-gauge, 2-in. needle, whereas the anterior end of the tube was attached to a bifurcated Teflon cannula (1.5 mm diameter) that had two 10-ml syringes attached. Needles, syringes, and cannula were prerinsed with collection media [Ham’s F12 media containing 1% (wt/vol) BSA (Sigma) and 10,000 IU penicillin and 10 mg streptomycin/ml] before granulosa cells were aspirated.

Once the largest follicle was located by ultrasound, the anterior end of the steel cannula was pushed by the operator through the ultrasound probe to enable the needle on the posterior end of the cannula to penetrate the vaginal wall and the dominant follicle. Note, by following a preset needle guide visible to the operator, the needle was positioned before aspiration to enter into the nonexposed surface of the dominant follicle to minimize leakage of bFF during cell recovery. Once the needle was observed in the antrum of the follicle, the follicular contents, which included bFF, granulosa cells, and blood cells, were quickly aspirated into one 10-ml syringe. Using the second syringe, the empty follicle was refilled with media (1 ml), which was then aspirated into the first syringe. The total follicular contents were then transferred to a 15-ml conical tube containing 1 ml collection media and kept on ice until granulosa cells were isolated and processed for culture. Total volume of bFF and flushing media were recorded, and volume of bFF was estimated based on two different diameter measurements of each follicle. The follicular aspirates were transported to the laboratory within 30 min of collection and centrifuged at 400 x g for 5 min. The supernatant was discarded, except for 1 ml that was stored at -20 C to determine intrafollicular concentration of estradiol. To separate granulosa from red blood cells, pelleted cells were resuspended into 2 ml fresh Ham’s F12 media and layered onto a 45% Percoll column. Each column was centrifuged for 20 min (400 x g, 4 C). Granulosa cells were recovered from the top 2 ml and washed two times with Ham’s F12 media. Number of granulosa cells was estimated with a Coulter Counter Particle Z1. Granulosa cell suspensions were diluted to a concentration of 200,000 cells/ml, and cell viability was assessed with Trypan blue exclusion dye. After aspiration and removal of red blood cells, number of granulosa cells recovered per follicle averaged 2.56 x 105 ± 0.67 [ (mean) ± SEM, n = 26 follicles from 26 cows]. Total elapsed time from granulosa cell aspiration until initiation of cell culture was approximately 2 h.

Serum-free culture procedure
Unless specified otherwise, granulosa cells obtained from each dominant or subordinate follicle were cultured in triplicate in serum-free media without hormonal or growth factor additives other than the addition of androgen substrate and the inhibin-related treatments, as previously described (47). Various numbers of granulosa cells (1 x 104/200 µl media, 1 x 105/200 µl, or 1 x 106/1000 µl) were cultured in Falcon Primaria plates (24- or 96-well plates, Becton Dickinson and Co., Lincoln Park, NJ) containing Ham’s F12 medium (200 or 1000 µl) supplemented with 19-hydroxyandrostenedione (1 or 2 µM, Sigma) and the various inhibin antibody or inhibin treatments previously equilibrated at 37 C. Within 2–3 h after addition of granulosa cells to media, they become weakly attached to the bottom of culture wells. Addition of fibronectin or vitronectin to improve plating does not alter capacity of granulosa cells to produce estradiol (data not shown). Thus, these agents were omitted in our studies. Granulosa cells were incubated at 37 C in a humidified atmosphere (5% CO2 and 95% air) for 18 h. After culture, spent medium was carefully removed from each well to avoid removal of granulosa cells, centrifuged (3000 x g for 1 min at room temperature) to remove debris, transferred to 1.5-ml microfuge tubes, and stored at -20 C until determination of estradiol concentration. Percent of viable cells remaining at the end of an experiment was estimated using Trypan blue exclusion dye, as previously explained (47). The Coulter Counter Particle Z1 was used to estimate number of cells at the end of culture.

RIA
Concentrations of estradiol and progesterone in unextracted bFF and concentration of estradiol in media were determined by RIA using commercially available kits (Diagnostic Products Corp., Los Angeles, CA), as previously validated (52). Sensitivity of the estradiol assay was 0.5 pg/ml, and intra- and interassay coefficients of variation were 6% and 7%, respectively. Sensitivity of the progesterone assay was 0.1 ng/ml, and intra- and interassay coefficients of variation were 5% and 9%, respectively.

Immunoblot
A previously validated ligand immunoblot procedure developed in our laboratory (31) was used to detect the different molecular weight forms of inhibin and inhibin {alpha}-subunits in spent media, bFF, or the bovine inhibin preparations used to treat granulosa cells. In brief, samples were subjected to 12% SDS-PAGE (53) under nonreducing or reducing conditions and then electrophoretically transferred from gels to Immobilon P membranes (Millipore Corp.; Ref.31). Each membrane was blocked with 0.01% Blotto, incubated with a mink antibovine {alpha}C1–26gly.tyr antiserum (1:1000), washed, incubated with radiolabeled {alpha}C1–26gly.tyr-125I (0.76 µCi/ml), rewashed, wrapped in plastic, and exposed 20 min to 72 h in a GS-250 Imaging Screen-BI (Bio-Rad Laboratories, Inc., Hercules, CA). Intensities of inhibin bands were determined with use of the GS-250 molecular imager (Bio-Rad Laboratories, Inc.) and Molecular Analyst software program (Bio-Rad Laboratories, Inc.). Molecular weights of each inhibin form were estimated using protein standards (Bio-Rad Laboratories, Inc.) after silver staining.

Protein
Protein concentrations of media or samples of bFF were determined with the use of DC protein assay kit (Bio-Rad Laboratories, Inc.) or DU-64 spectrophotometer (Beckman Coulter, Inc.).

Statistical analysis
Number of follicles and cows used in each cell culture experiment is explained in detail in Results and in the figure legends. In general, each cell culture experiment was replicated at least twice using granulosa cells primarily from individual follicles obtained from 68 cows at a local abattoir or 18 conscious Holstein dairy cows. Each treatment was replicated two to four times. Overall treatment effects were determined using the general linear model procedure of SAS (54). Concentrations of estradiol in media were log transformed to satisfy assumptions of normally distributed errors before statistical analyses, but actual values are reported. Overall treatment effects of P < 0.05 were considered significant. If treatment effects were significant, Bonferroni t test was used to determine whether individual means differed (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell viability and number
Viability of granulosa cells isolated from individual dominant or subordinate follicles obtained at an abattoir decreased (P < 0.05) from 63% ± 0.8% ( ± SEM, n = 100 follicles) at the beginning of culture to 55% ± 2% (n = 37) at the end of culture. Viability of granulosa cells isolated from dominant follicles of conscious cows averaged 60% ± 2.5% (n = 33 follicles) at the beginning of culture, which was similar to viability of granulosa cells obtained from follicles at an abattoir. Viability of granulosa cells from conscious cows at the end of culture was not determined. Treatment of granulosa cells with bIgG (n = 3 follicles), partially purified bovine anti-bINH antibodies (n = 6), or purified anti-bINH antibodies (n = 6) did not alter viability or number of cells, compared with untreated controls (n = 6, data not shown). Effect of bINH on cell viability or number was not examined.

Study 1: basal production of inhibin during culture of bovine granulosa cells
This study was designed to determine whether inhibin was produced during short-term serum-free culture of bovine granulosa cells. Granulosa cells and bFF were isolated from dominant nonovulatory follicles ( ± SEM, diameter = 15 ± 1.5 mm, estradiol = 50 ± 15 ng/ml bFF, n = 4 follicles) obtained from four cows at an abattoir. The granulosa cells from each follicle were cultured [1 x 106 cells (live + dead)/1000 µl per well, 24-well plates] in triplicate in serum-free Ham’s F12 supplemented with 2 µM 19-hydroxyandrostenedione, as explained in Materials and Methods. After 0 (<=10 min after initiation of culture) or 18 h in culture, 900 µl media were removed from each culture well and centrifuged to remove cell debris. To determine whether inhibin was basally produced during culture, 800 µl supernatant from each culture well was concentrated and desalted with use of a Microcon YM-3 centrifugal filter (Millipore Corp.). Each supernatant retentate (400 µl), and a matching sample of bFF (20 µg, control) from the same follicle that granulosa cells were isolated from, were then subjected to inhibin immunoblot analysis under reducing conditions. Intensity of inhibin precursor bands (59-kDa + 48- to 49-kDa bands combined) in bFF and spent media were determined by phosphor image analysis.

Immunoblot analysis detected predominant 59-kDa and 48- to 49-kDa inhibin {alpha}-subunit bands in bFF of each dominant follicle and in spent media after 18 h of culture of granulosa cells from each follicle (Fig. 1Go). Fainter 27-kDa and 24-kDa inhibin {alpha}-subunit bands were also detected in bFF of each follicle, whereas only a very faint 26-kDa inhibin {alpha}- subunit band was detected in media after 18 h of culture of granulosa cells from one follicle. From 0–18 h of culture for granulosa cells from all four follicles, the average concentration of estradiol in media increased (P < 0.05) 23-fold ( ± SEM, 0.08 ± 0.04 to 1.8 ± 0.6 ng/ml), amount of protein in media increased (P < 0.05) 2.7-fold (2.1 ± 0.4 to 5.7 ± 1.3 µg/400 µl), and amount of the inhibin precursor {alpha}-subunits in media increased (P < 0.06) 6.9-fold (12 ± 5 to 83 ± 43 U/400 µl).



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  		Figure 1. Representative immunoblot of inhibin produced in vivo by a dominant follicle and in vitro by granulosa cells from the same follicle. bFF and granulosa cells were isolated from four first-wave dominant nonovulatory follicles. Spent media were obtained after 0 (<=10 min after initiation of culture) or 18 h of serum-free culture of the bovine granulosa cells (1 x 106/1000 µl per well) from each follicle in Ham’s F12 media supplemented with 2 µM 19-hydroxyandrostenedione. After culture, spent media were desalted and concentrated, and spent media (400 µl) and the matching bFF sample from each follicle were subjected to SDS-PAGE under reducing conditions and inhibin immunoblot analysis. The immunoblot was exposed to phosphor imager screens for 72 h. This figure depicts a representative immunoblot of inhibin in bFF and spent media after culture of granulosa cells from one dominant follicle for 0 or 18 h. The average diameter of the dominant follicles, concentrations of estradiol in bFF for each follicle, and changes in concentration of estradiol and amount of protein in retentates and intensities of inhibin precursor {alpha}-subunit bands in media from 0–18 h of culture for all four follicles are described in the results for study 1.

 
Study 2: effect of bovine anti-bINH antibodies on capacity of bovine granulosa cells to produce estradiol
To determine whether inhibin had negative effects on estradiol production by granulosa cells isolated from follicles at different stages of differentiation, granulosa cells were obtained from individual healthy estrogen-active dominant follicles ( ± SEM, diameter = 12.6 ± 0.8 mm; estradiol = 99 ± 20 ng/ml bFF; progesterone = 36 ± 4 ng/ml bFF; n = 10 follicles from 10 cows), atretic estrogen-inactive dominant nonovulatory follicles (diameter = 11.5 ± 0.8 mm; estradiol = 16 ± 3 ng/ml bFF; progesterone = 52 ± 6 ng/ml bFF; n = 28 follicles from 28 cows), or subordinate follicles (diameter = 7.8 ± 1.1 mm; estradiol = 13 ± 4 ng/ml bFF; progesterone = 60 ± 16 ng/ml bFF; n = 4 follicles from 4 cows), as explained in Materials and Methods. Cells from each follicle were cultured [1 x 105 cells (live + dead)/200 µl per well, 96-well plates] in triplicate in serum-free media supplemented with 1 µM 19-hydroxyandrostenedione. At t = 0 h, granulosa cells were treated with 1 mg bovine anti-bINH antibodies or bIgG (control, Sigma). As depicted in Fig. 2Go, basal production of estradiol by granulosa cells from estrogen-active dominant follicles was 4- and 27-fold greater (P < 0.05), compared with granulosa cells from estrogen-inactive dominant or subordinate follicles. Nevertheless, treatment of granulosa cells with anti-bINH antibodies increased (P < 0.05) capacity of granulosa cells from estrogen-active and -inactive dominant follicles and subordinate follicles to produce estradiol 6-, 10-, and 17-fold, compared with controls for each follicle type (Fig. 2Go). Also note that despite the marked differences in stages of follicular differentiation and basal estradiol production by granulosa cells from the different follicle types, treatment of granulosa cells from each follicle type with anti-bINH antibodies increased (P < 0.05) estradiol production to similar (P > 0.30) levels.



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  		Figure 2. Effect of bovine anti-bINH antibodies on estradiol production by bovine granulosa cells from estrogen-active or -inactive dominant nonovulatory or subordinate follicles. Estrogen-active or -inactive dominant nonovulatory follicles and subordinate follicles were obtained from a local abattoir, as explained in Materials and Methods. Average diameter and estradiol and progesterone concentrations in bFF for each follicle type are in the results for study 2. Granulosa cells from each follicle were isolated and cultured (1 x 105 cells/200 µl media per well) in triplicate for 18 h in serum-free Ham’s F12 medium supplemented with 1 µM 19-hydroxyandrostenedione. At t = 0 h, granulosa cells from each follicle were treated with 1 mg bIgG (control) or bovine anti-bINH antibodies (treated). Bars represent the overall ± SEM values for concentrations of estradiol in media after culture of granulosa cells from 10 estrogen-active dominant follicles (n = 10 cows), 28 estrogen-inactive dominant follicles (n = 28 cows), or four subordinate (n = 4 cows) follicles. Different letters above bars indicate significant (P < 0.05) difference in means among control or treated, or for control vs. treated within each follicle type.

 
Study 3: dose-response effect of purified bovine anti-bINH antibodies on capacity of bovine granulosa cells to produce estradiol
Because relatively large quantities of anti-bINH antibodies were used to treat granulosa cells in study 2, bovine anti-bINH antiserum was purified by a combination of caprylic acid treatment and peptide affinity chromatography, as explained in Materials and Methods. The capacity of the purified bovine anti-bINH antibodies to immunoneutralize the inhibin produced during culture and enhance estradiol production by granulosa cells was then compared with the partially purified preparation of anti-bINH antibodies and bIgG used in study 2. For this study, granulosa cells were isolated from estrogen-active dominant nonovulatory follicles (n = 2 follicles from 2 cows) and cultured as explained in study 2. At t = 0 h, granulosa cells were treated with 12.5, 25, 50, or 100 µg of the purified bovine anti-bINH antibodies, partially purified bovine anti-bINH antibodies, or bIgG. As shown in Fig. 3Go, treatment of granulosa cells with purified anti-bINH antibodies increased (P < 0.05) estradiol production in a dose-response fashion. In contrast, comparable doses of the partially purified anti-bINH antibodies or bIgG (data not shown), which were 10- to 80-fold lower than the doses used in study 2, did not alter basal estradiol production. Hereafter, unless specified otherwise, purified anti-bINH antibodies were used to treat granulosa cells, whereas the partially purified anti-bINH antibody preparation was used as a control.



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  		Figure 3. Dose-response effect of purified bovine anti-bINH antibodies on estradiol production by bovine granulosa cells. Estrogen-inactive dominant nonovulatory follicles were obtained from a local abattoir, as explained in Materials and Methods. Granulosa cells from each follicle were pooled and cultured (1 x 105 cells/200 µl media per well) in triplicate for 18 h in serum-free Ham’s F12 medium supplemented with 1 µM 19-hydroxyandrostenedione. At t = 0 h, granulosa cells were treated with different doses of purified bovine anti-bINH antibodies (purified anti-bINH Ab) or partially purified bovine anti-bINH antibodies (anti-bINH Ab). Bars represent the ± SEM values for triplicate determinations of concentrations of estradiol in media after culture of a single pool of granulosa cells isolated from two follicles (n = 2 cows). Different letters above bars indicate significant (P < 0.05) difference in means.

 
Study 4: specificity of the bovine anti-bINH antibody-induced increase in estradiol production by bovine granulosa cells
The specificity of the bovine anti-bINH antibody-induced increase in estradiol production by bovine granulosa cells was evaluated in three separate studies. In the first study, the purified anti-bINH antibodies were adsorbed with bovine inhibin precursors (>34 kDa, see Fig. 6Go) or pro-{alpha}C (29 kDa, see Fig. 6Go) before treatment of granulosa cells to attempt to block the immunoneutralization capacity of the anti-bINH antibodies. Insufficient amounts of the 34-kDa inhibin form were available to test. For this study, granulosa cells were isolated from estrogen-active dominant nonovulatory follicles (three follicles from three cows) and cultured as explained in study 2. At t = 0 h, granulosa cells were treated with 25 µg purified bovine anti-bINH or 25 µg control antibodies (partially purified bovine anti-bINH antibodies) that had been previously (-30 min at 37 C in culture wells before addition of granulosa cells) incubated without (untreated) or with 3 µg BSA, 3 µg bovine 29-kDa pro-{alpha}C, or 3 µg bovine inhibin precursors (>34-kDa inhibin forms). As depicted in Fig. 4Go, treatment of granulosa cells with purified anti-bINH antibodies increased (P < 0.05) estradiol production 5-fold, compared with cells treated with control antibodies. However, incubation of purified anti-bINH antibodies with inhibin precursors or pro-{alpha}C suppressed (P < 0.05) capacity of the anti-bINH antibodies to enhance estradiol production 100% and 60%, compared with the control antibodies incubated with BSA.



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  		Figure 6. Dose-response effect of bovine inhibin on estradiol production by bovine granulosa cells. Bovine inhibin was isolated from bovine follicular fluid using immunoaffinity chromatography. An aliquot (20 µg) of immunoaffinity-purified bovine inhibin was subjected to SDS-PAGE under nonreducing conditions and inhibin immunoblot analysis, and the immunoblot was exposed to a phosphor imager screen for 20 min. To examine the effect of bovine inhibin on estradiol production, a single estrogen-inactive dominant nonovulatory follicle was obtained from a local abattoir, as explained in Materials and Methods. Granulosa cells were isolated and cultured (1 x 105 cells/200 µl media per well) for 18 h in serum-free Ham’s F12 medium supplemented with 1 µM 19-hydroxyandrostenedione. At t = 0 h, granulosa cells were untreated (0) or treated with different doses of affinity-purified bovine inhibin or BSA. Bars represent the ± SEM values for four replicate determinations of concentrations of estradiol in media after culture of granulosa cells from a single follicle. Different letters above bars indicate significant (P < 0.05) difference in means.

 


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  		Figure 4. Specificity of bovine anti-bINH antibodies on estradiol production by bovine granulosa cells. Estrogen-active dominant nonovulatory follicles were obtained from a local abattoir, as explained in Materials and Methods. Granulosa cells from each follicle were pooled and cultured (1 x 105 cells/200 µl media per well) in triplicate for 18 h in serum-free Ham’s F12 media supplemented with 1 µM 19- hydroxyandrostenedione. At t = 0 h, granulosa cells were treated with 25 µg partially purified bovine anti-bINH antibodies (control) or 25 µg purified bovine anti-bINH antibodies (treated), which had been previously (-30 min before addition of cells) treated without (untreated) or with 3 µg BSA, 3 µg bovine pro-{alpha}C (pro-{alpha}C), or 3 µg bovine inhibin precursors (inhibin precursors). Bars represent the overall ± SEM values for estradiol concentrations in media after culture of granulosa cells obtained from three follicles (n = 3 cows). Different letters above bars indicate significant (P < 0.05) difference in means.

 
In the second study, the effects of antibodies against TGF{alpha} on bovine granulosa cell estradiol production were examined because TGF{alpha} suppresses FSH-induced estradiol production, whereas anti-TGF{alpha} antibodies inhibit the suppressive effects of bFF on FSH-induced estradiol production by bovine granulosa cells (55). Anti-TGFß antibodies were also used to examine the specificity of the anti-bINH induced increase in estradiol because TGFß and inhibin belong to the same superfamily of growth factors (56). For this study, granulosa cells were isolated from two estrogen-inactive dominant nonovulatory follicle and cultured as explained in study 2. At t = 0 h, granulosa cells were treated with commercially purified pan-specific rabbit anti-TGFß antibodies (10–100 µg; R\|[amp ]\|D Systems Inc., Minneapolis, MN), goat antirecombinant human TGF{alpha} antibodies (60 µg; R&D Systems Inc.), or bIgG (10–100 µg). Treatment of granulosa cells with anti-TGFß or anti-TGF{alpha} antibodies had no effect on estradiol production, compared with bIgG (data not shown).

The third study was designed to determine whether the capacity of antiinhibin antibodies to enhance granulosa cell estradiol production was unique to the bovine anti-bINH antibodies. For this study, granulosa cells were isolated from estrogen-inactive dominant nonovulatory follicles and cultured as explained in study 2. At t = 0 h, granulosa cells were treated with 1 mg partially purified bovine anti-bINH antibodies (n = 11 follicles from 11 cows), rabbit anti-ap-bovine inhibin antibodies (generated in our laboratory, n = 5 follicles from 5 cows), porcine anti-bINH{alpha}C1–26gly.tyr antibodies (donated by Dr. Jack Britt, University of Tennessee, Knoxville; n = 5 follicles from 5 cows), goat anti-32-kDa bovine inhibin antibodies (donated by Dr. Y. Hasegawa, Kitasato University, Aomori, Japan; n = 5 follicles from 5 cows), or ovine antihuman INH{alpha}C1–32 antibodies (donated by Dr. Phil Knight, Reading University, Reading, England; n = 6 follicles from 6 cows). Before treatment of granulosa cells, each antiserum was treated with the protein G kit to isolate its IgG fraction, as explained in Materials and Methods. Treatment of granulosa cells with bovine anti-bINH antibodies or with nonhomologous antiinhibin antibodies increased (P < 0.05, ~10-fold) estradiol production similarly, compared with controls (data not shown).

Study 5: effect of purified bovine anti-bINH antibodies on capacity of granulosa cells isolated from dominant follicles of conscious cows to produce estradiol
Granulosa cells were obtained from dominant ovulatory follicles ( ± SEM diameter = 16.2 ± 0.7 mm; estradiol concentration in bFF = 196 ± 49 ng/ml bFF, n = 14 follicles from 14 cows) or first-wave dominant nonovulatory follicles (diameter = 15.2 ± 0.7 mm; estradiol in bFF = 38 ± 11 ng/ml, n = 12 follicles from 12 cows) of conscious cows using an ultrasound-guided transvaginal approach and cultured within 2 h of recovery, as explained in Materials and Methods. On the basis of the number of cells isolated from each cow, granulosa cells were cultured from either individual follicles (n = 2 preovulatory follicles from 2 cows) or different pools of granulosa cells from different cows (n = 3 pools of cells from 12 ovulatory follicles of 12 cows; n = 6 pools of cells from 12 first-wave dominant nonovulatory follicles of 12 cows). Cells from each follicle or pool were cultured [1 x 104 cells (live + dead)/200 µl per well; 96-well plates] as explained in study 2. At t = 0 h, cells were untreated or treated with 20 µg purified bovine anti-bINH antibody or 20 µg of the partially purified bovine anti-bINH antibodies (control). As shown in Fig. 5Go, treatment of granulosa cells isolated from dominant ovulatory or dominant nonovulatory follicles of conscious cows with anti-bINH antibodies increased (P < 0.05) estradiol production approximately 2-fold, compared with controls.



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  		Figure 5. Effect of bovine anti-bINH antibodies on estradiol production by bovine granulosa cells isolated from dominant ovulatory or dominant nonovulatory follicles of conscious cows. Granulosa cells were aspirated from dominant ovulatory follicles or first-wave dominant nonovulatory follicles using an ultrasound-guided transvaginal approach, as explained in Materials and Methods. Average diameter and estradiol concentrations for the different follicle types are in the results for study 5. Cells from each follicle or group of follicles were cultured (1 x 104 cells/200 µl media per well) in triplicate in serum-free Ham’s F12 media supplemented with 1 µM 19-hydroxyandrostenedione. At t = 0 h, granulosa cells were either untreated or treated with 20 µg partially purified bovine anti-bINH antibodies (control) or 20 µg purified bovine anti-bINH antibodies (treated). Because of the highly variable basal production of estradiol by granulosa cells from individual follicles or individual pools of granulosa cells from different follicles, results for each treatment are expressed as fold increase in estradiol concentrations over the untreated values. The average basal concentrations of estradiol in media for untreated granulosa cells from ovulatory or dominant nonovulatory follicles was 906 ± 409 pg/ml ( ± SEM, range = 281-2514 pg/ml, n = 5 follicles or pools) or 676 ± 289 pg/ml (range = 99–2039 pg/ml, n = 6 pools). Bars represent the overall average (± SEM) fold increase for estradiol concentrations in media over untreated control values after culture of granulosa cells isolated from 14 ovulatory (n = 2 follicles from 2 cows plus 3 different pools of cells from 12 follicles obtained from 12 cows) or 12 dominant nonovulatory follicles (n = 6 different pools from 12 cows). The number above each set of bars indicates total number of follicles or pools. Different letters above bars indicate significant (P < 0.05) difference in means.

 
Study 6: effect of bovine inhibin on capacity of bovine granulosa cells to produce estradiol
To test the direct effect of inhibin on estradiol production, granulosa cells were isolated from a single estrogen-inactive dominant nonovulatory follicle, and cells were cultured in quadruplicate, as explained in study 2. At t = 0 h, granulosa cells were untreated or treated with different doses of immunoaffinity-purified bovine inhibin (1, 10, or 100 µg) or BSA (100 µg, control; Serological Corp., Kankakee, IL). Immunoblot analysis revealed that the immunoaffinity-purified inhibin preparation contained a mixture of inhibin precursors (>34 kDa), the fully processed form of inhibin (34 kDa), and free inhibin {alpha}-subunits (29 kDa; Fig. 6Go). As shown in Fig. 6Go, treatment of granulosa cells with different doses of the immunoaffinity-purified inhibin preparation decreased (P < 0.05) estradiol production in a dose-response fashion. In contrast, an amount of BSA equivalent to the highest dose of inhibin had no effect on basal estradiol production.

Study 7: effect of a 28-amino acid ({alpha}C1–26 gly.tyr) peptide fragment of bovine inhibin’s {alpha}-subunit, bINH, on capacity of bovine granulosa cells to produce estradiol
The previous studies demonstrated that immunoneutralization of basally produced inhibin increased granulosa cell estradiol production, whereas treatment of granulosa cells with inhibin suppressed estradiol production. Inhibin {alpha}- subunits are hypothesized to act as antagonists of inhibin action (57). In support of this hypothesis, others show that inhibin {alpha}-subunits block FSH action in rat granulosa cells (58) or FSH-responsive cell lines (59). Consequently, this study was designed to evaluate whether treatment of granulosa cells during culture with a synthetic fragment of bovine inhibin’s {alpha}C-subunit would antagonize the negative local actions of basally produced inhibin on estradiol production. For this study, granulosa cells were isolated from estrogen-active (n = 3 follicles from 3 cows) or estrogen-inactive dominant nonovulatory follicles (n = 2 follicles from 2 cows) and cultured in duplicate or triplicate as explained in study 2. At t = 0 h, granulosa cells were untreated or treated with different doses (100, 500, or 1000 µg) of the bINH peptide (STPPLPWPWSPAALRLLQRPPEEPAAGY) or a nonsense 28-mer peptide randomly comprised of the same amino acids used to construct bINH (WPAPAQPWLPWLARLQEPEASATPAPGY). Note the addition of GY, which is not part of the amino acid sequence for inhibin’s {alpha}C1–26-subunit, which enabled bINH to be radioiodinated or conjugated to other proteins. As shown in Fig. 7Go, treatment of granulosa cells with different doses of the bINH peptide increased (P < 0.05) estradiol production in a dose-response fashion. In contrast, estradiol production was not altered by the control peptide.



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  		Figure 7. Dose-response effect of bovine inhibin’s {alpha}C1–26 gly.tyr subunit (bINH) on estradiol production by bovine granulosa cells. Estrogen-active and -inactive dominant nonovulatory follicles were obtained from a local abattoir, as explained in Materials and Methods. Granulosa cells isolated from each follicle were cultured (1 x 105 cells/200 µl media per well) in duplicate (bINH) or triplicate (nonsense peptide, control) for 18 h in serum-free Ham’s F12 media supplemented with 1 µM 19-hydroxyandrostenedione. At t = 0 h, granulosa cells were treated with different doses of the bINH peptide (bINH) or a nonsense 28-mer peptide (control), as explained in Materials and Methods. Bars represent the overall ± SEM fold increase in concentrations of estradiol in media after culture of granulosa cells from two estrogen-active and three estrogen-inactive dominant follicles (n = 5 cows). Because of highly variable basal values for untreated controls (range = 5–99 pg estradiol/ml in media; ± SEM = 35 ± 14 pg/ml), results are expressed as fold increase in estradiol concentration over the untreated control values. Different letters above bars indicate significant (P < 0.05) difference in means.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most significant outcome of our studies was the finding that immunoneutralization of basally produced inhibin by granulosa cells from individual estrogen-inactive dominant nonovulatory and subordinate follicles, which have very low basal capacities to produce estradiol and are destined for atresia, enhanced the capacity of these granulosa cells to produce estradiol to levels similar to granulosa cells from healthy estrogen-active dominant nonovulatory follicles. In addition, immunoneutralization of inhibin had a positive effect on capacity of granulosa cells isolated from dominant ovulatory follicles to produce estradiol. These in vitro observations, coupled with the distinct inverse relationship between intrafollicular concentrations of inhibin A and estradiol throughout development of dominant follicles in cattle (24, 30, 31, 32), whereas activin-A and follistatin remain relatively unchanged (24, 25), strongly imply that inhibin has a key intrafollicular role in suppression of aromatase activity and estradiol production as dominant and subordinate follicles develop during follicular waves. In vivo results also support an overall negative effect of inhibin on follicular estradiol production. Specifically, arterial infusion of human recombinant inhibin into ovaries of sheep suppresses estradiol and androstenedione secretion by preovulatory follicles with only minor decreases in FSH secretion and no alteration in follicular development (60). Because loss of intrafollicular capacity to produce estradiol precedes both onset of atresia of dominant nonovulatory and subordinate follicles (61) and ovulation of dominant ovulatory follicles (50, 62), the intrafollicular alterations in inhibin during growth of dominant and subordinate follicles (24, 30, 31, 32) may have an important role in regulation of the physiological fate of these follicles during follicular waves in cattle.

Although our study was not designed to examine the mechanism(s) associated with the antiinhibin antibody- induced increase in estradiol production by granulosa cells, previous studies show that activin increases basal and gonadotropin-induced estradiol production by granulosa cells (1, 63, 64) and inhibin antagonizes activin production (23) and activin binding to its receptors (6). Moreover, active immunization of sheep against inhibin enhances intrafollicular activin concentrations (23). Thus, it is reasonable to speculate that the increase in intrafollicular inhibin concentrations during development of dominant and subordinate follicles (24, 30, 31, 32) antagonizes the positive effects of activin on granulosa cell estradiol production (1, 63, 64) and explains why intrafollicular estradiol concentrations decrease as dominant and subordinate follicles develop in cattle.

Several independent lines of evidence in our study indicate that the basally produced inhibin during serum-free culture of bovine granulosa cells has a negative autocrine or paracrine role in regulation of estradiol production. First, immunoblot analysis of media obtained after culture of granulosa cells in our study detected the presence of relatively high amounts of inhibin precursors. This finding clearly demonstrated that inhibin was produced basally during short-term serum-free culture of bovine granulosa cells isolated from dominant follicles. Others (33, 34, 35, 36) also report that granulosa cells produce inhibin during cell culture.

Second, the highly specific, rapid, and marked increase in estradiol production by granulosa cells following immunoneutralization of the inhibin produced during cell culture provides compelling evidence for a negative autocrine or paracrine role for inhibin on estradiol production by bovine granulosa cells isolated from individual dominant or subordinate follicles. Others report that inhibin antiserum suppresses FSH-induced estradiol production by granulosa cells isolated from antral follicles of sheep (36) or whole follicles from immature rats (65). Both studies, however, not only used nondominant follicles from rats or sheep but also approaches strikingly different from our own. Specifically, granulosa cells or whole follicles were treated with inhibin antiserum rather than purified antibodies to immunoneutralize inhibin. Also, cultures were for 5–8 d in media that contained serum, FSH, insulin and/or IGFs, rather than short term and serum and additive free, to evaluate the effects of inhibin immunoneutralization. Although differences in antibody preparation and culture systems may explain these conflicting results, it is also possible that the effect of immunoneutralization of inhibin on estradiol production depends on stage of differentiation of granulosa cells.

The third line of evidence in our study supporting a negative effect of inhibin on estradiol production was shown by the direct inhibitory effect of a 1-µg dose of an immunoaffinity-purified preparation of bovine inhibin on estradiol production. Total intrafollicular concentrations of inhibin A are very high in large antral follicles of single-ovulating species [cattle, 6–18 µg/ml (24, 25); humans, 0.09–0.4 µg/ml (26)]. Thus, the 1-µg dose used in our study is in the physiological range for intrafollicular inhibin concentrations. In support of a negative effect of inhibin, others report that immunoaffinity purified porcine or human inhibin blocks FSH-induced cAMP production by a FSH-responsive cell line (59). Although previous studies have not examined the effects of inhibin on granulosa cells from dominant follicles, other studies show that porcine or human recombinant inhibin suppresses gonadotropin-induced estradiol production by granulosa cells isolated either from preantral-early antral follicles of immature rats (66) or small antral follicles of monkeys (64, 67). However, in contrast to our findings, inhibin treatments in these previous studies have no effect on basal estradiol production by granulosa cells. Moreover, in sharp contrast to the negative effects of inhibin on estradiol production demonstrated in our present study and studies by others (60, 64, 66, 67), one recent study reports that recombinant human inhibin has a positive effect on FSH- induced estradiol production by granulosa cells from antral follicles of sheep (36), but another study reports that bovine inhibin A has no effect on basal or FSH-induced estradiol production by granulosa cells from preantral-antral follicles of immature rats (63).

In unpublished studies from our laboratory, treatment of bovine granulosa cells with different doses (1–1000 ng) of individual bovine inhibin precursors, the fully processed form of inhibin, or pro-{alpha}C resulted in inconsistent effects on estradiol production, similar to the aforementioned controversial results of others. The reason for these inconsistent in vitro results among laboratories is unclear. Nevertheless, granulosa cells may have high-affinity inhibin binding sites or receptors (2, 5, 7, 8, 9, 68) and produce relatively high and variable basal amounts of inhibin during culture, as shown in our study and studies by others (33, 34, 35, 36). Thus, inhibin produced during culture would be expected to rapidly bind its receptor and mask or at least modify effects of inhibin treatments on granulosa cells. It is possible, therefore, that the variable results among laboratories that examined the direct effect of inhibin on granulosa cell function (36, 63, 64, 66, 67, 69), including our own, are attributable to the differences in the stage of differentiation of granulosa cells used for culture, length of culture, and hormonal or growth additives during culture, which may have altered basal inhibin production and/or number of inhibin receptors and thus responsiveness of granulosa cells to inhibin treatment during culture.

The fourth line of evidence in our study supporting a negative autocrine or paracrine role for basally produced inhibin was demonstrated by the marked dose-response increase in estradiol production by granulosa cells treated with a short ({alpha}C1–26gly.tyr) synthetic peptide fragment of bovine inhibin’s {alpha}-subunit, bINH. Although several interpretations of these results are possible, it is likely that pharmacological doses of the synthetic bINH peptide antagonize the negative autocrine or paracrine actions of inhibin (57) on granulosa cells for several reasons: 1) treatment of granulosa cells with bINH mimicked the stimulatory effect of anti-bINH antibodies on estradiol production by bovine granulosa cells; 2) previous reports also demonstrate an antagonistic role for inhibin {alpha}-subunits on FSH action (58, 59); and 3) the bINH peptide contains a 26-amino-acid sequence that corresponds to the same sequence in each inhibin form in the immunoaffinity-purified bovine inhibin preparation. Yet bINH did not mimic the negative effect of bovine inhibin on granulosa cells in our study. Rather, bINH enhanced capacity of granulosa cells to produce estradiol. Taken together, these findings strongly support the likelihood that the synthetic bINH peptide acts as an antagonist to the negative local actions of inhibin, which in turn explains why bINH enhanced, but inhibin suppressed, estradiol production by bovine granulosa cells in our study.

A key unanswered question is whether free {alpha}-subunits, such as pro-{alpha}C, {alpha}N{alpha}C, or {alpha}N that are in bFF (31, 45, 70, 71, 72, 73) and potentially produced during culture of bovine granulosa cells (our study), act as natural antagonists of inhibin action, as previously hypothesized (57). Several lines of reasoning indicate that inhibin precursors or the fully processed 32- to 34-kDa inhibin form, rather than inhibin {alpha}-subunits, have physiologically important inhibitory effects on estradiol production by granulosa cells. First, several studies report that inhibin precursors and the fully processed 32- to 34-kDa inhibin form are biologically active in pituitary bioassays, whereas free {alpha}-subunits are inactive (45, 70, 73, 74). Second, the 32- to 34-kDa inhibin form is the primary inhibin form that varies inversely with estradiol concentrations during development of dominant follicles in cattle (24, 30, 31, 32). Third, antibodies generated against synthetic peptide fragments of inhibin’s {alpha}C-subunit, such as bINH, have a much higher affinity (~20-fold) for free inhibin {alpha}-subunits (such as pro-{alpha}C) than for inhibin or inhibin precursors (45). Nevertheless, in our study, inhibin precursors were more effective blockers of antiinhibin antibody-induced increases in estradiol production by granulosa cells, compared with pro-{alpha}C. This finding implies that inhibin precursors or the fully processed 32- to 34-kDa inhibin rather than {alpha}-subunits have the major negative autocrine or paracrine effects on estradiol production by granulosa cells. Fourth, in contrast to the relatively small amounts (600 nM) of the purified antiinhibin antibodies needed to enhance estradiol production by granulosa cells in our study, pharmacological amounts (2.5–5 mM) of the putative synthetic inhibin {alpha}-subunit peptide antagonist, bINH, were required for a similar positive response. Others also report that pharmacological doses of recombinant or synthetic inhibin {alpha}-subunits are required to inhibit FSH action on granulosa cells (58) or an FSH-responsive cell line (59). However, relatively high doses (0.1–1 µg) of bovine pro-{alpha}C, which is a free {alpha}-subunit found in high concentrations in bFF (72), have no effect on basal or gonadotropin-induced estradiol production by granulosa cells (64).

Collectively, these findings imply that under physiological conditions free {alpha} subunits do not act as potent natural antagonists to the autocrine or paracrine effects of inhibin on granulosa cells. Thus, the enhancement in estradiol production following treatment of bovine granulosa cells with anti-inhibin antibodies in our study is most likely the result of immunoneutralization of inhibin precursors or the fully processed 32- to 34-kDa inhibin form rather than inhibin {alpha}-subunits.

In summary, bovine granulosa cells isolated from dominant follicles basally produced inhibin during serum-free culture; immunoneutralization of basally produced inhibin increased capacity of bovine granulosa cells isolated from dominant preovulatory, dominant nonovulatory, or subordinate follicles to produce estradiol; treatment of bovine granulosa cells with an immunoaffinity-purified preparation of bovine inhibin suppressed estradiol production; and treatment of granulosa cells with a short ({alpha}C1–26gly.tyr) synthetic fragment of bovine inhibin’s {alpha}-subunit enhanced capacity of bovine granulosa cells to produce estradiol. On the basis of these results, we concluded that both the synthetic {alpha}C1–26gly.tyr fragment of inhibin’s {alpha}C-subunit and antiinhibin antibodies blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells, and inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant and subordinate follicles to produce estradiol.


   Footnotes
 
This work was supported by grants from United States Department of Agriculture (90-27240-5508; 200002391), Select Sires, and Research Excellence Funds from Michigan State University (to J.J.I.).

Abbreviations: bFF, Bovine follicular fluid; bIgG, bovine IgG; bINH, bovine inhibin; HAG, human {alpha}-globulin; PGF2{alpha}, prostaglandin F2{alpha}; , mean.

Received October 17, 2002.

Accepted for publication January 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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