Leydig Cell Protein Synthesis and Steroidogenesis in Response to Acute Stimulation by Luteinizing Hormone in Rats¹

^c Division of Reproductive Biology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 ^d Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

	ABSTRACT

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We examined the temporal relationship between protein synthesis and testosterone production by rat Leydig cells in primary culture. Leydig cells were isolated from adult control Sprague-Dawley rats and from rats that had received LH-suppressive testosterone and estradiol (TE) implants in vivo for 10 days. The cells were incubated for 1–4 h with [³⁵S]methionine in the presence or absence of maximally stimulating ovine LH, and newly synthesized proteins were examined by two-dimensional PAGE autoradiography. Approximately 800–900 newly synthesized polypeptides were readily visible on all autoradiograms, most of which did not differ in the cells from intact control and TE-treated rats. Incubation of cells from the control and treated rats with maximally stimulating LH for 4 h in both cases resulted in significant increases in testosterone production and in three newly synthesized polypeptides. These polypeptides, along with two others that changed little in response to LH, were similar in apparent molecular mass, 30 kDa, but differed in isoelectric point. Time-course studies revealed a temporal relationship between stimulation of the three 30-kDa proteins and of testosterone production. Western blot analysis identified the 30-kDa proteins as steroidogenic acute regulatory protein (StAR). The results of these studies, for the first time utilizing primary cultures of highly purified, testosterone-producing Leydig cells, provide further correlative evidence of a role for StAR protein in the acute regulation of Leydig cell testosterone biosynthesis by LH.

	INTRODUCTION

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LH has both long-term (trophic) and short-term (acute) effects on Leydig cells. With respect to the former, Leydig cells require LH to maintain their fully differentiated structure and function [1]. Suppression of gonadotropins by steroid administration over time causes Leydig cell atrophy, reductions in the steroidogenic enzymes and in the cellular organelles in which they are sequestered, and reductions in the ability of the cells to produce testosterone [2–4]. Although the exact mechanisms by which LH acts acutely continue to be debated, it is generally accepted that LH binds to its receptor, thereby initiating a cascade of events that includes activation of adenylate cyclase, increased intracellular cAMP levels, activation of cAMP-dependent protein kinase, phosphorylation of proteins, and, ultimately, transport of cholesterol to the inner mitochondrial membrane and thus to the mitochondrial P450 side-chain cleavage enzyme [5].

The mechanism by which cholesterol moves to the inner mitochondrial membrane has received considerable attention. In this regard, a number of studies have reported that steroid production in response to hormonal stimulation requires the synthesis of new proteins, and that newly synthesized proteins participate in sterol translocation to the inner mitochondrial membrane [5–8]. Indeed, the transport of cholesterol to the inner mitochondrial membrane is now considered by many to be the rate-limiting step in steroidogenesis, though this step, by itself, does not assure maximal steroidogenesis.

Particular mitochondrial proteins have been shown to be synthesized rapidly in response to acute stimulation by LH [9], hCG [10], or dibutyryl cAMP [11–14] in many steroidogenic cells. There is now considerable evidence that these proteins, synthesized as 37-kDa precursors and subsequently processed to 32-kDa intermediates and ultimately to 30-kDa forms [15], are integrally involved in the delivery of cholesterol to the inner mitochondrial membrane, and thus represent an essential component of the conversion of cholesterol to pregnenolone. This 30-kDa protein family has been named steroidogenic acute regulatory protein, or StAR [16]. The recent discovery that mutations in the gene encoding StAR are associated with congenital lipoid adrenal hyperplasia, a condition characterized by impaired adrenal and gonadal steroid hormone production, provides compelling evidence that StAR represents an essential mediator of the acute actions of tropic hormones, at least in some steroidogenic cells [17]. Indeed, StAR has been shown to occur in a variety of steroidogenic cells, including Leydig, luteal, and adrenal cells [18], though it is not found in placental cells [19]. It seems reasonable to conclude from this that there may be StAR-independent as well as StAR-dependent mechanisms for steroid synthesis [20, 21].

In vitro studies of MA-10 mouse and R2C rat Leydig tumor cells have suggested that StAR is involved in steroidogenesis by these cells [10, 22]. Consistent with this, a recent study showed that injection of mice with lipopolysaccharide caused rapid decreases in serum testosterone concentration and in StAR protein levels in isolated Leydig cells [23], further suggesting a role for StAR protein in Leydig cell steroidogenesis. It should be noted, however, that MA-10 and R2C Leydig cells are transformed and that their major steroidogenic product is progesterone, not testosterone. Moreover, changes in serum testosterone levels are not necessarily reflective of changes in Leydig cell testosterone production. To our knowledge, there have been no reports in which the synthesis of StAR polypeptides has been related temporally to testosterone production by Leydig cells in primary culture. Such an analysis is now feasible because techniques are available to isolate very pure populations of Leydig cells that retain their ability to produce testosterone at high levels for up to 3 days [24, 25]. The temporal relationship between protein synthesis and steroidogenesis in such cells in response to acute stimulation by LH thus can be readily examined.

In the present study, we examined this relationship in primary culture of Leydig cells isolated from Sprague-Dawley rat testes and cultured in the presence or absence of maximally stimulating LH. We show that StAR proteins are synthesized by these cells in response to acute stimulation by LH and that, indeed, there is a temporal relationship between their synthesis and testosterone production. These studies further support a role for StAR in the acute regulation of Leydig cell testosterone production by LH.

	MATERIALS AND METHODS

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Animals

This study used a total of 60 adult male Sprague-Dawley rats (275–325 g BW), purchased from Charles River Laboratories (Wilmington, MA). Rats were housed in a temperature-controlled room (22°C) and allowed access to food and water ad libitum. All experimental protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Hygiene and Public Health.

Chemicals

Testosterone and estradiol-17ß were purchased from Steraloids (Wilton, NH). Ovine LH-26 was a gift from the National Hormone and Pituitary Program, NIDDK (Rockville, MD).

[1,2,6,7,16,17-N-³H]Testosterone (specific activity 140.9 Ci/mmol) was obtained from New England Nuclear (Wilmington, DE). Rabbit testosterone antiserum was obtained from ICN Biomedicals (Costa Mesa, CA). All other chemicals were of analytical grade.

Administration of Testosterone and Estradiol Implants

Testosterone and estradiol were administered via s.c. polydimethylsiloxane (Silastic; Dow Corning, Midland, MI) implants. Details of the fabrication of the implants have been described previously [26]. Briefly, rats received implants of 2.5-cm testosterone and 0.1-cm estradiol placed s.c. into the interscaplular region; 10 days later, rats were killed by guillotine.

Purification of Leydig Cells

Leydig cells from control and implanted rats were isolated and purified by centrifugal elutriation and Percoll density gradient centrifugation as previously described [24, 25]. The purity of cell preparations from adult rats was assessed by determining the percentage of cells that stained histochemically for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) [24, 25]. The purity achieved consistently was > 95%.

Testosterone RIA

Testosterone concentration in cell culture medium was determined in 10-µl aliquots by RIA according to the method described by Schanbacher and Ewing [27]. The assay sensitivity was 10 pg/tube.

Metabolic Labeling with [³⁵S]Methionine

Freshly isolated Leydig cells (2 x 10⁶ cells/ml) were placed in tissue culture dishes and labeled for 4 h at 34°C in serum-free Dulbecco's Modified Eagle's medium (DMEM) lacking methionine and cysteine and containing 50 µCi/ml of [³⁵S]methionine (specific activity > 1000 Ci/mmol; cat. no. SJ 1015; Amersham, Arlington Heights, IL). At 1–4 h thereafter, the medium was removed and the cells were washed twice with ice-cold PBS containing a protease inhibitor mixture (0.2 mM phenylmethysulfonyl fluoride; 2 mg/ml leupeptin; 1 mg/ml aprotinin). The cells were harvested from the dishes with the aid of a plastic cell lifter (Costar no. 3008; Cambridge, MA), transferred into centrifuge tubes, and pelleted by gentle centrifugation (600 x g) for 5 min.

Two-Dimensional PAGE

Cell pellets were suspended in 150 µl of lysis buffer A [28] and sonicated on ice (three bursts of 15 sec at 60-sec intervals) using a Micro Ultrasonic Cell Disrupter (Fisher Scientific, Pittsburgh, PA) at maximum power. Sonicated samples were centrifuged at 14 000 rpm for 20 min at 4°C. Duplicate aliquots of the supernatant (5 µl) were precipitated in trichloroacetic acid to determine the extent of [³⁵S] incorporation. Typically, 10 µl of sample containing 6–9 x 10⁵ cpm and 5–10 µg of protein was applied to each gel.

2D-PAGE was performed as described previously [28]. Briefly, the first-dimensional separation was performed at room temperature under isoelectric focusing conditions (constant power 20 mW/tube, 13 500 Vh total) in 4% (w:v) polyacrylamide gels (180 mm x 1.5 mm) containing 2% carrier ampholytes (1.6% pH 4–8, 0.4% pH 3.5–10). First-dimensional tube gels were extruded from the basic end directly onto the surface of a second-dimensional, 1.5-mm thick, 7–15% polyacrylamide gradient slab gel, and electrophoresis was performed using a Bio-Rad (Hercules, CA) Protean II xi multicell electrophoresis apparatus. After electrophoresis (4–4.5 h), gels were dried and exposed to Kodak XAR-5 (Eastman Kodak, Rochester, NY) x-ray film for autoradiography. Autoradiograms were analyzed using the Elsie 5 semiautomated computer analysis system, as described previously [29]. Quantities of individual polypeptides are presented as the percentage of total integrated intensities after correction for differences in the loading of the gel.

Immunoblot Analysis

Immunoblot analysis of StAR proteins was conducted according to methods described by Clark et al. [16]. Briefly, freshly isolated Leydig cells from untreated (control) and testosterone and estradiol (TE)-implanted adult rats were incubated for 4 h in the absence or presence of maximally stimulating ovine LH (100 ng/ml) at 34°C. The 30-kDa polypeptides present in 1 x 10⁶ cells were resolved on 2D minigels and analyzed by Western blot using rabbit anti-StAR and chemiluminescence.

Statistical Analysis

One-way ANOVA was used to detect significant effects among groups. Scheffé's multiple range test was used to identify differences between groups. Group means were considered to be significantly different at p < 0.05.

	RESULTS

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Leydig cells isolated from control rats and from rats that had received LH-suppressive TE implants for 10 days are shown in Figure 1. In both cases, approximately 95% of the isolated cells stained for 3ß-HSD activity. The Leydig cells from the implanted rats (Fig. 1B) were smaller in size than those from intact controls (Fig. 1A) and contained less total protein (101.4 ± 8.0 µg/10⁶ cells vs. 117.5 ± 9.6 µg/10⁶ cells, respectively). When incubated with maximally stimulating LH, Leydig cells from the implanted rats produced less than 5% of the testosterone of cells from intact controls.

View larger version (61K): [in this window] [in a new window]   FIG. 1. Light micrographs of freshly isolated Leydig cells from control rats (A) and from rats that had received TE implants for 10 days (B). The cells are stained for 3ß-HSD. Magnification x1300 (reproduced at 56%).

Figure 2 shows two-dimensional (2D)-PAGE autoradiograms of polypeptides synthesized by Leydig cells that were isolated from intact control or TE-implanted rats and incubated with [³⁵S]methionine for 4 h in the absence (panels A and C) or presence (panels B and D) of LH. Approximately 800–900 spots were readily visible on the autoradiograms. Despite the substantial differences in the volume and protein content of Leydig cells from control and TE-treated rats, qualitative differences in newly synthesized proteins were not apparent when the cells were cultured in the absence (compare panels A and C) or presence (compare panels B and D) of LH.

View larger version (142K): [in this window] [in a new window]   FIG. 2. 2D-PAGE autoradiograms of [35S]methionine-labeled polypeptides synthesized by Leydig cells that were isolated from control (A, B) or TE-implanted (C, D) rats and incubated with 50 µCi/ml [35S]methionine for 4 h in the absence (A, C) or presence (B, D) of maximally stimulating LH (100 ng/ml). Equivalent counts (7 x 105 cpm) were loaded. Arrows identify a family of 30-kDa polypeptides, numbered 1–5.

In response to maximally stimulating LH, proteins of approximately 30-kDa molecular mass were synthesized in cells from both control (Fig. 2B) and implanted (Fig. 2D) rats. In particular, three 30-kDa polypeptides (numbered 1, 2, and 3) increased considerably in the LH-stimulated cells, while two polypeptides (4 and 5), with the same molecular mass as polypeptides 1, 2, and 3 but different charge, changed little. Figure 3 shows the results of quantification of polypeptides 1–3 and 4–5 from the 2D autoradiograms. After 4 h of culture in the absence of LH, newly synthesized polypeptides 1–3 represented a higher percentage of the total protein in control cells as compared to cells from TE-treated rats. Incubation of the control Leydig cells with LH in vitro for 4 h resulted in a 4-fold increase in polypeptides 1–3. Incubation of Leydig cells from TE-treated rats with LH for 4 h resulted in a 30-fold increase in the three polypeptides, a far greater increase than elicited by LH treatment of the control cells. In contrast, newly synthesized polypeptides 4 and 5 were similar in cells from control and TE-treated rats whether or not the cells were cultured with LH.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Quantitative analysis of 2D-PAGE autoradiograms of 30-kDa proteins. Cells isolated from control (C) or TE-implanted (TE) rats were incubated in the absence (-LH) or presence (+LH) of LH for 4 h. Protein quantities are expressed as the percentage of total radioactive proteins on the 2D autoradiograms. The bars represent typical results from two separate experiments.

Incubation of Leydig cells from intact control or TE-treated rats with maximally stimulating LH for 1–4 h in both cases resulted in time-dependent increases in testosterone production (Fig. 4). This, together with the observation that LH stimulated increases in members of the family of 30-kDa polypeptides by these cells (Figs. 3 and 4), suggested that there might be a functional relationship between testosterone production and the synthesis of particular 30-kDa polypeptides. To begin to address this possibility, the temporal relationship between testosterone production and the synthesis of the 30-kDa proteins (polypeptides 1–5) in response to LH was examined in cells from the TE-implanted rats. We chose to use these cells for these studies in part because their steroidogenic response to LH was rapid and substantial, and in part because their reduced protein content increased the ability to detect even low levels of newly synthesized proteins. Three separate studies were performed, with consistent results obtained. Figure 5 shows the results of one of these studies. Polypeptides 4 and 5, but not 1, 2, and 3, were synthesized by freshly isolated Leydig cells (Fig. 5A). With 1–2 h of LH stimulation (Fig. 5, B and C, respectively), polypeptides 1 and 2 increased. Polypeptide 1 changed little thereafter, but polypeptide 2 was more prominent at 4 h (Fig. 5D) than at 2 h (Fig. 5C). In contrast, the synthesis of polypeptides 4 and 5 decreased somewhat over time after LH stimulation of the cells. Quantitative analyses of autoradiograms (Fig. 6) revealed progressive time-dependent increases in polypeptides 1–3 in response to LH, and a slight decrease in polypeptides 4 and 5.

View larger version (39K): [in this window] [in a new window]   FIG. 4. In vitro testosterone production by Leydig cells isolated from control (C) or TE-implanted (TE) rats. Cells were incubated for 4 h with maximally stimulating LH (100 ng/ml) for 0, 1, 2, or 4 h. For each sample, Leydig cells were isolated from 22 testes, and incubations were conducted in triplicate. Bars represent mean ± SEM of triplicate.

View larger version (141K): [in this window] [in a new window]   FIG. 5. 2D-PAGE autoradiograms of newly synthesized 30-kDa polypeptides (1–5) by Leydig cells isolated from rats that received TE implants. The cells were incubated with 500 µCi/ml [35S]methionine in the presence of maximally stimulating LH (100 ng/ml) for 0 (A), 1 (B), 2 (C), or 4 (D) h. Equivalent radioactive counts (2 x 106 cpm) were loaded.

View larger version (45K): [in this window] [in a new window]   FIG. 6. Quantitative analysis of 2D-PAGE autoradiograms showing time course of LH effects on 30-kDa proteins synthesized by Leydig cells isolated from TE-implanted rats. Protein quantities are expressed as the percentage of total radioactive proteins on the 2D autoradiograms. The bars are representative.

The molecular mass of the LH-responsive proteins (30 kDa), coupled with the observation that they responded rapidly to LH, suggested that the proteins might be StAR. Western blot analysis, using rabbit anti-StAR, identified polypeptides 2–5 in cells from both intact (Fig. 7A) and TE-treated (Fig. 7B) rats as StAR. Polypeptide 1 was not detected by the anti-StAR antibody, suggesting that this may be a new LH-induced protein or a StAR intermediate. Treatment of rats with TE in vivo caused reduced StAR (Fig. 7B) as compared to that in controls (Fig. 7A). Incubation of cells from TE-treated rats with LH caused obvious increases (50-fold) in polypeptides 2 and 3, with no apparent change in polypeptides 4 and 5 (Fig. 7C). Treatment of cells with alkaline phosphatase (overnight) revealed that polypeptides 2 and 3 were phosphoproteins (Fig. 7D).

View larger version (82K): [in this window] [in a new window]   FIG. 7. Western blots of 30-kDa polypeptides of Leydig cells from control (A) and TE-implanted rats (B), and Leydig cells from TE-implanted rats incubated with LH (C). The 30-kDa polypeptides present in 1 x 106 cells were resolved on 2D minigels, using rabbit anti-StAR polyclonal antibody and chemiluminescence. Treatment of cells with alkaline phosphate overnight prior to 2D-PAGE revealed that polypeptides 2 and 3 are phosphoproteins (D).

	DISCUSSION

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Suppression of endogenous LH in rats with steroid-filled Silastic implants or by hypophysectomy has been shown to result in rapid (days) reductions in Leydig cell testosterone production and, temporally associated with this, reductions in Leydig cell volume, smooth endoplasmic reticulum surface area, and steroidogenic enzyme activities [1, 4]. With respect to the latter, LH suppression has been shown to cause reductions in the activities of each of the steroidogenic enzymes involved in converting pregnenolone to testosterone [3]. Additionally, we recently found that TE treatment of rats results in dramatic, rapid reductions in P450 cholesterol side-chain cleavage mRNA activity and protein (unpublished results). LH stimulation of the cells, over the course of days to weeks, can result in complete restoration of structure and steroidogenic function [1, 4].

In the present study, TE treatment of rats for 10 days was accompanied by decreases in the total protein content of the cells, which would be expected of cells with reduced volume. Interestingly, there were no obvious qualitative differences in the major proteins able to be newly synthesized by these cells in comparison to cells from control rats. The predominant constituent proteins in Leydig cells from intact and TE-treated rats (as seen on silver-stained gels; not shown) also did not differ qualitatively. In response to LH stimulation of Leydig cells either from control or from TE-treated rats, however, obvious increases were seen in a family of three polypeptides, each of approximately 30-kDa molecular mass. Orme-Johnson [11, 12], Stocco [10, 18], and their colleagues first described a family of 30-kDa mitochondrial proteins, now called StAR proteins, that are synthesized by many (but not all) steroidogenic cells in response to acute stimulation of the cells by their trophic hormones [9–12] or by dibutyryl cAMP [11, 12], and whose appearance is correlated with acute increases in steroidogenesis. In the present study, anti-StAR antibody recognized two of the three LH-responsive 30-kDa proteins and, in addition, two members of the family seen in cells cultured in the presence or absence of LH. It is not known whether or not the polypeptide not recognized by the anti-StAR antibody, polypeptide 1, belongs to the StAR family.

In previous studies, the timing of StAR protein synthesis in relationship to steroidogenesis was reported for transformed, progesterone-producing cells [10, 22], but this relationship was not established in primary cultures of cells in which differentiated steroidogenic function (i.e., testosterone production) was retained. In the present studies, we show that three polypeptides, at least two of which are StAR proteins, increased by 1 h after incubation of Leydig cells from TE-treated rats with LH, and that testosterone production also increased. This timing is consistent with, but does not prove, a cause-effect relationship. The three polypeptides further increased through 2 and 4 h, as did testosterone production. These results suggest that increases in StAR may be required in order for increases in steroidogenesis to occur.

Clearly, however, factors in addition to StAR quantity are required to achieve maximal steroidogenesis. First, the immediate effects of StAR may be related less to StAR quantity than to StAR phosphorylation [30]. Second, testosterone production cannot be maximal under conditions in which there is diminished steroidogenic enzyme activity, as in Leydig cells from the TE-treated rats [1–4]. Third, there are other candidate proteins that have been suggested to be integrally involved in the acute regulation of steroidogenesis, including the peripheral benzodiazepine receptor (PBR) [31–34] and its endogenous ligand, diazepam-binding inhibitor (DBI) [35]. For example, in a recent publication, Papadopoulos et al. [34] reported that despite the presence of high levels of the 30-kDa StAR protein in the mitochondria of PBR-negative R2C rat tumor cells, the cells produced low amounts of steroids. Importantly, stable transfection of these cells with PBR cDNA resulted in the recovery of steroidogenic function. At this juncture, the relationship between StAR and PBR/DBI, and their respective roles in steroidogenesis, remain uncertain.

	ACKNOWLEDGMENTS

 
We are very grateful to Dr. Peter Wirth (NCI) for his assistance with image analysis and to Ms. Janet Folmer of the Hopkins Core Morphology Laboratory for assistance with photography.

	FOOTNOTES

 
¹ >This work was supported by NIH grants AG08321 (B.R.Z.), HD17481 (D.M.S.), and HD06268 (Hopkins Population Center).

² Correspondence. FAX: (410) 614–2356; lindiluo{at}welchlink.welch.jhu.edu

Accepted: March 12, 1998.

Received: December 1, 1997.

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