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Biology of Reproduction 64, 100-106 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Follicle-Stimulating Hormone Regulation of Inhibin {alpha}- and ßB-Subunit and Follistatin Messenger Ribonucleic Acid in Cultured Avian Granulosa Cells1

Adam J. Davis3,a, Carrie F. Brooksa, and Patricia A. Johnson2,a

a Department of Animal Science, Cornell University, Ithaca, New York 14853

ABSTRACT

FSH regulation of inhibin {alpha}-, ßB-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F4 + F5 follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P < 0.05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin {alpha}-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin {alpha}-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P < 0.05) mRNA levels for the inhibin {alpha}-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F4 + F5 granulosa cells. The effects of FSH on follistatin and ßB-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin {alpha}-subunit in small prehierarchical follicles.

activin, follicle, follicular development, follistatin, granulosa cells, inhibin

INTRODUCTION

Inhibin is a dimeric glycoprotein hormone that contains the inhibin-specific {alpha}-subunit as well as a ß-subunit in its structure. It acts in an endocrine manner to suppress pituitary FSH secretion. The ß-subunit exists as ßA or ßB and, depending upon which of the ß-subunits is bound with the {alpha}-subunit, inhibin exists as either inhibin-A ({alpha}-ßA) or inhibin-B ({alpha}-ßB). Activin is a dimeric protein hormone that stimulates pituitary FSH secretion. It is synthesized as a homo- or heterodimer of the ß-subunits (ßA or ßB), which combine to form activin-A (ßA-ßA), activin-B (ßB-ßB), or activin-AB (ßA-ßB). Activin and inhibin, along with their binding protein follistatin, have been shown to have diverse endocrine as well as paracrine/autocrine functions that affect a wide array of reproductive and nonreproductive cell types [1–5]. Follistatin is a soluble binding protein capable of binding activin and, with less affinity, inhibin [6, 7]. The biological significance of follistatin binding, especially with regard to inhibin, is not completely understood. Many of the biological actions of activin [8], although not all [9], are neutralized by the binding of activin to follistatin.

The ovary of the laying hen contains five to eight large yolk-filled preovulatory follicles. These large follicles are arranged in a hierarchy according to size, with the largest follicle designated as the F1 follicle and the second largest designated as the F2 follicle and so on for the other follicles. The development of these hierarchical follicles is tightly regulated, with an interval of 24–26 h between each consecutive ovulation. After ovulation of the F1 follicle, the F2 follicle becomes the new F1 follicle, succeeding follicles each advance one place in the hierarchy, and an additional follicle is recruited from the pool of small nonhierarchical follicles. The small nonhierarchical follicles consist of a pool of small yellow follicles (SYF) that are about 5–10 mm in diameter and a pool of white follicles that are less than 5 mm in diameter. The granulosa and theca cells form distinct layers in the developing follicles that can be easily and cleanly separated from one another.

Initial studies with the domestic chicken demonstrated the existence of bioactive inhibin in testes preparations [10] and ovarian granulosa cells [11, 12]. Based on plasma immunoreactive {alpha}-inhibin concentrations and selective follicle removal experiments, the largest follicles of the hen ovary have been shown to be the major source of plasma immunoreactive inhibin [13, 14]. Using a specific two-site ELISA, Lovell et al. [15] examined the content of inhibin-A and activin-A in the theca and granulosa cells of the four largest follicles. They reported that inhibin-A production was largely confined to the granulosa cells and activin-A was more abundant in the theca cells. The content of inhibin-A was reported to increase about 40-fold from the F3 granulosa cells to the F1 granulosa cells.

The mRNA expression pattern of the inhibin/activin subunits in the granulosa layer from hen preovulatory follicles [16, 17] is consistent with the data reported by Lovell et al. [15]. The mRNA for the inhibin {alpha}-subunit is abundant in the large hierarchical follicles and undetectable by Northern blot analysis in the small (<5 mm) nonhierarchical follicles [17]. Expression of the inhibin/activin ßA-subunit is much greater in the F1 follicle than in the other large preovulatory follicles [16, 17] and is not detectable by Northern blot analysis in nonhierarchical follicles [17]. Expression of the mRNA for follistatin and the inhibin/activin ßB-subunit is highest in the smaller (F6–F8) hierarchical and nonhierarchical follicles and is not detectable by Northern blot analysis in the largest preovulatory follicles [17].

Addition of LH or FSH to granulosa cell cultures from the large preovulatory follicles of domestic hens has been reported to stimulate immunoreactive inhibin {alpha}-subunit production [18, 19] and support higher levels of mRNA for the inhibin {alpha}-subunit [19]. Gonadotropin regulation of the mRNA for the inhibin/activin subunits and follistatin has not been investigated in the small follicles of the hen ovary, even though there are dramatic changes in the expression of these transcripts as these follicles develop [17]. Furthermore, the granulosa cells of small hierarchical and nonhierarchical follicles of the hen ovary are FSH sensitive [20–22], and this sensitivity may be higher in the SYF because the expression of mRNA for the FSH receptor is greater in granulosa cells from these follicles than in those cells from the largest preovulatory follicles [23]. We examined the effect of FSH on the growth of granulosa cells from hierarchical (F4 + F5) and nonhierarchical (SYF, large white follicles [LWF]) follicles. In addition, we examined the FSH regulation of the mRNA for the inhibin {alpha}-subunit and immunoreactive inhibin {alpha}-subunit. The inhibin/activin ßB-subunit, the binding protein follistatin, and progesterone production were also investigated in granulosa cells from hierarchical and nonhierarchical follicles. Our focus in this study was on the small follicles; therefore, we did not examine expression of the ßA-subunit because expression of this subunit is restricted to the hierarchical follicles [17].

MATERIALS AND METHODS

Animals

Single-comb White Leghorn hens of the Babcock B300-strain were individually caged, and egg records were maintained at 2-h intervals during daylight. The hens had free access to water and a commercial layer diet and were maintained on a lighting schedule of 14L:10D (lights-on at 0600 h). Hens between 22 and 36 wk of age that were laying regular sequences were selected and killed at 1.5–2 h after oviposition for collection of follicles. All animal procedures were approved by the Institutional Animal Care and Use Committee of Cornell University.

Cell Culture

The F4 and F5 follicles, SYF, and LWF (2–5 mm in diameter) were removed from five or six birds for each experiment and placed in ice-cold Krebs solution. The granulosa layer was isolated from each F4 and F5 follicle and pooled. Granulosa cells from the F4 + F5 follicles [19] and from the SYF and LWF [24] were dispersed and washed as previously described. A hemacytometer and trypan blue staining were used to estimate the number of viable cells. Cell viability was always >95%.

Experiment 1

Highly purified ovine FSH (NIH oFSH-19-SIAFP, generously provided by the National Hormone and Pituitary Program of the NIDDK, NICHHD, and U.S. Department of Agriculture) was added at doses of 0 and 50 ng/ml of culture medium to evaluate its effect on granulosa cell growth. This FSH dose was maximally effective in stimulating {alpha}-subunit expression in cultured granulosa cells from hierarchical follicles [19]. Cells from each follicle size were plated at a density of 625,000 cells/well (1 ml total volume per well) and incubated in 24-well tissue culture plates (Corning Costar, Cambridge, MA) as previously described [19, 24]. The granulosa cell cultures for each follicle type were terminated at 24 and 48 h after plating. Each treatment consisted of two wells, and the cells from each well were washed from the wells using a 0.1% collagenase solution of M199. The collected cells were pelleted by centrifugation (150 x g at 4°C for 5 min), the pellet was resuspended in M199, and cells in an aliquot of the solution from each well were counted using a hemacytometer. The number of cells from the two duplicate wells was averaged for each follicle type and treatment. Viability was determined using trypan blue staining. This experiment was repeated three times for a total of four replicate experiments, each consisting of two duplicate wells for each treatment and follicle type.

Experiment 2

The protocol for experiment 2 was similar to that of experiment 1. To have enough cells for RNA extraction and subsequent Northern blot analysis, the cells were plated at a density of 2.75 x 106 cells/well (4 ml total volume per well) and incubated in six-well tissue culture plates (Corning Costar). The cultures were terminated at 4 and 24 h after plating. Each treatment consisted of three triplicate wells and the cells from these wells were combined prior to RNA extraction. At each termination time, medium from the triplicate wells was combined and saved for progesterone and immunoreactive inhibin {alpha}-subunit RIA. This experiment was repeated two times (three replicate experiments).

RNA Extraction and Northern Blot Analysis

Total RNA was extracted from the cells using a guanidine isothiocynate/phenol-chloroform method [25], and 20 µg of total RNA per sample was run on an agarose/formaldehyde gel and then transferred to a nylon membrane as previously described [17]. Chicken inhibin {alpha}-subunit, inhibin/activin ßB-subunit, follistatin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA clones were prepared for Northern blot analysis as previously described [17]. The hybridization and densitometry procedures were also the same as those described previously [17].

RIA

Medium progesterone (experiment 2) and inhibin {alpha}-subunit concentrations (experiment 2) were determined by RIA procedures that have been described previously [19]. The antibody used for the inhibin {alpha}-subunit RIA detects both free {alpha}-subunit and intact inhibin [19]. Intra-assay coefficients of variation for a high- and a low-granulosa-cell culture medium pool were <10% for both the inhibin {alpha}-subunit and progesterone RIA.

Statistics

Analysis of variance was performed for each follicle type using the general linear model procedure of SAS (SAS, Cary, NC) or Minitab, with replicate, dose and time as variables. Protected least significant difference was used to determine differences between samples. Differences were considered significant when P values were <0.05.

RESULTS

Experiment 1

The mean ± SEM (n = 4) number of cells per well for the untreated and FSH-treated cultures for each follicle size is presented in Table 1. Viability of the treated and untreated granulosa cells from all three follicle types was >95% at both 24 and 48 h. All of the untreated and FSH-treated cell cultures at 24 h had a significant (P < 0.05) increase in cell number when compared with time zero. At 24 h of culture, there was a clear trend (P <= 0.1) that indicated FSH-enhanced granulosa cell proliferation for all these follicle types. The differences in cell proliferation at 48 h between the untreated and FSH-treated cultures for all follicle types, however, were all significant (P < 0.05).


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  		TABLE 1. Effect of FSH on the growth of cultured granulosa cellsa obtained from follicles of different sizes

Experiment 2

The expression of the mRNA for the inhibin {alpha}-subunit and GAPDH in the F4 + F5 follicles, SYF, and LWF for one of the three replicate experiments is shown in Figure 1. The mean ± SEM (n = 3) relative intensity values of the mRNA for the inhibin {alpha}-subunit, after quantification by densitometry and correction for RNA loading and transfer with GAPDH, are shown in Figure 2. Expression of the inhibin {alpha}-subunit was increased by FSH treatment of the LWF granulosa cell cultures. The addition of FSH to the cell culture medium also enhanced expression of the inhibin {alpha}-subunit at 4 and 24 h in the SYF granulosa cells and at 24 h in the F4 + F5 granulosa cell cultures. Expression of the inhibin {alpha}-subunit mRNA significantly decreased with time in both the treated and untreated granulosa cell cultures from the F4 + F5 follicles and the SYF.



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  		FIG. 1. Autoradiographs from one of the three replicates of the Northern blot analysis of the inhibin {alpha}-subunit and GAPDH. Addition of FSH to the granulosa cell cultures enhanced expression of the mRNA for the inhibin {alpha}-subunit



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  		FIG. 2. Relative density (mean ± SEM) of the mRNA for the inhibin {alpha}-subunit in cultured granulosa cells from F4 + F5 follicles, SYF, and LWF after FSH treatment. * Indicates values that are significantly different (P < 0.05) from the corresponding untreated value. Some error bars were too small to be illustrated

The mean ± SEM (n = 3) accumulation of immunoreactive inhibin {alpha}-subunit protein in the medium of the cell cultures is presented in Figure 3. Addition of FSH to the granulosa cell cultures increased the accumulation of immunoreactive inhibin {alpha}-subunit protein at 24 h of culture for all three follicle types. For both the untreated and FSH-treated F4 + F5 granulosa cell cultures, immunoreactive inhibin {alpha}-subunit protein increased significantly with time. In the SYF granulosa cell culture, immunoreactive inhibin {alpha}-subunit protein increased significantly with time for only the FSH-treated cultures. The F4 + F5 granulosa cell cultures produced significantly more (P < 0.05) immunoreactive inhibin {alpha}-subunit protein than did the granulosa cell cultures from the other two follicle types at both time points and for both treatments.



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  		FIG. 3. Mean ± SEM accumulation of immunoreactive inhibin {alpha}-subunit protein in FSH-treated and untreated granulosa cells obtained from F4 + F5 follicles, SYF, and LWF. * Indicates values that are significantly different (P < 0.05) from the corresponding untreated value. The differences in the expression of the mRNA for the inhibin {alpha}-subunit at 4 h (Fig. 2) were not detected at the secreted protein level until 24 h

The mean ± SEM (n = 3) relative intensity of follistatin mRNA for each follicle type is shown in Figure 4. The level of the mRNA for follistatin was significantly reduced (P < 0.05) at 4 h for the FSH-treated F4 + F5 and SYF cultures. FSH treatment of the F4 + F5 granulosa cells, however, increased follistatin mRNA levels at 24 h of culture. The amount of follistatin mRNA decreased significantly with time in both the treated and untreated granulosa cell cultures from all three follicle types.



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  		FIG. 4. Mean ± SEM relative density of the mRNA for follistatin in cultured granulosa cells from F4 + F5 follicles, SYF, and LWF after FSH treatment. * Indicates values that are significantly different (P < 0.05) from the corresponding untreated value. Some error bars are too small to be illustrated

The mean ± SEM (n = 3) relative intensity of the mRNA for the inhibin/activin ßB-subunit for each individual follicle type is shown in Figure 5. The amount of inhibin/activin ßB-subunit mRNA decreased significantly with time in both the treated and untreated granulosa cell cultures from all three follicle types. Addition of FSH to the F4 + F5 granulosa cell cultures suppressed expression of the inhibin/activin ßB-subunit at 4 h.



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  		FIG. 5. Mean ± SEM relative density of the mRNA for the inhibin/activin ßB-subunit in cultured granulosa cells from F4 + F5 follicles, SYF, and LWF. * Indicates values that are significantly different (P < 0.05) from the corresponding untreated value. Some error bars are too small to be illustrated. Treatment of granulosa cells with FSH had minimal effect on the expression of the mRNA for the inhibin ßB-subunit

Progesterone accumulation (mean ± SEM) in medium from F4 + F5 untreated and treated granulosa cell cultures was 17 ± 3 ng/ml and 91 ± 22 ng/ml, respectively, at 4 h and 24 ± 4 ng/ml and 185 ± 34 ng/ml, respectively, at 24 h. The progesterone values for the F4 + F5 granulosa cell cultures did not significantly increase over time for the untreated cells but did increase for the FSH-treated cells. Addition of FSH to the granulosa cell cultures significantly increased production of progesterone in these cells at both time points when compared with untreated controls. In the SYF and LWF granulosa cell medium, mean progesterone concentrations from the untreated and treated cultures at 4 and 24 h were all <2 ng/ml, although FSH significantly increased progesterone production at both time points in the SYF cultures and at 24 h in the LWF cultures.

DISCUSSION

Our novel findings are 1) FSH stimulates the proliferation of cultured granulosa cells obtained from the F4 + F5 follicles, the SYF, and the LWF, 2) the mRNA for inhibin {alpha}-subunit is increased in granulosa cells from LWF in response to FSH, and 3) immunoreactive inhibin {alpha}-subunit secretion from granulosa cells isolated from small hierarchical and nonhierarchical follicles is increased in response to FSH. In addition, the effects of FSH on the mRNA expression of the inhibin/activin ßB-subunit and follistatin in the cultured granulosa cells were variable and related to the maturity of the follicles from which the granulosa cells were isolated.

Previous reports [26, 27] had indicated that chicken granulosa cells isolated from 4- to 8-mm-diameter follicles and maintained in suspension culture with no serum undergo significant apoptosis at 6 h of culture. As previously reported [24], we established an isolation method and culture system that included serum and allowed for the successful culture of granulosa cells from SYF and LWF for up to 48 h with a high degree of cell viability. There was significant proliferation of these cultured granulosa cells, and this proliferation was enhanced by the addition of FSH to the culture medium. The increased secretion of progesterone and inhibin {alpha}-subunit protein with increasing culture duration was another indication of the overall health of these granulosa cell cultures.

In mammalian species the stimulation of immunoreactive inhibin secretion from granulosa cells by gonadotropins, especially FSH, has been well reported [28–33]. In the domestic hen, the addition of FSH to granulosa cell cultures from F1, F3, and F4 follicles resulted in increased secretion of immunoreactive inhibin {alpha}-subunit [18, 19] and increased levels of mRNA for the inhibin {alpha}-subunit [19]. In this earlier study [19], we were unable to determine whether the increased levels of mRNA for the inhibin {alpha}-subunit detected at 24 and 48 h of culture were the result of increased transcription or of increased stability of the {alpha}-subunit mRNA. This inability was due to the lack of any difference in inhibin {alpha}-subunit mRNA level at 4 h of culture between the FSH-treated and untreated cultures followed by the overall sharp decline in {alpha}-subunit mRNA with increased culture duration, which was more pronounced in the untreated cultures. In the present study, although the levels of {alpha}-subunit mRNA again declined with culture duration for the F4 + F5 and SYF cultures, it appears that FSH stimulated {alpha}-subunit mRNA transcription in the LWF cultures at 4 and 24 h; no mRNA for the inhibin {alpha}-subunit was detected in freshly dispersed granulosa cells (data not shown) or in the untreated granulosa cells at 4 or 24 h of culture.

The lack of any detectable inhibin {alpha}-subunit mRNA by Northern blot analysis in the untreated LWF granulosa cell cultures was not surprising because we previously were unable to detect any of this species of mRNA in a combined theca and granulosa total RNA sample from these follicles [17]. We also previously reported that inhibin {alpha}-subunit mRNA levels increased rapidly in granulosa cells as they matured from SYF to hierarchical follicles [17]. Safi et al. [34] subsequently confirmed by quantitative competitive reverse transcription polymerase chain reaction that the amount of the mRNA for the inhibin {alpha}-subunit in the LWF is beyond the detection limits of Northern blot analysis and that the mRNA for the inhibin {alpha}-subunit increased 40-fold as granulosa cells matured from LWF to SYF. In the current study, the production of immunoreactive inhibin {alpha}-subunit also increased as the maturity of the follicles from which they were collected increased. Thus, increasing the expression of the inhibin {alpha}-subunit mRNA and protein may be a key event in the early maturation of chicken follicles. It may be that the LWF that acquire enough FSH receptors and can respond to a FSH stimulus by producing inhibin {alpha}-subunit protein are the ones that mature into SYF. The production of inhibin {alpha}-subunit may provide a biological sink that reduces the dimerization of the inhibin/activin ßB-subunits to form activin in those follicles that express ßB-subunit mRNA [17]. We have previously demonstrated [17] that the ßB-subunit is expressed in high amounts in the LWF and SYF. If activin acts as a local regulator preventing further maturation of an LWF, reducing the local concentration of activin by expression of {alpha}-subunit could be what allows an LWF to mature to an SYF.

Although the production of immunoreactive inhibin {alpha}-subunit protein mirrors the pattern of mRNA expression for the inhibin {alpha}-subunit, cell number may explain some of the difference in the production of this protein between the untreated and treated cultures. At 4 h of culture, there were no differences in cell number between the two treatments for any follicle type (data not shown). At 24 h however, the number of cells in the FSH-treated granulosa cultures was greater, although not significantly greater than in the untreated controls for all follicle types. Thus, if the concentrations of immunoreactive inhibin {alpha}-subunit protein and the concentration of progesterone were corrected for cell number, the differences in production between the untreated and treated cell cultures would be less. This does not minimize the overall biological effect that FSH is exerting to increase total output of {alpha}-subunit and progesterone. In addition, the specific effect on stimulation of {alpha}-subunit mRNA is clear.

In rat granulosa cell cultures, follistatin mRNA [35] and secreted immunoreactive follistatin [28] were both reported to be stimulated in a dose-dependent manner by FSH. Although our findings are different, Tuuri and Ritvos [36] reported a rapid increase and decline in follistatin mRNA levels in cultured human granulosa cells treated with FSH during a 24-h period, implying that rapid changes in follistatin mRNA may occur in vitro. The fact that the mRNA level for follistatin in the LWF cultures remained unchanged may be a reflection of the developmental stage of these cells compared with those obtained from the more mature SYF and F4 + F5 follicles. The granulosa cells from the LWF, however, were sensitive to FSH based on its stimulation of cell proliferation and progesterone production in the LWF cultures. Taken together, these results may indicate that FSH is not having a direct effect on follistatin mRNA levels in the more mature granulosa cells but is instead regulating another molecule, which results in the suppressed follistatin mRNA levels.

The lack of significant change in the expression of the ßB-subunit in response to FSH is not surprising given that the promoter for the ßB-gene does not have a cAMP-responsive element [37]. Recent data suggest that the oocyte factor GDF-9 may be involved in regulation of ßB-subunit expression in granulosa cells [38].

In summary, as in our previous study with granulosa cells from hierarchical follicles [19], we found that FSH increased expression of the inhibin {alpha}-subunit in the granulosa cells from the small growing follicles. More significantly, FSH dramatically increased the expression of the {alpha}-subunit mRNA in LWF. These data indicate that FSH may support the maturation and growth of follicles from small nonhierarchical follicles to larger hierarchical follicles by increasing inhibin {alpha}-subunit production.

FOOTNOTES

First decision: 11 July 2000.

1 Supported by USDA grant 97-35203-4979. Back

2 Correspondence: Patricia Johnson, 202 Morrison Hall, Department of Animal Science, Cornell University, Ithaca, NY 14853. FAX: 607 255 9829; paj1{at}cornell.edu Back

3 Current address: Department of Poultry Science, University of Georgia, Athens, GA 30602. Back

Accepted: August 16, 2000.

Received: June 8, 2000.

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