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Endocrinology Vol. 138, No. 11 4783-4791
Copyright © 1997 by The Endocrine Society

ARTICLES

Stimulating Effect of Both Human Recombinant Inhibin A and Activin A on Immature Porcine Leydig Cell Functions in Vitro1

Hervé Lejeune, Franck Chuzel, Pascale Sanchez, Philippe Durand, Jennie P. Mather and José M. Saez

INSERM-INRA U-418 and IFR D’Endocrinologie, Hôpital Debrousse (H.L., F.C., P.S., P.D., J.M.S.), 69322 Lyon; and Clinique Endocrinologique, Hospices Civils de Lyon, Hôpital de l’Antiquaille (H.L.), 69321 Lyon, France; and Genentech (J.P.M.), South San Francisco, California 94080

Address all correspondence and requests for reprints to: Dr. J. M. Saez, INSERM-INRA U-418, Hôpital Debrousse, 69322 Lyon, France. E-mail: pdurand{at}univ-lyon1.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to the regulation of FSH secretion, it has been clearly shown that inhibin and activin have paracrine/autocrine effects in the gonads. We have studied the effect of human recombinant inhibin A and human recombinant activin A on immature porcine Leydig cells in vitro. Leydig cells were prepared by collagenase digestion of testes from 3-week-old piglets, purified on Percoll gradient, then cultured in a chemically defined medium. The cells were treated with increasing amounts of inhibin A or activin A (0.5–200 ng/ml). Direct application of either inhibin A or activin A on Leydig cells for 4 or 48 h did not stimulate basal testosterone secretion. Conversely, treatment of the cells for 48 h with either factor resulted in a dose-dependent increase in hCG-stimulated testosterone secretion (10-9 M hCG, 2 h) with a maximal effect of 2.40 ± 0.37- and 2.43 ± 0.37-fold increases for inhibin A and activin A, respectively, and these changes were associated with a slight increase in LH/hCG-binding sites (1.37 ± 0.19- and 1.24 ± 0.11-fold increases). In addition, both inhibin A and activin A enhanced messenger RNA (mRNA) levels of LH/hCG receptor (2.75 ± 0.40- and 2.53 ± 0.60-fold increases) and cytochrome P450 17{alpha}-hydroxylase (6 ± 1- and 3.5 ± 0.6-fold increases), but had no effect on side-chain cleavage cytochrome P450 or cytochrome P450 aromatase mRNAs. 3ß-Hydroxysteroid dehydrogenase mRNA levels were increased (3.1 ± 1.3-fold increase) by activin A, but not by inhibin A. However, inhibin A blocked the stimulatory action of activin A. In keeping with these changes in the steroidogenic enzyme mRNAs, both peptides enhanced the conversion of exogenous 22R-hydroxycholesterol and progesterone, but only activin A increased the conversion of dehydroepiandrosterone into testosterone. In conclusion, our findings demonstrate that both inhibin A and activin A have a stimulatory effect on immature porcine Leydig cell differentiated function in vitro. As inhibin has a stimulatory and activin has an inhibitory effect on rat Leydig cell function in vitro, the effects of these factors on Leydig cells seem to be species dependent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INHIBIN and activin are biochemically related proteins composed of two subunits joined by disulfide bonds (1). Inhibins are heterodimers composed of an {alpha}-subunit and one of the two homologous, but distinct, ß-subunits (ßA or ßB), whereas activins are homodimers of the ß-subunits. Both proteins belong to the transforming growth factor-ß (TGFß) superfamily, which includes TGFß, anti-Mullerian hormone, bone morphogenic proteins, the product of the drosophila decapentaplegic gene complex, and the vg1 gene product of Xenopus (2). Inhibin/activin subunits are expressed in a variety of tissues, including ovary (3), testis (4), placenta, adrenal, kidney, brain, and pituitary gland (5, 6). In addition to their opposite effects on FSH secretion, inhibins and activins have been reported to regulate diverse physiological functions, including ACTH and GH secretion, neuronal survival, hypothalamic oxytocin secretion, erythropoiesis, early embryonic development (1), and gonadal functions (7).

In rodents (7) as well as in nonhuman primate and man (8), there is evidence for the presence of inhibin/activin subunits ({alpha}, ßA, and ßB) in several testicular cell types, including Leydig and Sertoli cells, although, at least in rodents, the relative expression levels and the pattern of expression appear to change throughout sexual development and during the stage of the seminiferous cycle (9, 10, 11). In situ ligand binding studies have shown that [125I]inhibin A binds specifically to Leydig cells, whereas [125I]activin-binding sites were located in the basal compartment of the seminiferous tubules regardless of the age of the animals as well as on round spermatids in stages VII–VIII of the seminiferous tubule (12). Moreover, activin type II receptor (ActRII) messenger RNA (mRNA) is expressed in Sertoli cells and at a lower level in Leydig cells (13). In situ hybridization with an ActRIIB probe revealed localization in Leydig cells (14).

Further studies have shown that inhibins and activins act as regulators of testicular functions. Using staged segments of rat seminiferous tubules, it has been shown that activin increased DNA synthesis in preleptotene spermatocytes and intermediate spermtogonia, whereas inhibin decreased DNA synthesis in these spermatogonia (15). Similarly, using a coculture of immature rat germ cells-Sertoli cells, activin stimulated cellular aggregation and spermatogonial proliferation (16). Although inhibin produced no detectable effect in this system, in an earlier study it was shown that intratesticular injection of inhibin reduced spermatogonial number in adult mice and Chinese hamster (17). Controversial data have been reported concerning the effects of inhibin and activin on Leydig cell steroidogenesis (18, 19, 20, 21, 22).

In the present work, using purified immature Leydig cells, we have studied the effects of recombinant inhibin A and activin A on hCG-induced testosterone production as well as on the expression of several genes involved in Leydig cell-specific functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human recombinant inhibin A and human recombinant activin A were produced by Genentech (South San Francisco, CA) as previously described (23). They were handled in polypropylene tubes and kept at 4 C as a 50 µg/ml solution in NaCl-Tris buffer; thus, successive freezing-thawings were avoided. They were diluted in the medium just before use. The hCG (batch CR-125; 13,000 IU/mg) used for iodination was a gift from Dr. R. E. Canfield (New York, NY). hCG used to stimulate testosterone production was obtained from Organon (Paris, France). BSA, deoxyribonuclease, soybean trypsin inhibitor, transferrin, insulin, vitamin C, vitamin E, 22R-hyroxycholesterol, progesterone, and dehydroepiandrosterone were purchased from Sigma Chemical Co. (St. Louis, MO). Ham’s F-12, DMEM, penicillin-streptomycin, nystatin, and trypsin-EDTA solution were purchased from Life Technologies (Grand Island, NY); collagenase was obtained from Serva (Heidelberg, Germany); Percoll and Ultrogel ACA 54 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden); and Iodogen was obtained from Pierce Chemical Co. All chemicals were of analytical grade.

Leydig cell preparation and culture
Leydig cells were isolated from 3-week-old pig testes as previously described (24). Briefly, piglet testes (3–4 weeks) were obtained fresh from a local farm and placed immediately in ice-cold Ham’s F-12 and DMEM (1:1) supplemented with 1.2 mg/ml sodium bicarbonate, 16 µg/ml gentamicin, 10 µg/ml penicillin, 100 U/ml streptomycin, 0.1% nystatin, 8 µg/ml fortum, and 15 mM HEPES, pH 7.4 (F12/DME). The testes were decapsulated, minced with a razor blade, and incubated under agitation for 90 min at 33 C in F12/DME containing 750 µg/ml collagenase, 1 µg/ml trypsin inhibitor, and 10 µg/ml deoxyribonuclease. The dispersed cells were filtered through a 100-µm nylon screen and pelleted by centrifugation at 180 x g for 5 min at 4 C. The cell pellet was resuspended, and two successive sedimentations of 5 min and 30 min, respectively, were performed to grossly eliminate the seminiferous tubules. The crude suspension of Leydig cells was recovered, and the final pellets were pooled and resuspended to apply 108 to 1.5 x 108 cells on a 32-ml discontinuous Percoll density gradient (density = 1.032, 1.040, 1.048, 1.057, and 1.082 g/ml) and centrifuged at 1250 x g for 30 min at 4 C. The cells recovered on Percoll gradient with a density between 1.057–1.082 were collected, washed in medium, and counted in a Coulter counter (model ZBI, Coulter Electronics, Hialeah, FL). Their viability was measured by trypan blue exclusion. More than 90% of cells were viable, and between 80–90% of cells were identified as Leydig cells by cytochemical staining for 3ß-hydroxysteroid dehydrogenase {Delta}5-{Delta}4-isomerase (3ßHSD) (25).

The Leydig cells were plated in 12- or 6-multiwell culture dishes at a concentration of 1 or 2 x 106 cells/well, respectively, and cultured in a controlled humidified atmosphere of 5% CO2 in air in HEPES-buffered F12/DME supplemented with 0.2% FCS, insulin (10 µg/ml), transferrin (10 µg/ml), vitamin C (10-4 M), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1% nystatin for 72 h to allow the cells to attach. The medium was removed and replaced by fresh medium without FCS for 24 h. Then inhibin A, activin A, or both were diluted in fresh medium and applied to the cells at concentrations ranging from 0.5–200 ng/ml. Cells cultured in medium without inhibin A and activin A were used as controls.

Leydig cell functions
hCG binding assay.
Purified hCG was radioiodinated with Na125I by the Iodogen method and separated by gel filtration chromatography on a column of Ultrogel ACA 54 equilibrated with 0.1 M phosphate buffer, pH 7.4, containing 0.2% BSA to separate the labeled hormone from free iodine. The specific activity of the biologically active fraction of [125I]hCG was approximately 100–150 µCi/µg. Cultured Leydig cells were washed with cold saline (0.9% NaCl and 0.2% BSA). The binding assays were performed by adding [125I]hCG for 4 h at 33 C. Then, the cells were cooled to 4 C, and the binding reaction was terminated by discarding the medium. The cells were then washed three times with ice-cold saline and dissolved in 0.5 M NaOH-0.4% sodium deoxycholate. The radioactivity was measured in a {gamma}-counter with 75% efficiency. Specific binding was determined by subtracting from the total binding the radioactivity associated with cells incubated in the presence of a large excess of cold hormone (3 IU; Organon). The specific binding is expressed as counts per minute bound per mg protein.

Testosterone measurement.
Testosterone was measured in the medium by RIA as previously described (26). Testosterone production was expressed as nanograms per 106 cells.

Northern blot analysis.
After 48 h of treatment, the culture medium was removed, and total RNA was extracted following a modification of the method of Chomczynski and Sacchi (27) as previously described (28). Total cytoplasmic RNAs (20 µg) were denatured by heating (95 C for 2 min), resolved by electrophoresis through 1% agarose gel containing 10% formaldehyde, and transferred onto Hybond-N nylon membrane (Amersham France, Les Ulis, France). RNAs were then cross-linked to the membranes by irradiating for 2 min with UV light and baking at 80 C for 2 h. Prehybridization was performed for at least 2 h at 42 C in 50% formamide, SSPE (0.75 M NaCl; 20 mM NaPO4, pH 7.5; and 1 mM EDTA), 5 x Denhardt’s solution (0.1% Ficoll 400, 0.1% polyvinylpyrolidone, and 0.1% BSA), 0.1% SDS, 10% dextran sulfate, and 100 mg/ml denatured salmon sperm DNA. Hybridization was carried out overnight at 42 C in the same prehybridization buffer containing 106 dpm/ml 32P-labeled probe. The blots were hybridized with the following probes, labeled to a specific activity of 109 dpm/µg DNA, with [{alpha}-32P]deoxy-CTP by the random priming method (Megaprime DNA labeling kit, Amersham France): porcine LH/hCG receptor complementary DNA [cDNA; 2.1 kilobases (kb); gift from E. Milgrom] (29), human 3ßHSD cDNA (1.5 kb; gift from F. Labrie) (30), bovine cytochrome P450 17{alpha}-hydroxylase (P450c17) cDNA (1.8 kb) (31), bovine side-chain cleavage cytochrome P450 (cytochrome P450scc) cDNA (0.9 kb; gifts from M. Waterman) (32), and human cytochrome P450 aromatase cDNA (2.5 kb; gift from E. Simpson) (33). Nylon membranes were washed twice in 2 x SSC (1 x SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.4)-0.1% SDS at room temperature for 15 min each, then twice in 1 x SSC-0.1% SDS at 65 C for 15 min each time, once in 0.5 x SSC-0.1% SDS at 65 C for 15 min, and once in 0.1 x SSC-0.1% at 65 C for 5 min. Autoradiographs were obtained after a 4- to 8-day exposure at -70 C to Hyperfilm MP (Amersham) with intensifying screens. Autoradiographs and 28S RNA ethidium bromide fluorescence photographs were submitted to densitometry scanning and analyzed with the software NIH-IMAGE. For LH/hCG mRNAs, regulation was similar for the bands of 6.7, 4.7 4.0, 2.6, and 1.4 kb (34); data are presented as the averaged values for the five bands.

Statistical analysis
All results for testosterone production and hCG binding sites were normalized by the number of cells at the end of each experiment. The dose-response relationships were studied according to a linear regression model of the fold increase relative to the control condition on the decimal logarithm of the dose of inhibin A or activin A. The ED50 values were determined from the inverse linear regression of the decimal logarithm of the dose on the fold increase. Comparisons between control and inhibin A- or activin A-treated cells were performed by multifactorial ANOVA, taking into account the different independent cultures and the experimental conditions. Comparisons of the amplitudes of the maximal effects obtained with inhibin A, activin A, or both together were performed by multifactorial ANOVA followed by Bonferroni-Dunn posttest. P < 0.05 was considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct application of either inhibin A (up to 200 ng/ml) or activin A (up to 200 ng/ml) on Leydig cells for 4 or 48 h did not stimulate testosterone secretion or cell multiplication evaluated by [3H]thymidine incorporation during the last 4 h of culture (data not shown). Treatment of the cells for 48 h with either inhibin A or activin A resulted in a dose-dependent increase in acute hCG-stimulated testosterone secretion for both inhibin A (r = 0.672; P < 0.0001) and activin A (r = 0.634; P < 0.0001; Fig. 1Go). The maximal effect, obtained with the 100 ng/ml dose for inhibin A or activin A, was, respectively, 2.40 ± 0.37- and 2.43 ± 0.37-fold increases (mean ± SEM); the difference was not significant (P = 0.9136). The ED50 was 10.5 and 8.5 ng/ml for inhibin A and activin A, respectively.



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  		Figure 1. Dose-response effects of human recombinant inhibin A and activin A on hCG-induced testosterone secretion by immature porcine Leydig cells. The chronological design of the study is indicated in the upper panel. Testosterone was measured in triplicate for three culture wells for each condition in each independent culture. Results expressed as the fold increase over the control value (without inhibin A or activin A; 8.75 ± 1.22 ng/2 h·106 cells) are the mean ± SEM of three to eight independent cultures.

 
To determine the mechanism by which inhibin A and activin A enhanced hCG-stimulated testosterone production, the effects of both proteins on specific Leydig cell functions were analyzed. LH/hCG-binding sites were increased in a dose-dependent manner (0.5–200 ng/ml) by both inhibin A (r = 0.621; P < 0.0001) and activin A (r = 0.524; P = 0.0006; Fig. 2Go). The maximal effects caused by inhibin A and activin A were 1.37 ± 0.19- and 1.24 ± 0.11-fold increases (mean ± SEM), respectively (P = 0.118 between both peptides). This effect on LH/hCG receptor number was associated with a parallel dose-dependent increase in LH/hCG receptor mRNA levels (Fig. 3Go; inhibin A: r = 0.648; P = 0.0003; activin A: r = 0.495; P = 0.0063). The amplitude of this effect was 2.75 ± 0.4- and 2.53 ± 0.6-fold increases (mean ± SEM) for inhibin A and activin A, respectively, values that are similar (P = 0.7383). Half-maximum stimulation of hCG-induced testosterone production, LH/hCG receptor number, and mRNA was obtained with 11.7 ± 1.6 and 10.6 ± 1.1 ng/ml inhibin A and activin A, respectively.



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  		Figure 2. Dose-response effects of human recombinant inhibin A and activin A on [125I]hCG binding to immature porcine Leydig cells. The chronological design of the study is indicated in the upper panel. Specific [125I]hCG bound to the cells was measured in three culture wells for each condition in each independent culture. Results expressed as the fold increase over the control value (without inhibin A or activin A) are the mean ± SEM of three independent cultures.

 


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  		Figure 3. Dose-response effects of human recombinant inhibin A and activin A on LH/hCG receptor mRNA levels in immature porcine Leydig cells. Percoll-purified immature porcine Leydig cells were treated for 48 h with inhibin A or activin A at the indicated doses before total RNA extraction. In the upper panel, results expressed as the fold increase over the control value (without inhibin A or activin A) are the mean ± SEM of three independent cultures. Autoradiographs of Northern blots of one representative experiment are presented in the lower panel.

 
In addition to these effects on LH/hCG receptor, both peptides positively regulated the expression of some ste-roidogenic enzymes. Thus, inhibin A and activin A increased in a dose-dependent manner P450c17 mRNA levels (inhibin A: r = 0.82; P < 0.0001; activin A: r = 0.689; P = 0.0001; Fig. 4Go). Half-maximal effects were observed at 3.7 and 3.5 ng/ml, respectively. However, the maximal effect of inhibin A (6 ± 1-fold increase) was significantly higher than that produced by activin A (3.5 ± 0.6-fold increase; P = 0.003). In contrast, activin A increased dose-dependently 3ßHSD mRNA levels (r = 0.55; P = 0.007), with a maximal effect of a 3.1 ± 1.3-fold increase, whereas inhibin A had no effect (Fig. 5Go). Neither inhibin A nor activin A had any effect on P450scc or P450 aromatase (Fig. 6Go).



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  		Figure 4. Dose-response effects of human recombinant inhibin A and activin A on cytochrome P450c17 mRNA levels in immature porcine Leydig cells. Percoll-purified immature porcine Leydig cells were treated for 48 h with inhibin A or activin A at the indicated doses before total RNA extraction. In the upper panel, results expressed as the fold increase relative to the control condition (without inhibin A or activin A) are the mean ± SEM of three to six independent cultures. Autoradiographs of Northern blots of a representative experiment are presented in the lower panel.

 


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  		Figure 5. Dose-response effect of human recombinant inhibin A and activin A on 3ßHSD mRNA levels in immature porcine Leydig cells. Percoll purified immature porcine Leydig cells were treated for 48 h with inhibin A or activin A at the indicated doses before total RNA extraction. In the upper panel, results expressed as the fold increase over the control value (without inhibin A or activin A) are the mean ± SEM of three to five independent cultures. Autoradiographs of Northern blots of one representative experiment are presented in the lower panel.

 


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  		Figure 6. Cytochrome P450scc (upper panel) and cytochrome P450 aromatase (lower panel) mRNA levels in immature porcine Leydig cells after treatment with human recombinant inhibin A and activin A. Percoll-purified immature porcine Leydig cells were treated for 48 h with inhibin A or activin A at the indicated doses before total RNA extraction. Autoradiographs of Northern blots of one representative experiment of three independent cultures are shown.

 
To verify that changes in steroidogenic enzyme mRNA levels were associated with changes in steroidogenic enzyme activities, conversion of several precursors into testosterone was studied after treatment of Leydig cells with inhibin A or activin A for 48 h. As shown in Fig. 7Go, pretreatment of the cells with both inhibin A (100 ng/ml) and activin A (100 ng/ml) enhanced the conversion of exogenous 22R-hydroxycholesterol (P = 0.001 for inhibin A; P = 0.0001 for activin A) and progesterone (P = 0.016 for inhibin A; P < 0.0001 for activin A) into testosterone. The conversion of dehydroepiandrosterone into testosterone was significantly enhanced by activin A (P = 0.007), but not by inhibin A (P = 0.09).



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  		Figure 7. Effects of human recombinant inhibin A (100 ng/ml) and activin A (100 ng/ml) on 22R-hydroxycholesterol (10-5 M)-, progesterone (10-5 M)-, or dehydroepiandrosterone (10-5 M)-supported testosterone secretion by immature porcine Leydig cells. The chronological design of the study is indicated in the upper panel. Testosterone was measured in triplicate for three culture wells for each condition in each independent culture. Results expressed as the fold increase over the control value (without inhibin A or activin A) are the mean ± SEM of two independent cultures. *, P < 0.05. The amounts of testosterone produced by control cells incubated with 22R-hydroxycholesterol, progesterone, and dehydroepiandrosterone were 7.65 ± 1.37, 15.18 ± 1.45, and 77.50 ± 5.75 ng/2 h·106 cells (mean ± SEM), respectively.

 
As in some systems inhibin antagonizes the effects of activin, we investigated whether this was the case in our model. When the cells were treated with inhibin A and activin A together at doses that induced a maximal effect (Fig. 8Go), hCG-induced testosterone secretion (mean ± SEM, 3.41 ± 0.54-fold increase) was higher than the values obtained with inhibin A alone (2.40 ± 0.27-fold increase; P < 0.0001) or activin A alone (2.45 ± 0.35-fold increase; P < 0.0001). The maximal effect was not different for [125I]hCG-binding sites with inhibin A and activin A alone or together. LH/hCG receptor mRNA levels were slightly higher when the cells were treated with both inhibin A and activin A together (2.39 ± 0.25-fold increase) than with when they were treated with inhibin A alone (2.15 ± 0.28-fold increase) or activin A alone (2.02 ± 0.12-fold increase); however, these differences did not reach significance (by ANOVA, P = 0.14). Cytochrome P450c17 mRNA levels were significantly higher with both inhibin A and activin A together (6.24 ± 0.60-fold increase) than with activin A alone (3.0 ± 0.22-fold increase; P < 0.0001) and were slightly higher than with inhibin A alone (5.40 ± 0.91-fold increase); however, this latter difference did not reach significance (P = 0.08). For 3ßHSD mRNA levels, although inhibin A alone had no effect in dose-response experiments, it had an antagonistic effect, preventing the increase in 3ßHSD mRNA levels induced by activin A. The values obtained with inhibin A and activin A together (1.24 ± 0.16-fold increase) were clearly lower than those obtained with activin A alone (2.43 ± 0.28; P = 0.0001) and sightly higher than those with inhibin A alone (0.82 ± 0.8-fold increase); nevertheless, this latter difference did not reach significance (P = 0.11).



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  		Figure 8. Maximal effects of human recombinant inhibin A and activin A, alone or together, on immature porcine Leydig cell functions. Percoll-purified immature porcine Leydig cells were treated for 48 h with inhibin A, activin A, or both, each at a dose that induced the maximal effect in the dose-response experiments (200 ng/ml). Results expressed as the fold increase over the control value (without inhibin A or activin A) are the mean ± SEM of three independent cultures. A, hCG-induced testosterone secretion; B, [125I]hCG binding; C, LH/hCG receptor mRNA levels and autoradiographs of Northern blots of a representative experiment; D, cytochrome P450c17 mRNA levels and autoradiographs of Northern blots of a representative experiment; E, 3ßHSD mRNA levels and autoradiographs of Northern blots of a representative dose-response experiment. *, P < 0.05. ns, Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to regulating FSH secretion at the pituitary level, inhibins and activins appear to be involved in a wide range of biological processes in both reproductive (7) and nonreproductive tissues (1). Because both peptides are expressed not only in the gonad but also in other tissues (5, 6), their effects might be exerted through either endocrine or paracrine/autocrine models.

Leydig cell differentiated functions are regulated by several growth factors through endocrine, paracrine, and autocrine mechanisms (35). This study was designed to address the involvement of both inhibin and activin as potential autocrine/paracrine regulators of Leydig cell functions, using Percoll-purified immature porcine Leydig cells in primary culture and recombinant human inhibin A and activin A. Neither inhibin A nor activin A had a direct steroidogenic effect in either short or long term incubations, but both induced a reproducible dose-dependent enhancement of acute hCG-induced testosterone secretion after 48 h of treatment. The amplitude of this effect and the dose giving half-maximum stimulation were similar for both factors. This enhancement of Leydig cell responsiveness to LH/hCG appeared to result from a specific pattern of regulation of Leydig cell-specific gene expression. Inhibin A and activin A induced a dose-dependent stimulation of LH/hCG receptor-binding sites and mRNA levels, with similar amplitude and half-maximum stimulatory dose for both factors. The pattern of regulation of steroidogenic enzyme gene expression appeared to be more complex. Both inhibin A and activin A enhanced cytochrome P450c17 mRNA levels, although the amplitude of this effect was higher for inhibin A than for activin A. The regulation of 3ßHSD gene expression appeared to be even more factor specific. Activin A stimulated 3ßHSD mRNA levels; inhibin A alone had no effect, but inhibited the stimulatory effect of activin A when both factors were added together. In contrast, cytochrome P450scc and aromatase mRNAs were not modified by inhibin A or activin A. These results suggest that inhibin and activin must be considered in the list of the potential regulators of Leydig cell differentiated function. Table 1Go summarizes the effects of inhibin A and activin A along with several other growth factors on some markers of immature porcine Percoll-purified Leydig cell differentiated functions as observed in vitro with the same conditions of culture in our laboratory (36, 37). Thus, inhibin A, activin A, and insulin-like growth factor I (IGF-I) enhanced Leydig cell steroidogenic responsiveness, LH/hCG receptor number, and mRNA and P450c17 mRNA levels, whereas TGFß, epidermal growth factor, and basic fibroblast growth factor had opposite effects. The amplitudes of the effects of inhibin A and activin A on steady state P450c17 mRNA levels were as high or even higher (for inhibin A) than those induced by hCG (37) or IGF-I (2- to 3-fold increase for both) (36). Although all of these factors regulate, positively or negatively, P450c17 mRNA levels, the regulation of the other steroidogenic enzymes appeared to be more factor specific, i.e. P450scc and 3ßHSD mRNA levels were increased by IGF-I and activin A, respectively, but not by the other growth factors studied.


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  		Table 1. Summary of the effects of inhibin A, activin A, and growth factors on some markers of immature porcine Leydig cell differentiated functions in vitro

 
Contradictory results have been reported concerning the effects of inhibin and activin on Leydig cell functions. Using inhibin purified from ovine rete testis fluid, activin purified from porcine follicular fluid, and mixed testis cell cultures from hypophysectomized rats, it has been reported that inhibin increased, but activin decreased, LH-stimulated testosterone production; neither factor affected basal testosterone production (18). In contrast, other studies using cultures of purified Leydig cells from intact adult rats have reported either no effect of either peptide on basal and LH-stimulated testosterone production (20) or an inhibition by purified activin; this effect was reversed by purified inhibin (19). In the R2C rat Leydig tumor cell line, recombinant activin A decreased progesterone secretion, whereas inhibin purified from ovine rete testis had no effect (22). In another study using cultured purified immature pig Leydig cells, it has been reported that human recombinant activin A had no effect (after 48 h of treatment) or increased (after 72 h of treatment) hCG-induced testosterone production, reduced hCG-stimulated dehydroepiandrosterone, and increased the conversion of exogenous pregnenolone and dehydroepiandrosterone to testosterone (21). The reasons for the discrepancies between these results and ours are unknown, but can be related to interspecies differences, differences in the purity and/or maturation of the Leydig cells used, and differences in the origin (extractive or recombinant) of the peptides used. Careful handling of recombinant peptides to minimize degradation, nonspecific adsorption to the tube, and aggregation are crucial to obtain reproducible results. Taking these precautions, we have obtained reproducible results using more than 10 cell preparations over 3 yr.

In target tissues to both factors the effects of inhibin and activin are often opposite. This is the case not only for FSH secretion by gonadotropes (1), but also for other tissues or cells. In rat ovary, inhibin enhanced follicular recruitment, whereas activin promotes atresia and blocks follicular development (38). Opposite effects have been also described on DNA synthesis in rat thymocytes (stimulated by inhibin and inhibited by activin) (39) and in rat intermediate spermatogonia and preleptotene spermatocytes in staged seminiferous segments in vitro, but in these cells activin was stimulatory, whereas inhibin was inhibitory (16). However, lack of inhibin A action has been observed in a coculture system in which activin A induced aggregation of germ cells and Sertoli cells and spermatogonial DNA synthesis (17). Interestingly, even in some cases in which inhibin alone had no effect, it is able to block the effects of activin, as observed in R2C rat Leydig tumor cells (23) and in our study of 3ßHSD. In pig Leydig cells, the effects of inhibin A and activin A on LH/hCG receptor number and mRNA as well as on P450c17 mRNA levels are clearly not opposite, but are very similar. Although not reaching significance, the effects of both peptides together on LH/hCG receptor mRNA and P450c17 are slightly higher than those of either peptide alone. These findings together with the fact that inhibin A could not completely block the stimulatory effect of activin A on 3ßHSD mRNA could explain the enhanced steroidogenic responsiveness of cells treated with both peptides compared with that of cells treated with each peptide alone.

The molecular basis of the differential effects of inhibin and activin is unknown. However, the effects of inhibin A and activin A on Leydig cells, as observed in our in vitro study, are consistent with the specific binding of [125I]inhibin A on Leydig cells observed throughout rat testis development (13). Although binding of [125I]activin was detected mainly in the basal compartment of the seminiferous tubules as well as on round spermatids in stages VII–VIII of the spermatogenic cycle (13), type II activin receptor mRNAs were detected not only in the seminiferous tubule, but also in Leydig cells, as shown by Northern blot of both mature and immature rat testis mRNAs (14) and in situ hybridization (15); this latter study suggested than Leydig cells expressed mainly ActRIIB receptor.

The potential roles of inhibin, activin, and activin receptors in mouse Leydig cell development and function in vivo have been investigated by targeting inactivation of these genes (40). Knockout of activin ßB-subunit, producing mice deficient in activin B, activin AB, and inhibin B, results in males with normal reproductive capacity (41). Activin ßA-deficient mice develop to term, but die within 24 h secondary to multiple craniofacial abnormalities, but without apparent abnormalities of the external or internal genitalia. Similarly, mice deficient in both activins ßA and ßB display the defects of both activins ßA and ßB mutant mice, but no additional defects (42). Only inactivation of one of the two ActRII caused a marked reduction in testicular weight associated with a delay in fertility of about 3 weeks compared with that in mice heterozygous for such a mutation (43). However, this abnormality is probably secondary to the very low plasma FSH levels, as similar findings have been observed in mice with targeting inactivation of FSH ß-subunit (44). Finally, homozygous {alpha}-inhibin deficient mice were initially healthy and had normal genitalia, but were infertile (45). This was due to the development of gonadal sex cord-stromal tumors (granulosa/Sertoli cell tumors) in both sexes as early as 4 weeks of age. However, spermatogenesis, as well as the number of Leydig cells were normal in male from 5–7 weeks, but a regression of both parameters occurred in parallel with enlargement of the tumor mass. Interestingly, inhibin-deficient mice have very high plasma levels of both activin A and B (46), but these high levels of activins are not responsible for the gonadal sex cord-stromal tumor development (40, 47). Thus, the clear-cut conclusion for all of the above studies is that inhibins function as tumor suppressors in both gonads and adrenal cortex (40, 46), but that inhibins and activins A and B are not critical for Leydig cell development and function, at least in the mouse.

The discrepancies between the clear effect of inhibin and activin on cultured pig Leydig cells, as demonstrated by our study, and the lack of action, as suggested by the knockout experiments, may be related to differences between species or between the model used, to the possibility that the lack of ßA- and ßB-subunits may be compensated by the recently described ßC (48)- and ßD (49)-subunits and/or to the complex interactions between activins and the heparan sulfates proteoglycans and follistatin that might be different in vivo and in vitro.

Although in the male, inhibin B and activin B are the predominant circulating isoforms (1), these peptides were not available to us for studying their biological activity in our model. However, it has been reported that the two isoforms of inhibins (1) as well as those of activins (50) are equipotent in several mammalian cell assays.

In conclusion, the present work clearly demonstrates that both inhibin A and activin A enhanced Leydig cell hCG responsiveness, by regulating the expression of genes encoding specific proteins involved in Leydig cell functions. As the expression of both factors in testicular cells is gonadotropin dependent, it is likely that both peptides participate in a fine-tuning of Leydig cell regulation.


   Acknowledgments
 
We thank Drs. E. Milgrom, F. Labrie, E. Simpson, and M. Watermann for their generous gift of cDNAs, and Dr. M. G. Forest for the antitestosterone antibody.


   Footnotes
 
1 This work was supported in part by the Reseau de Recherche Clinique INSERM (Grant 493010) and University Claude Bernard (Lyon, France). Back

Received June 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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