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Endocrinology Vol. 140, No. 4 1681-1686
Copyright © 1999 by The Endocrine Society

ARTICLES

Growth Hormone Regulates Steroidogenic Acute Regulatory Protein Expression and Steroidogenesis in Leydig Cell Progenitors1

Masanori Kanzaki and Patricia L. Morris

Population Council (M.K., P.L.M) and The Rockefeller University (P.L.M.), New York, New York 10021

Address all correspondence and requests for reprints to: Dr. Patricia L. Morris, Center for Biomedical Research, Population Council and The Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: p-morris{at}popcbr.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gonadal development and differentiation is dependent in part on GH, as GH deficiency has been implicated as a cause of lowered fertility and spermatogenic cessation in humans and some biological models. In this study, we demonstrate that GH receptor messenger RNA (mRNA) is preferentially expressed in progenitor Leydig cells (PLCs) isolated and purified from 21-day-old rats. GH induces significant increases in the levels of steroidogenic acute regulatory protein (StAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD) expression, and androgen production in PLCs. Additionally, the cytokine interferon-{gamma} (IFN{gamma}) markedly inhibits GH-stimulated StAR mRNA and protein levels. When cells are cultured with both GH and IFN{gamma}, IFN{gamma} decreases the stimulating effect of GH on androgen production. Treatment of PLCs with cycloheximide does not prevent the GH-induced StAR mRNA, indicating that GH induction of StAR transcripts does not require de novo protein synthesis. In contrast, the induction of 3ß-HSD mRNA by GH is altered by cycloheximide treatment. H7, a serine/threonine kinase inhibitor, completely abrogates the increases in StAR mRNA by GH, whereas the tyrosine kinase inhibitor genistein does not. Moreover, GH further enhances StAR and 3ß-HSD mRNA expression in isolated adult rat Leydig cells despite their increased basal expression subsequent to maturational acquisition of these steroidogenic components. These data provide the first demonstration of the direct effects of GH on testicular steroidogenesis during progenitor Leydig cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE POTENTIAL involvement of GH in testicular function is suggested by both experimental and clinical data. In humans, either isolated GH deficiency or GH resistance is associated with delayed puberty and poor responsiveness to hCG stimulation (1, 2). In the immature hypophysectomized rat model, LH, PRL, and GH treatment are all capable of increasing precursor mesenchymal cells (3). These data imply that GH is involved in the functional development of Leydig cells in vivo. Although many of the actions of GH are thought to be secondary to increases in insulin-like growth factor I (IGF-I) in vivo (4, 5), we recently reported a direct effect of GH on primary immature Leydig cells, demonstrating that GH activates the signal transducer and activator of transcription-5b (STAT5b) (6).

During the prepubertal period, three well defined developmental stages of Leydig cells are observed in the rat testis (7, 8). Mesenchymal stem cells proliferate and differentiate into progenitor Leydig cells (PLCs), which predominate in the testis from days 14–21. In vivo, PLCs subsequently acquire the characteristics of immature Leydig cells (ILCs), which produce preferentially the androgen, androstane-3{alpha},17ß-diol (3{alpha}-DIOL), rather than the testosterone reflective of the adult Leydig cell (ALC). ILCs then undergo an additional round of proliferation and begin to terminally differentiate into nondividing ALCs. ALCs are distinguished from their immature precursors by a higher abundance of LH receptors (LHR), the predominant expression and activity of {Delta}5-3ß-hydroxysteroid dehydrogenase (3ß-HSD) rather than the 3{alpha}-HSD characteristic of PLCs and ILCs, and increased production of testosterone. These maturational characteristics of PLC, ILC, and ALC are typified at 21, 35, and 55–90 days in the rat, respectively.

The acute response of steroidogenic cells to tropic hormone stimulation is a rapid increase in the rate of steroid hormone biosynthesis (9). The process involves at least two mechanisms. The first part of the process is transport of cholesterol substrate to the inner mitochondrial membrane, a process that involves steroidogenic acute regulatory protein (StAR), the peripheral benzodiazepine receptor and its ligand, and potentially other mediators. The second part of the process is the metabolism of cholesterol into testosterone, which requires several different steroidogenic enzymes, including cytochrome P450 side-chain cleavage (P450scc) and 3ß-HSD enzymes activities. P450scc converts transported cholesterol to pregnenolone, and 3ß-HSD is involved in the conversion of pregnenolone to progesterone. In adrenal cells, 3ß-HSD is expressed not only in smooth endoplastic reticulum but also in the mitochondria, where it is associated with StAR and P450scc (10). StAR messenger RNA (mRNA) and protein are induced concomitantly via a cAMP-mediated mechanism in the MA-10 Leydig cell line (9, 11). In the hypophysectomized sheep corpus luteum in vivo, StAR gene expression is stimulated by treatment with either of the tropic hormones LH or GH (12). Chronic treatment (48 h) with IGF-I is required to increase StAR expression in cultured porcine granulosa cells (13). Recent studies showed that pretreatment with the cytokines interferon-{gamma} (IFN{gamma}) (24 h) and tumor necrosis factor-{alpha} (48 h) decreased hCG-induced StAR expression in primary Leydig cells (14, 15).

In this study, we examined the ontogeny of GH receptor (GHR) and StAR mRNA expression in purified Leydig cells at different maturational stages, and the effect of GH on the level of StAR expression and androgen production in vitro. To our knowledge, these data provide the first evidence for a direct effect of GH on testicular steroidogenesis and specific steroidogenic components during Leydig cell development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparations
PLCs and ILCs were prepared from 21- and 35-day-old Sprague-Dawley (SD) rats [Crl:CD(SD)BR-CD, Charles River Laboratories, Inc., Kingston, NY], respectively, according to the procedures described previously (16). After collagenase dissociation, a fraction enriched in PLCs or ILCs was isolated using a Percoll density gradient separation. ALCs were prepared from SD rats (55–65 days of age) and purified by Percoll gradient and centrifugal elutriation as previously reported (6). The purity of three developmentally defined Leydig cell preparations was assessed using 3{alpha}-HSD immunocytochemistry and 3ß-HSD histochemistry as we previously described (6). PLCs were 45% 3{alpha}-HSD positive and 42% 3ß-HSD positive after 2 days of culture. Both cultured ILCs and ALCs were found to average over 90% and 97% 3ß-HSD positive, respectively. Leydig cells were cultured for 2 days in serum-free medium supplemented with 2.5 µg/ml insulin (Sigma Chemical Co., St. Louis, MO), 5 µg/ml transferrin (Calbiochem, La Jolla, CA), and 10 µg/ml bacitracin (Sigma Chemical Co.). Cells were rinsed and then pretreated with or without cycloheximide (CHX; Calbiochem) or kinase inhibitors (H7 or genistein; Calbiochem) as indicated. After removal of the pretreatment-containing media and several rinses with the addition of fresh serum-free media, cells were stimulated with hCG (13,000 mIU/mg; a gift from Y. Y. Tsong, The Population Council), ovine GH (NIDDK oGH-15), rat PRL (NIDDK rPRL-B8SIAFP), or rat IFN{gamma} (Genzyme Corp., Cambridge, MA) for the indicated times and dosages. The media were then collected and stored at -20 C until the measurement of 3{alpha}-DIOL and testosterone by specific RIAs with either anti-3{alpha}-DIOL (3% cross-reactivity with testosterone; Miles Scientific, Naperville, IL) or anti-testosterone (<1% cross-reactivity with 3{alpha}-DIOL; gift from Dr. G. D. Niswender, Colorado State University, Boulder, CO) antiserum (17). Purified steroids for use as RIA standards were obtained from Steraloids (Wilton, NH). Procedures involving the use of animals strictly followed the Guidelines for the Care and Use of Laboratory Animals set forth by the NIH. The MA-10 cell line (provided by M. Ascoli, University of Iowa, Iowa City, IA) used in these experiments was maintained as previously reported (6).

Northern blot analysis
RNA isolation and Northern blot analyses were performed using methods we previously described (18). Mouse 3ß-HSD (892-bp) or rat GHR (533-bp) complementary DNA (cDNA) was amplified by the RT-PCR method using previously described primers as reported (19, 20, 21) and the cDNAs cloned into pPCR-Script plasmid (Stratagene, La Jolla, CA). 3ß-HSD is a member of a multigene family, and the probe used in this study was designed to recognize several of the rat isoforms, including the type I and type II isoforms expressed exclusively in the testis (19, 22). The sequences of mouse 3ß-HSD cDNA probe (GenBank M58567) is 91% identical to both rat type I (M38178) and type II (M38179) 3ß-HSD mRNA. Mouse StAR (1.5 kb; provided by Dr. D. M. Stocco), mouse 3ß-HSD, rat GHR, or human G3PDH (CLONTECH Laboratories, Inc., Palo Alto, CA) cDNAs were labeled with [{alpha}-32P]deoxy-CTP (Amersham, Arlington Heights, IL) using random hexamers. The filters were exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY), and signals were evaluated using PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analyses.

Immunoblot analysis
Mitochondrial protein was isolated from cultured PLCs treated with or without reagents using TSE buffer containing 0.25 M sucrose, 10 mM Tris (pH 7.4), and 0.1 mM EDTA as previously described (11). The protein concentration was determined by the Bradford assay. Mitochondrial proteins (50 µg/lane) were subjected to SDS-PAGE using 12% polyacrylamide gels and transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). The membrane was probed with a specific mouse polyclonal antiserum raised against a 10-amino acid peptide (88–98) of the StAR protein (provided by Dr. D. M. Stocco). Blots were developed using an enhanced chemiluminescence Western blotting system (Amersham).

Data analysis
All experiments were repeated at least three times using separate primary cell preparations. The results presented are typical for each experiment illustrated. The significance of the results was determined using Student’s t test and ANOVA, followed by a posteriori testing using Dunnett’s or Tukey-Kramer multiple comparison test as required. P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We isolated and purified three different maturational types of rat Leydig cells (PLCs, ILCs, and ALCs). Using immunocytochemistry and histochemistry, 3{alpha}- and 3ß-HSD have been shown to be appropriate markers of the maturational status of Leydig cell (6, 7, 8, 16, 17). Therefore, detection of 3ß-HSD mRNA was used as one parameter of the developmental and steroidogenic status of the cultured Leydig cells in this study. As anticipated, the levels of 3ß-HSD mRNA (1.7 kb) increased with the age of the rats used and paralleled Leydig cell differentiation. The same Northern filters were stripped and rehybridized with StAR, GHR, and G3PDH cDNA probes. Two species of mRNA (3.8 and 1.7 kb) from Leydig cells hybridized to the StAR cDNA probe. PLCs demonstrated low signals for StAR transcripts, whereas intense signals appeared in ILCs and ALCs. In rodents, GHR mRNA exists in two forms, one encoding the hormone-binding domain common to the GHR and the other encoding the soluble GH-binding protein (GHBP). Hybridization of GHR cDNA showed that a 1.3-kb band corresponding to GHBP mRNA was clearly detected, whereas a GHR band (4.3-kb) was faintly observed in PLCs. In contrast, low levels of GHBP mRNA were demonstrated in ILCs and ALCs (Fig. 1Go).



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  		Figure 1. Expression of 3ß-HSD, GHR, and StAR mRNAs in three different types of Leydig cells isolated from 21-day-old (PLC), 35-day-old (ILC), and adult (ALC) rats. Leydig cells were cultured for 2 days in serum-free medium. RNA from the MA-10 mouse Leydig tumor cells was loaded onto the same gel for comparison. RNA samples (20 µg) were then subjected to sequential Northern blot hybridization with labeled 3ß-HSD, GHR, StAR, and glyceraldehyde-3-phosphate dehydrogenase cDNAs.

 
To examine the ability of hormones and cytokines to modulate the expression of StAR, we studied the kinetics of their effects in PLCs (Fig. 2AGo). GH (100 ng/ml) as well as hCG (100 ng/ml) increased StAR transcripts within 3 h, whereas 100 ng/ml PRL had no effect within 18 h. By rehybridizing the same membrane with a 3ß-HSD cDNA probe, we determined the levels of 3ß-HSD. Induction of 3ß-HSD transcripts was also seen after both GH and hCG treatment, but the effect of PRL treatment was not different from that of the vehicle blank control treatment. Next, various concentrations of GH (0.1–500 ng/ml) were added for 3 h to the PLCs, and changes in StAR mRNA levels were determined. The addition of GH at increasing doses resulted in a dose-dependent increase in the levels of StAR mRNAs (Fig. 2BGo). Although IFN{gamma} (10 ng/ml) had no effect on the basal level of StAR gene expression (Fig. 2AGo), IFN{gamma} inhibited GH-induced StAR mRNA expression within 3 h (Fig. 2CGo). Western blot analyses showed that GH as well as hCG induces StAR protein expression, and that IFN{gamma} inhibits the stimulation by GH, data consistent with the changes in mRNA levels from Northern blot analyses (Fig. 2DGo).



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  		Figure 2. Effects of GH and IFN{gamma} on StAR and 3ß-HSD gene expression in PLCs. After 2 days of culture, PLCs were treated with hormones and/or IFN{gamma}. A, PLCs were treated with hCG (100 ng/ml), GH (100 ng/ml), PRL (100 ng/ml), or IFN{gamma} (10 ng/ml) for the times indicated. RNA samples (20 µg) were subjected to sequential Northern blot hybridization with labeled StAR, 3ß-HSD, and G3PDH cDNAs. B, Various doses (0.1–500 ng/ml) of GH were added to PLCs, and the cells were cultured for 3 h. RNA samples (20 µg) were subjected to sequential Northern blot hybridization with labeled StAR and G3PDH cDNAs. C, Effect of IFN{gamma} on GH-induced StAR gene expression in PLCs. PLCs were cultured with GH (100 ng/ml) and/or IFN{gamma} (10 ng/ml) for either 3 or 6 h. RNA samples (20 µg) were subjected to sequential Northern blot analyses as described in B. D, PLCs were cultured with hCG (100 ng/ml), GH (100 ng/ml), and/or IFN{gamma} (10 ng/ml) for 6 h, after which time mitochondrial protein was isolated for immunoblotting analyses with anti-StAR polyclonal antibody (1:1000 dilution).

 
As there is a direct relationship between StAR function and steroidogenesis (9), we investigated androgen production (testosterone and 3{alpha}-DIOL) in PLCs treated with hCG (100 ng/ml), GH (100 ng/ml), and/or IFN{gamma} (10 ng/ml) for 18 h (Fig. 3Go). GH as well as hCG increased steroid production (P < 0.01 compared with the control value). IFN{gamma} alone appeared to decrease androgen production, but not at a statistically significant level (P > 0.05 compared with the control value). When cells were cultured with both GH and IFN{gamma}, IFN{gamma} decreased the stimulating effect of GH on PLC androgen production (P < 0.05 compared with the value with GH alone treatment).



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  		Figure 3. Effects of GH and IFN{gamma} on steroid production in PLCs. PLCs were cultured in serum-free medium for 2 days. After medium change, cells were stimulated with or without hCG (100 ng/ml), GH (100 ng/ml), or IFN{gamma} for 18 h. 3{alpha}-DIOL and testosterone were measured in the supernatants by separate RIAs. The results normalized to control levels represent the mean of the total androgens (3{alpha}-DIOL and testosterone) ± SE of four different replicate samples. *, P < 0.01 compared with the value in control cells. **, P < 0.05 compared with the value in GH-treated cells.

 
To further characterize the effect of GH on transcription of the StAR gene in Leydig cells, PLCs were treated for 1 h with the protein synthesis inhibitor, CHX, or the kinase inhibitors, genistein (23) and H7 (24), before GH treatment for 5 h. Although pretreatment of the cells with CHX or the tyrosine kinase inhibitor, genistein, significantly reduced the increase in 3ß-HSD mRNA expression caused by GH, neither treatment abrogated the GH-induced StAR mRNA expression in PLCs. Interestingly, pretreatment with CHX or genistein showed stimulating effects on GH-induced StAR transcription. In contrast, the serine/threonine kinase inhibitor H7 completely blocked the GH induction of both StAR and 3ß-HSD mRNAs (Fig. 4Go).



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  		Figure 4. PLCs were pretreated for 1 h with or without CHX (10 µg/ml), genistein (50 mM), or H7 (50 mM). After several rinses, PLCs were treated with GH (100 ng/ml) for 5 h. RNA was then isolated, and samples (20 µg) were subjected to sequential Northern blot hybridization with labeled StAR, 3ß-HSD, and glyceraldehyde-3-phosphate dehydrogenase cDNAs.

 
We next examined the regulation of StAR and 3ß-HSD mRNA expression in ALCs (Fig. 5Go). ALCs were cultured for 2 days, and then treated with either 100 ng/ml hCG or GH. Both GH and hCG increased the levels of StAR and 3ß-HSD gene expression in ALCs. Although temporally GH initially induced the 1.7-kb band of StAR in PLCs (Fig. 2Go), increases in the 3.8-kb transcript were first seen at 3 h after GH or hCG treatment in ALCs (Fig. 5Go).



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  		Figure 5. Effects of GH on StAR and 3ß-HSD gene expression in ALCs. After 2 days of culture in vitro, ALCs were treated with hCG (100 ng/ml) or GH (100 ng/ml) for the times indicated. RNA samples (10 µg) were used for sequential Northern blot hybridization with labeled StAR, 3ß-HSD, and glyceraldehyde-3-phosphate dehydrogenase cDNAs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Herein we describe the developmental changes in the levels of GHR and StAR mRNAs in isolated and purified Leydig cells and further characterize the effect of GH on StAR and 3ß-HSD expression during Leydig cell differentiation. Three types of Leydig cells (PLCs, ILCs, and ALCs) were evaluated by Northern blot analyses using a 3ß-HSD cDNA probe. Developmentally related patterns were observed, consistent with the findings of previous studies (7, 8, 16). Lower levels of 3ß-HSD mRNA were seen in PLCs compared with those of ILCs and ALCs. The age-related increase in the level of testosterone production during development is dependent on the balance between androgen-metabolizing enzymes (3{alpha}-HSD and 5{alpha}-reductase) and testosterone biosynthetic enzymes (3ß-HSD, P450scc, and P450c17) (9). In an ALC, testosterone production is 150 times greater than that in a PLC and 5 times greater than that in an ILC (8). In addition to changes in these enzymes, we now show that basal levels of StAR gene expression are greatly increased in ILCs and ALCs compared with those in PLCs. Other recent studies reported that StAR protein is undetectable in freshly isolated immature Leydig cells, but is easily detectable in those prepared from adult rats (25). Differences in the findings between this study and ours may reflect the methodologies used. The increasing capacity for testosterone production in developing Leydig cells may be concordant with rising levels of StAR protein, which regulate cholesterol transport in mitochondria.

Our present data imply that GH is involved in the functional development of Leydig cells in vivo. However, direct or indirect effects of GH have never been clearly established in the Leydig cell. This remains unresolved due in part to issues associated with defining the actions of GH on the Leydig cell, namely the ability of human GH to bind to both somatogenic (GH) and lactogenic (PRL) receptors. In this study, ovine GH, but not rat PRL (both in 100 ng/ml doses), stimulated StAR and 3ß-HSD mRNAs in PLCs despite the presence of PRL receptors in the immature rat testis (26, 27, 28). Many of the actions of GH are thought to be mediated by IGF-I. Although a previous study showed that bovine GH increased testicular IGF-I mRNAs in immature hypophysectomized rats (4), these investigators were unable to detect GHR in the rat testis and suggested that the effects of GH were indirect. A later immunohistochemical study showed the localization of GHR protein in the Leydig and Sertoli cells of the adult rat testis (29). We now demonstrate the expression of GHR mRNA in purified progenitor, immature, and adult Leydig cells. Taken together with our recent study (6), these findings are suggestive of a functional GH signaling pathway in the Leydig cell. Additionally, pretreatment of PLCs with CHX did not prevent the GH-stimulated elevation in the levels of StAR mRNA, indicating that GH induction of StAR transcripts is direct and does not require de novo protein synthesis. GHR is preferentially expressed in PLCs compared with the mRNA levels in ILCs and ALCs. In contrast, the levels of LHR mRNA and hCG-binding sites in ILCs and ALCs are higher than those in PLCs (30). The relative scarcity of LHR in PLCs indicates that additional hormones and factors may be important in the initial phase of Leydig cell differentiation in vivo. In male rats, episodic GH secretion is established during the prepubertal period (at about 22 days of life) with the presence of peaks of GH of low amplitude (31). In rats, 5{alpha}-reduced metabolites, 3{alpha}-DIOL, and androsterone are abundant in the circulation between days 20–40 postpartum when the testosterone level is still low (32). These data imply that GH may play an important role in Leydig cell differentiation or proliferation during the prepubertal period. Additionally, our data suggest that the GHR is functional in ALCs despite the low level of its expression, and that testosterone production can be further increased upon GH stimulation.

Several studies imply that IFN{gamma} plays a negative role in testicular steroidogenesis (14, 33, 34, 35). We recently showed that the mRNA and protein for IFN{gamma} receptor (IFN{gamma}R) {alpha}- and ß-chains are expressed in isolated rat Leydig and Sertoli cells (18). In cultured porcine Leydig cells, IFN{gamma} treatment inhibits gonadotropin-stimulated testosterone production as well as the level of mRNAs for steroidogenic enzymes (33). Inhibitory effects of IFN{gamma} can be partially or completely reversed by the addition of cholesterol analogs that can be used as substrate for P450, indicating that IFN{gamma} also affects early events in cholesterol transport before the side-chain cleavage of cholesterol (14, 33). In addition, pretreatment of rat ALCs by IFN{gamma} for 24 h inhibits hCG-stimulated Leydig cell steroidogenesis by the down-regulation of StAR expression (14). Our data demonstrate that IFN{gamma} has an acute inhibitory effect on GH-induced StAR mRNA and protein levels. Chronic administration of IFN{gamma} to mice results in reduced testicular weights, decreased sperm count and concentration, and abnormalities in sperm morphology (35). Our previous findings showed that IFN{gamma}Rs are functional in rat Sertoli cells (36, 37). Taken together, these findings suggest that chronic IFN{gamma}-induced effects on spermatogenesis are mediated by both Leydig and Sertoli cells in vivo. Furthermore, CHX or genistein pretreatment enhances the GH induction of StAR mRNA in this study, indicating the putative involvement of inhibitors that require tyrosine phosphorylation and/or new protein synthesis, respectively, on StAR expression.

In this study, H7, a broad-based serine/threonine kinase inhibitor, altered GH-induced StAR mRNA in PLCs. H7 inhibits protein kinase A, C, and G and myosin light chain kinase. Phosphorylation of StAR protein on a threonine residue is required for the acute induction of steroidogenesis in MA-10 mouse Leydig tumor cells (38). We recently showed that GH can act directly on rat Leydig cells by phosphorylation of STAT-5b. GH stimulated DNA binding of STAT-5b in immature (18-day-old) Leydig cells, but not in ALCs (6). However, StAR mRNAs were induced in both PLCs and ALCs. Furthermore, genistein pretreatment did not block the induction of StAR transcripts in PLCs, while tyrosine phosphorylation of the Janus kinase (JAK)/STAT pathway is required for STAT-5b activation by GH in Leydig cells (6). Therefore, it is less likely that the effect of GH on StAR expression is mediated by the JAK/STAT-5b cascade. In addition to the STAT, mitogen-activated protein kinase, and Ras-dependent pathways that are linked to the tyrosine phosphorylation of JAK2, GH signals are transduced via voltage-dependent Ca2+ channels, phospholipase C, protein kinase C (PKC), insulin receptor substrate 1, and cytosolic phospholipase A2 (39, 40, 41, 42). Treatment with phorbol 12-myristate 13-acetate, a pharmacological activator of PKC, eliminates the FSH induction of StAR mRNA in luteinized porcine granulosa cells, indicating that PKC is unlikely to be an intermediary in the GH-dependent stimulation of the StAR gene (13). Similar to the steroid hydroxylase genes, consensus cAMP-responsive elements are not present in the StAR promoter region (43). The roles of LH and subsequent increases in cAMP in mediating StAR gene transcription by steroidogenic factor 1, an orphan nuclear receptor, have been investigated (44, 45). Further work is needed to determine the signaling pathway(s) activated by GH during the induction of the StAR gene in Leydig cells.

In conclusion, GH directly increases the levels of StAR mRNA and protein expression through GHR, but not PRL receptor, in Leydig cells. Additionally, IFN{gamma} inhibits GH-stimulated StAR levels and steroid production in both basal and GH-stimulated PLCs. Thus, our studies are consistent with an important role for an additional pituitary hormone, namely GH, in the pubertal acquisition of steroidogenic ability.


   Acknowledgments
 
The authors express their appreciation for the excellent technical skills of L. R. Mitchell and the editorial assistance of J. E. Schweis. We gratefully acknowledge the gift of purified hCG from Dr. Y. Y. Tsong (The Population Council), anti-testosterone antibody from Dr. G. D. Niswender (Colorado State University), ovine GH and rat PRL from the NIDDK hormone program, and StAR cDNA and antibody from Dr. D. M. Stocco (Texas Tech University).


   Footnotes
 
1 This work was supported by NIH Grant R01-HD-16149 (to P.L.M.). Fellowship support for M.K. was provided by The Andrew W. Mellon Foundation. Back

Received October 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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