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Endocrinology Vol. 138, No. 6 2544-2551
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of Follistatin Production by Rat Granulosa Cells in Vitro1

Yasumi Shintani2, Mitzilee Dyson, Ann E. Drummond and Jock K. Findlay

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Prof. J. K. Findlay, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: jock.findlay{at}med.monash.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aims of this study were to apply enzyme-linked immunosorbent assays (ELISA) for human follistatins (FS) to measure total immunoreactive (ir-) rat FS and free rat FS, and investigate the regulation of production of total ir-FS and free FS by rat granulosa cells (GC) in vitro. Production of ir-inhibin was monitored as an index of GC function.

The ELISAs for total ir-FS, based on an immunoradiometric assay developed recently for human FS, and free FS, based on capture of FS by a monoclonal antibody and detection by activin A binding, had sensitivities of 0.4 and 0.8 ng recombinant human (rh-) FS 288/ml, respectively, and did not cross-react with inhibin A, rLH, or FSH. rh-Activin did not cross react in the total ir-FS ELISA, but interfered with the measurement of free FS. Dilutions of GC-conditioned medium were parallel to the standard curve of rh-FS 288 for each assay. The values obtained in the free FS assay were 10- to 20-fold higher than those in the total ir-FS ELISA, suggesting that rat FS may be recognized by the antibodies differently than the human standard.

Both total ir-FS and free FS production by undifferentiated GC from diethylstilbestrol (DES)-treated, immature rats increased with cell number and time in culture and were stimulated dose dependently by FSH, rh-activin A (except free FS, which was not measured because of interference), forskolin, and phorbol 12-myristrate. The effects of FSH and activin on FS production by undifferentiated GC were additive.

There were significant effects of degree of differentiation of GC on basal FS production and responsiveness to FSH, LH, and rh-activin A. Both total ir-FS and free basal FS production increased up to 4-fold with the degree of differentiation of GC, produced by treating rats in vivo with DES (undifferentiated), DES plus FSH (partially differentiated), or DES plus FSH plus hCG (fully differentiated). The addition of FSH in vitro increased FS production by undifferentiated and partially differentiated GC, but not by fully differentiated GC. The only detectable effect of LH on FS production was on partially differentiated GC. Activin A stimulated total ir-FS production by undifferentiated and partially differentiated GC, but inhibited total ir-FS production by fully differentiated GC.

Ir-inhibin production in these experiments was similar to that of FS with the following exceptions; phorbol 12-myristrate inhibited ir-inhibin production by undifferentiated GC, basal ir-inhibin decreased in fully differentiated GC, FSH stimulated ir-inhibin only in undifferentiated GC, and rh-activin A stimulated ir-inhibin at all stages.

It is concluded that 1) FS protein production by cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways; 2) increasing GC differentiation is associated with a significant increase in basal FS production by rat GC and a change in the hormonal regulation of FS production; and 3) FS and ir-inhibin production by cultured rat GC can be differentially regulated. The results are consistent with the hypothesis that activin tone decreases within follicles as they develop due to increased production of the activin-binding protein FS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLISTATIN (FS) is an activin-binding protein that neutralizes the actions of activin by forming an inactive complex (1, 2, 3). It is encoded by a single gene in all species examined, and the primary structure of FS is highly conserved (>97%) (4, 5). FS exists as a number of different isoforms that arise from a combination of alternate splicing of the heteronuclear RNA and degrees of proteolytic cleavage and glycosylation during posttranslational processing of the proteins (6, 7, 8, 9, 10). FS 344 messenger RNA (mRNA) encodes the precursor of a protein of 344 amino acids, which can be proteolytically cleaved to form proteins of 315 and 300 amino acids. FS 317 mRNA encodes a carboxyl-truncated form of 317 amino acids that lacks 27 amino acids of exon 5 and can be further processed to a protein of 288 amino acids (6, 7, 8). The various forms of FS differ in their relative amounts and abilities to suppress FSH secretion (9, 10).

The only sites of production of FS mRNA in the ovary are the granulosa cells (GC) and early luteal cells (4, 11). Regulation of the expression of FS mRNA has been studied in situ and in vitro in several species, including the rat. The mRNA for FS 344 is the major form, with FS 317 being at or below the level of detection, and there was no detectable change in the relative abundances of the alternately spliced forms (FS 344 and FS 317) in rat ovarian cells with various hormonal treatments in vivo or in vitro (12). The steady state level of FS mRNA was increased in rat ovaries by PMSG (2) or PMSG plus hCG treatment (12). FS mRNA was first detected by in situ hybridization in GC of secondary follicles during the rat estrous cycle, with maximum expression in preovulatory follicles and early corpora lutea (11). FSH had dose- and time-dependent stimulatory and inhibitory effects on steady state levels of FS mRNA in rat GC, whereas LH, PRL, GH, and insulin-like growth factor I had little or no effect (13). Activin had a biphasic effect on FS mRNA, being inhibitory at low doses and stimulatory at high doses, in the presence or absence of FSH (13). Both cAMP and phorbol ester stimulated FS mRNA levels (14), whereas epidermal growth factor inhibited FSH-stimulated FS mRNA levels in rat GC (13). These studies showed that steady state levels of FS mRNA in rat GC are activin and FSH dependent, rather than LH dependent, and are regulated via both the protein kinase A and C pathways.

Less is known about regulation of the FS protein production in the rat, primarily because it has been difficult to measure with current assays. Using immunocytochemical analysis, detection of FS protein was confined to healthy dominant preovulatory follicles and a subpopulation of tertiary follicles in rats entering estrus, compared with FS mRNA, which was first detected in secondary follicles (11). Saito et al. (15) used an affinity gel assay to measure activin-binding activity in medium conditioned by rat GC. This assay detects activin-binding proteins that have mol wts and immunoblots consistent with multiple forms of FS, but it is not particularly sensitive (>1 ng/ml), and it probably measures only free FS, because FS bound to activin does not readily dissociate (16) and, therefore, would not bind labeled activin. Nevertheless, it was shown that the activin-binding activity in the conditioned medium increased with cell number and time, and was FSH, but not LH, dependent. We used a RIA to show that FS in medium conditioned by bovine GC was under similar regulation in vitro (17).

The aims of this study were 1) to develop an enzyme-linked immunosorbent assay (ELISA) for total immunoreactive (ir-) rat FS based on an immunoradiometric assay (IRMA) developed recently for human FS (18) and an activin-binding assay for free FS based on immobilization of FS by a specific monoclonal antibody (19), and 2) to apply these assays to investigate the regulation of production of FS by rat GC in vitro. We used GC at various stages of differentiation induced by hormonal treatments of immature rats in vivo and monitored the production of ir-inhibin as an index of GC function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and reagents
Recombinant human (rh-) FS 288, rat FSH, and LH were provided by the National Hormone and Pituitary Program and the NIDDK, NIH (Baltimore, MD). Rh-activin A was prepared by Dr. A. J. Mason, and purified bovine FS and anti-bovine FS polyclonal antibody were provided by Dr. D. M. Robertson (Prince Henry’s Institute of Medical Research, Clayton, Australia). Human FSH (Metrodin) and hCG (Chorulon) were purchased from CSL (Parkville, Australia) and Intervet (Sydney, Australia), respectively. Forskolin (FK) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals
Immature female Sprague-Dawley rats were obtained from Monash University Central Animal House (Clayton, Australia) and kept under standard lighting and feeding conditions. At 21 days of age, the rats received sc SILASTIC brand implants (Dow Corning, Midland, MI) containing 10 mg diethylstilbestrol (DES; Sigma) and were killed 4 days later at 25 days of age. To obtain undifferentiated GC, no additional treatments were given to the animals. Differentiated GC were obtained according to the protocol described by Miró et al. (20). Partially differentiated GC were produced by treating DES-treated rats with human FSH (10 IU) injected sc 48, 36, 24, and 12 h before ovariectomy. Fully differentiated (preovulatory) GC were obtained from DES-treated animals similarly injected sc with hFSH at 48, 36, and 24 h, followed by injection with hCG (20 IU) at 12 h. The animal experiments were approved by the institutional ethics committee and conformed with the Code of Practice for Experiments on Animals approved by the National Health and Medical Research Council of Australia.

Granulosa cell culture
The animals were killed on day 25, and ovaries were collected into ice-cold McCoy’s 5A medium (Life Technologies, Melbourne, Australia). GC were harvested into ice-cold McCoy’s 5A medium containing 10 mg/liter deoxyribonuclease by puncturing the ovaries with a fine needle. When obtaining various stages of differentiated GC, 50 U/ml heparin sodium (Multiparin, Fisons, Melbourne, Australia) were added to prevent blood clotting. The viability of the cells was estimated using trypan blue and usually averaged 50–70%. The dispersed cells were plated in 48-well plates (Costar, Cambridge, MA) in McCoy’s 5A medium supplemented with 5 mg/liter transferrin (Sigma), 2 mmol/liter L-glutamine (CSL), and antibiotics (100 U/ml penicillin, 100 pg/ml streptomycin, and 250 ng/ml fungizone; CSL). The cells were then cultured with or without added reagents at a concentration of 1–8 x 105 cells/well for different times, as indicated in the text. The standard culture was carried out at a concentration of 4 x 105 cells/well for 72 h. At the end of culture, medium was collected and stored frozen until the determination of FS and inhibin concentrations. After the collection of media, the number of adherent cells cultured in 48-well plates was assayed using the supravital stain neutral red (3-amino-7-dimethylamino-2-methyl-phenazime hydrochloride; Sigma) to confirm the uniformity of the number of viable cells at the end of culture, as described previously (21).

Total immunoreactive FS ELISA
To measure total ir-FS concentrations in culture medium, an ELISA for rat FS was developed based on a recently developed human FS immunoradiometric assay (18). The antibodies generated against rh-FS 315 were confirmed to bind with both free FS and activin-FS complex equally (18) and, therefore, approximate the measurement of total FS. It was evident that these reagents can react with rat FS protein (22).

Ninety-six-well mictotiter plates (MaxiSorp, Nunc, Roslilde, Denmark) were coated with 100 µl anti-FS monoclonal antibody (4–6D9; 2 mg/liter) in 100 mmol/liter sodium bicarbonate buffer (pH 9.6) at 4 C overnight and blocked with 0.5% Block Ace (Teikoku Zoki Pharmaceutical Co., Tokyo, Japan) for 6 h at room temperature. After three washes with washing buffer (6.5 mmol/liter Tris-0.9% sodium chloride-0.05% Tween-20), 50 µl rh-FS 288 standard solution or sample, and 50 µl anti-FS polyclonal IgG (1:500) diluted in assay buffer (50 mmol/liter PBS, pH 7.4, containing 5 mmol/liter EDTA, 0.1% Tween-20, 0.01% Thimerosal (Sigma) , 1% goat serum, and 1% Block Ace) were incubated overnight at 4 C. After three washes, bound antibody was detected by the following treatments with biotinylated goat antirabbit IgG (H+L) antibody (1:2000; Zymed Laboratories, San Francisco, CA) for 1 h and streptavidin-biotinylated horseradish peroxidase complex (1:1000; Amersham, Aylesbury, UK) for 30 min. After extensive washing, 100 µl ELISA substrate (2 mg/ml O-phenylenediamine in 0.1 mol/liter sodium citrate-citric acid buffer, pH 5.5, containing 0.03% hydrogen peroxide) were added, the color development was stopped after 10 min with 50 µl 2 mol/liter sulfuric acid, and the absorbances at 490 nm were determined with a plate reader.

Free FS ELISA
For the determination of free rat FS in the culture media, an activin-binding assay using an immobilized monoclonal antibody and biotinylated activin A was adapted from an assay for free FS (19). The procedure of this ELISA was similar to that of the total ir-FS ELISA described above, except that another antihuman FS 315 monoclonal antibody (4–17G12) was immobilized on the plates for capturing FS, and biotinylated rh-activin A was used as a detector ligand. The biotinylation of rh-activin A was carried out as follows. Rh-activin A (50 µg) was dissolved in 0.1 mol/liter sodium bicarbonate (pH 8.5), and 3 µl 0.1 mol/liter biotin hydroxysuccinimide (Sigma) dissolved in dimethylsulfoxide was added. After incubation at room temperature for 90 min, 5 mg glycine in 20 µl distilled water were added to stop the reaction, and biotinylated activin A was purified on a PD-10 column (Pharmacia, Uppsala, Sweden).

After plates were coated with 4–17G12 (2 mg/liter) and blocked, 50 µl standard solution or sample, and 50 µl biotinylated rh-activin A (1:150) were incubated overnight at 4 C. After washing, 100 µl streptavidin-biotinylated horseradish peroxidase complex (1:1000) were incubated for 30 min, and the specific signal was generated as described above. As previously documented (19), the FS recovery was dose dependently reduced by the addition of activin, indicating that this system could detect only activin-unbound (free) FS. As treatment with activin A in cultures significantly reduced the free FS recovery and made it difficult to interpret the data, the results from experiments using exogenous activin A were not included in this study.

Inhibin RIA
To monitor GC function, conditioned medium was subjected to an {alpha}-subunit inhibin RIA (Monash assay) using the partially purified rat ovarian inhibin standard, as described previously (23, 24, 25). The sensitivity of the assay was 0.34 U/ml, and the intra- and interassay coefficients of variation (CV) were less than 15%.

Statistical analysis
All values in the figures are expressed as the mean ± SD of three or four replicates. Parallelism between dilution curves was determined by a comparison of the variance associated with the linear regression analysis after a log-log dose transformation. Results from different treatment groups were subjected to one-way ANOVA, and Bonferroni/Dunn’s post-hoc tests were applied to the evaluation of statistical significance. Each experiment was repeated at least twice, and representative examples are shown in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Total ir-FS and free FS ELISA
The standard curve of the total ir-FS ELISA is shown in Fig. 1AGo. The sensitivity of this assay was 0.4 ng rh-FS 288/ml, and the dilution curves of GC culture medium were all parallel to the standard curve regardless of the addition of rh-activin A. This assay did not cross-react with rh-activin A, rh-inhibin A (<0.1%), or rLH and FSH (<0.5%). Intra- and interassay CVs were 5.4% and 12.2%, respectively, and the recovery rates of ir-FS from GC culture medium at 1, 2, and 5 ng/ml were between 87–91%. The addition of various doses of rh-activin A negligibly affected ir-FS recovery (Table 1Go), and ir-FS recovery was conserved over 80% even in the presence of 500 ng/ml rh-activin A. Bovine FS standard was not detectable in this ELISA, but was detectable when antibovine FS antibody was used as a detector ligand (data not shown), suggesting that the anti-hFS polyclonal antibody has some species specificity.



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  		Figure 1. Standard curve of rh-FS 288 and dilutions of rat granulosa cell culture medium (rGCCM) in total immunoactive FS ELISA (A) and free FS ELISA (B). •, Rh-FS 288 standard; {circ}, rGCCM without rh-activin A; {blacksquare}, rGCCM with 20 ng/ml rh-activin A; {square}, rGCCM with 100 ng/ml rh-activin A. Each value represents the mean ± SD of triplicate determinations. A, The slopes of the dilution curves for rGCCM without rh-activin A (slope = 1.36), rGCCM with 20 ng/ml rh-activin A (slope = 1.18), and rGCCM with 100 ng/ml rh-activin A (slope = 1.26) were statistically parallel to the standard curve (slope = 1.21). B, The slope of the dilution curves for rGCCM without rh-activin A (slope = 1.27) and rGCCM with 20 ng/ml rh-activin A (slope = 1.25) were parallel to the standard curve (slope = 1.31). All of the values of the dilution curve for rGCCM with 100 ng/ml rh-activin A added were below the detectable limit of the free FS ELISA.

 

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  		Table 1. Effects of activin A on follistatin recovery in total ir-FS and free FS ELISAs

 
The standard curve of the free FS ELISA is shown in Fig. 1BGo. The sensitivity of this assay was a little less (0.8 ng/ml) than that of the total ir-FS ELISA, and the dilution curve of GC medium was parallel to the standard curve. As shown in Fig. 1BGo and Table 1Go, the addition of rh-activin A dose dependently reduced the recovery of FS, showing that this ELISA can measure only free FS. Surprisingly, the values obtained in this assay were 10–20 times higher than those measured in total ir-FS ELISA, suggesting that the low rat FS concentrations measured in the human FS ELISA are due to species specificity of the antibodies employed. In contrast to the total ir-FS ELISA, bovine FS was reactive in this assay, indicating that the free FS assay is not species specific. No significant cross-reaction with other related peptides was observed. Intra- and interassay CVs were 3.4% and 7.2%, respectively, and the recovery rates of free FS at 2, 5, and 10 ng/ml from GC culture medium averaged 108%.

Secretory profiles of FS and inhibin from granulosa cells; effects of FSH and activin
The time course of FS secretion from cultured rat GC is shown in Fig. 2Go, A and B. FS secreted into the medium became detectable after 1 day of incubation and gradually accumulated with time of culture in both FS assays. Although total ir-FS showed a linear increase during 5 days of culture, free FS reached a plateau around 3 days. Ir-inhibin was also detectable after 1 day of incubation and showed a secretory profile similar to that of free FS (Fig. 2CGo). Basal FS production was generally proportional to GC number after 3 days of culture and was significantly stimulated in the presence of FSH (Figs. 2Go, A and B, and 3, A and B). Ir-inhibin secretion was similarly cell density dependent and responded to FSH treatment (Figs. 2CGo and 3CGo).



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  		Figure 2. Time course of total ir-FS (A), free FS (B), and ir-inhibin (C) secretion from cultured rat GC. GC from DES-treated immature rats (undifferentiated) were cultured in serum-free McCoy’s 5C medium at a cell density of 2 x 105 or 4 x 105 cells/well with or without treatment with 20 ng/ml FSH, and the medium was collected after 1, 2, 3, and 5 days of culture, respectively. {circ}, 2 x 105 cells/well without FSH; •, 2 x 105 cells/well with FSH; {square}, 4 x 105 cells/well without FSH; {blacksquare}, 4 x 105 cells/well with FSH. Values represent the mean ± SD (n = 4). Comparison was performed at each time point, and significant differences between groups are indicated by different letters (P < 0.05).

 


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  		Figure 3. Effects of cell density and FSH on total ir-FS (A), free FS (B), and ir-inhibin (C) secretion from cultured rat GC. Rat GC were cultured in serum-free McCoy’s 5C medium at four different densities from 1 x 105 to 8 x 105 cells/well for 72 h with ({blacksquare}) or without ({square}) 20 ng/ml FSH. Values are expressed as the mean ± SD of four replicates, and significant differences (P < 0.05) between groups are shown by different letters.

 
The addition of FSH to the culture medium resulted in a dose-dependent increase in both total ir-FS and free FS production, with a maximal response at 20 ng/ml (Fig. 4Go, A and B). Activin A alone also stimulated total ir-FS secretion dose dependently (Fig. 5AGo). Ir-inhibin secretion was similarly stimulated dose dependently by FSH and rh-activin A treatment (Figs. 4CGo and 5BGo). FSH in combination with rh-activin A had additive effects on total ir-FS and ir-inhibin secretion from GC (Figs. 6Go and 7Go).



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  		Figure 4. Dose-dependent effects of FSH on total ir-FS (A), free FS (B), and ir-inhibin (C) secretion from cultured rat GC. Rat GC were cultured in serum-free McCoy’s 5C medium at a cell density of 4 x 105 cells/well for 72 h in the presence of FSH (0–40 ng/ml). Values represent the mean ± SD (n = 4). Significant differences between treatment groups are indicated by different letters (P < 0.05).

 


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  		Figure 5. Dose-dependent effects of rh-activin A on total ir-FS (A) and ir-inhibin (B) secretion from cultured rat GC. Rat GC were cultured in serum-free McCoy’s 5C medium at a cell density of 4 x 105 cells/well for 72 h in the presence of rh-activin A (0–100 ng/ml). As the treatment with exogenous activin A interfered with free FS assay, the result of free FS assay is not presented. Values represent the mean ± SD (n = 3). Significant differences between treatment groups are indicated by different letters (P < 0.05).

 


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  		Figure 6. Effects of FSH in combination with rh-activin A on total ir-FS (A) and ir-inhibin (B) secretion from cultured rat GC. Rat GC were cultured in serum-free McCoy’s 5C medium at a cell density of 4 x 105 cells/well for 72 h with various doses of FSH (0–20 ng/ml) in the presence of 20 ng/ml rh-activin A. As the treatment with exogenous activin A interfered with free FS assay, the result of free FS assay is not presented. Values represent the mean ± SD (n = 3). Significant differences between treatment groups are indicated by different letters (P < 0.05).

 


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  		Figure 7. Effects of rh-activin A in combination with FSH on total ir-FS (A) and ir-inhibin (B) secretion from cultured rat GC. Rat GC were cultured in serum-free McCoy’s 5C medium at a cell density of 4 x 105 cells/well for 72 h with various doses of rh-activin A (0–100 ng/ml) in the presence of 5 ng/ml FSH. As the treatment with exogenous activin A interfered with free FS assay, the result of free FS assay is not presented. Values represent the mean ± SD (n = 3). Significant differences between treatment groups are indicated by different letters (P < 0.05).

 
Effects of FK and PMA on FS secretion from granulosa cells
The effects of FK and PMA on FS secretion were investigated in a time-course study (Fig. 8Go). Treatment with 10 mmol/liter FK or 100 nmol/liter PMA similarly stimulated both ir-FS and free FS secretion from GC (Fig. 8Go, A and B). The effect was apparent at 24 h, and the accumulated FS continued to increase until the end of culture (72 h). On the other hand, ir-inhibin secretion was stimulated with FK, but suppressed in the presence of PMA (Fig. 8CGo).



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  		Figure 8. Effects of FK and PMA on total ir-FS (A), free FS (B), and ir-inhibin (C) secretion from cultured rat GC. Rat GC were cultured in serum-free McCoy’s 5C medium at a cell density of 4 x 105 cells/well for 72 h in the presence of FK or PMA. {circ}, Control culture; {blacksquare}, 10 mmol/liter FK; {blacksquare}, 100 nmol/liter PMA. Values represent the mean ± SD (n = 3). Comparison was performed at each time point, and significant difference between treatment groups are indicated by different letters (P < 0.05).

 
Effects of degree of granulosa cell differentiation on FS secretion
The effects of GC differentiation on FS and inhibin secretion are shown in Fig. 9Go. The basal secretion of total ir-FS and free FS was remarkably accelerated with increasing GC differentiation and showed maximal production from fully differentiated GC. Both total ir-FS and free FS secretion from undifferentiated and partially differentiated GC were stimulated with FSH, but no further stimulation occurred in fully differentiated GC. Activin A also showed a stimulatory effect on total ir-FS secretion from undifferentiated GC and had no effect on partially differentiated cells, but, conversely, inhibited total ir-FS production from fully differentiated GC. Although LH did not show any effect on total ir-FS and free FS secretion in undifferentiated and fully differentiated GC, partially differentiated GC showed a stimulatory response to LH in both FS assays.



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  		Figure 9. Effects of FSH, LH, and rh-activin A on total ir-FS (A), free FS (B), and ir-inhibin (C) secretion from cultured rat GC obtained at various stages of differentiation. Rat GC were collected from immature female rats with different in vivo treatments (see Materials and Methods) and cultured in serum-free McCoy’s 5C medium at a cell density of 4 x 105 cells/well for 72 h in the presence or absence of FSH (20 ng/ml), LH (20 ng/ml), or rh-activin A (100 ng/ml). Values represent the mean ± SD (n = 3 or 4), and significant differences (P < 0.05) between groups are shown by different letters. *, As the treatment with activin A interfered with the free FS assay, the free FS level in the presence of 100 ng/ml rh-activin A became undetectable.

 
In contrast, basal ir-inhibin secretion from GC did not increase with increasing GC differentiation and even decreased in fully differentiated GC (Fig. 9CGo). The stimulatory effect of FSH was observed only in undifferentiated GC, not in partially or fully differentiated GC. Activin A had a stimulatory effect on ir-inhibin secretion at all stages of GC differentiation, but LH did not cause any significant change in ir-inhibin secretion at any stage.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we applied two different ELISAs to detect total ir-FS and free FS, respectively, to examine FS protein secretion into the medium from cultured rat GC. FS protein production from rat GC increased with cell number and time of culture, and was stimulated dose dependently by FSH and activin A. Furthermore, FS secretion from GC was stimulated by FK and PMA, and increased significantly with the advance of GC differentiation.

The total ir-FS and free FS assays were both sufficiently sensitive (0.4 and 0.8 ng/ml equivalents of rh-FS 288 standard, respectively) and specific to FS in medium conditioned by rat GC, and dilutions of the conditioned medium were parallel to the standard. In the absence of pure rat FS standards, it was not possible to determine the cross-reactivity of rat FS in the total ir-FS assay, but it is likely that using rh-FS 288 as standard in this assay underestimates the total FS present in the medium, based on the relative levels of free FS measured in the binding assay, which were 20-fold higher. The availability of rat FS standard may well change the absolute levels of FS measured in the total ir-FS assay, but it should not alter the patterns of response in view of the parallelism between dilutions of conditioned medium and the rh-FS 288 standard. The addition of exogenous activin at concentrations above 20 ng/ml to GC cultures significantly interfered with the free FS assay, so data from these experiments are not included.

The forms of FS protein identified in rat ovary and released by rat GC in culture range from 32–38 kDa (1), but the relative contribution of each form to the total ir-FS and free FS measured in these assays is not known. All of the forms identified to date have equal activin-binding capacities (9, 10), but differ in their heparin- and GC-binding capacities, with the truncated FS 288 form having a higher affinity than the other forms. Given this and the predominance of the FS 344 mRNA over the FS 317 mRNA in rat ovary (12) and GC (13), it seems likely that most of the FS protein detected by these assays will be glycosylated forms of FS 315 and 300.

The data show that rat GC are a major site of production of total ir-FS and free FS in vitro, which is both time and cell number dependent and is similar to patterns obtained using the affinity gel binding assay (15). Furthermore, FSH had time- and dose-dependent stimulatory effects on total ir-FS and free FS production by undifferentiated GC, similar to previous findings for FS mRNA and protein in rat (13, 14) and bovine GC (17), supporting the proposal that increases in FS mRNA lead to increases in FS protein. Under our culture conditions using 400,000 cells/well, maximal production of both total ir-FS and free FS occurred after 72 h in the presence of 20 ng/ml FSH. GC obtained from rats treated with FSH for 2 days in vivo (partially differentiated GC) produced more total ir-FS and free FS in response to LH in vitro than controls, whereas LH did not stimulate FS production by undifferentiated or fully differentiated GC above control levels. LH had no detectable effect on FS mRNA levels in undifferentiated rat GC (13), although to our knowledge the effects of LH on FS mRNA at later stages of GC differentiation have not been examined. Overall, this indicates that FS production is primarily FSH dependent, but during the early antral phase of folliculogenesis, FS production by GC may also be LH sensitive, coinciding with the first detection of FS protein in GC of tertiary follicles (11). FS production by fully differentiated GC was maximal and was not stimulated further by FSH or LH treatment.

The data demonstrate for the first time that activin had dose-dependent stimulatory effects on total ir-FS and had additive effects with FSH on FS production by undifferentiated GC. Activin also stimulated total ir-FS production by partially differentiated GC, but inhibited production by fully differentiated GC. This is similar to the changing effects of activin on progesterone production by rat GC in vitro (20). It also underlines the capacity of activin to regulate the levels of its own binding protein and, hence, its biological effectiveness, which had been foreshadowed by similar effects of activin on steady state levels of FS mRNA in rat GC (13, 14).

The fact that FK and PMA both stimulated total ir-FS and free FS production by GC shows that the regulation of FS production can be via the protein kinase A and C pathways. These effects on FS protein mimic the influence of FK and PMA on steady state levels of FS mRNA in GC (14), but with a later time course. Although FSH is likely to act through cAMP and the protein kinase A pathway, it may also act via a non-cAMP-dependent pathway or by direct activation of ion channel(s) (26), or via production of activin (27, 28, 29). This is compatible with the previous studies demonstrating that activin subunit mRNA expression in GC is stimulated by treatment with FSH in vitro (27, 28) and in vivo (29). However, there is also a recent report (30) demonstrating that activin dimer production measured by double ligand blotting is stimulated by PMA, but not by FSH or FK. The mechanisms by which activin influences basal FS production are not known; it could facilitate the effect of FSH on FS by up-regulating the FSH receptor (31, 32, 33).

We used the production of ir-inhibin as a marker of GC function, as previously described in this laboratory (23, 24, 25). The RIA detects primarily inhibin forms containing the {alpha}-subunit and is not specific for dimeric inhibin. The patterns of production of ir-inhibin in these experiments confirmed previous work (23, 24, 25, 34, 35) and mirrored the production of FS protein with two exceptions. Whereas PMA stimulated FS production, it inhibited ir-inhibin production by undifferentiated GC, generally in accordance with previous reports (34, 35). Whereas basal FS production increased with increasing GC differentiation, basal ir-inhibin production by fully differentiated GC decreased, and activin inhibited FS and stimulated ir-inhibin production by these cells. This could represent a change in the forms of inhibin produced by GC that are detected by the inhibin RIA. It could also indicate differential regulation of these proteins at different stages of GC development.

An important observation was the increasing levels of total ir-FS and free FS with increasing degrees of GC differentiation, with fully differentiated cells showing no further response to FSH or LH stimulation. This would be consistent with the hypothesis of Hillier (36) that there is a decreasing activin "tone" within follicles as they develop, due in this case to higher levels of FS. The capacity of GC to increase FS production in vitro over 3-fold as they become more differentiated has not been reflected in the circulating concentrations of FS in sheep or humans during the late follicular phase (19, 37, 38), although FS concentrations were reported to rise after GnRH/gonadotropin treatment of women to stimulate folliculogenesis (39). This suggests that the increased capacity of GC to produce FS is related to its paracrine role in folliculogenesis.

In summary, this study demonstrated for the first time that FS protein secretion from cultured undifferentiated rat GC is up-regulated by FSH and activin, possibly via both protein kinase A and C pathways, and that increasing GC differentiation is associated with a significant increase in basal FS production from rat GC and a change in hormonal regulation. These findings generally support the hypothesis that activin tone within follicles decreases with follicular development due to increased production of FS, but the exact roles and regulatory mechanisms of activin and FS during folliculogenesis remain to be studied further.


   Acknowledgments
 
We thank Dr. Yuzuru Eto (Ajinomoto Central Research Laboratories, Kawasaki, Japan) for providing rh-FS 315, Drs. Anthony J. Mason and David M. Robertson (Prince Henry’s Institute of Medical Research, Clayton, Australia) for rh-activin A and bovine FS and its antibody, respectively, Dr. Masahiro Abe (First Department of Internal Medicine, University of Tokushima School of Medicine, Tokushima, Japan) for anti-FS monoclonal antibodies, and Mrs. Faye Coates for assistance with the manuscript.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council of Australia (Program Grant Regkey 943208) and a Grant-in-Aid for Scientific Research (no. 04772049) from the Ministry of Education, Science, and Culture of Japan. Presented in part at the Serono Symposium on Inhibin, Activin, and Follistatin–Recent Advances and Future Views, Tokushima, Japan, 1996. Back

2 Present address: First Department of Internal Medicine, University of Tokushima School of Medicine, 3–18-15 Kuramoto-cho, Tokushima 770, Japan. Back

Received November 25, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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