Role of proteoglycans on testosterone synthesis by purified Leydig cells from immature and mature rats.

In order to characterize an involvement of proteoglycans (PG) in the regulation of Leydig cell function, we have examined the effects of para-nitrophenyl-beta-D-xyloside (PNPX), a specific inhibitor of PG synthesis and para-nitrophenyl-beta-D-galactoside (PNPG), an inefficient structural analogue, on testosterone production by purified Leydig cells from immature and mature rats, in the presence or not of various concentrations of hCG during 24 h. Whatever the age, the addition of PNPX induces a decrease of [35S] and [3H] incorporations into cell layer associated-PG; these latter being less numerous (-50 and -25%, respectively in immature and mature rat), and less sulfated (-40%) when compared to control Leydig cells. In immature Leydig cells, the inhibition of PG synthesis decreases both the basal and weakly stimulable-hCG or -(Bu)2cAMP or -LH testosterone synthesis. In mature Leydig cells, the PG inhibition has no effect on testosterone production both in the absence of hCG and in the presence of weak amounts of hCG but increases it in the presence of subsaturating hCG concentrations. Whatever the age, the inhibition of PG synthesis is ineffective in the presence of saturating amounts of either hCG or (Bu)2cAMP. These effects are maintained in the presence of MIX, PMA, but are not observed in the presence of 22R-hydroxycholesterol. Therefore, our results suggest that in rat Leydig cells, the inhibition of PG synthesis affects the signal transduction at a step distal to cyclic AMP and more precisely, the cholesterol supply to the mitochondria by acting on its cellular distribution (free and esterified cholesterol).     

Corticotropin-releasing factor receptors and actions in rat Leydig cells

Rat Leydig cells possess functional high affinity receptors for corticotropin-releasing factor (CRF). CRF inhibited human chorionic gonadotropin (hCG)-induced androgen production in cultured fetal and adult Leydig cells in a dose-dependent manner, but it had no effect on basal testosterone secretion. Comparable inhibitory effects of CRF were observed in the presence or absence of 3-isobutyl-1-methylxanthine. CRF treatment caused a marked reduction of steroid precursors of the androgen pathway (from pregnenolone to testosterone) during gonadotropin stimulation, but it did not influence their basal levels. The inhibitory action of CRF on hCG-induced steroidogenesis was fully reversed by 8-bromo-cAMP but was not affected by pertussis toxin. The action of CRF was rapid; and it was blocked by coincubation with anti-CRF antibody. CRF caused no changes in hCG binding to Leydig cells, and in contrast to other target tissues, CRF did not stimulate cAMP production, indicating that CRF receptors are not coupled to Gs in Leydig cells. These studies have demonstrated that CRF-induced inhibition of the acute steroidogenic action of hCG is exerted at sites related to receptor/cyclase coupling or cAMP formation. The inhibitory effects of CRF in the Leydig cell do not occur through the Gi unit of adenylate cyclase, but could involve pertussis toxin-insensitive G protein(s). These observations demonstrate that CRF has a novel and potent antireproductive effect at the testicular level. Since CRF is synthesized in the testis and is present in Leydig cells, it is likely that locally produced CRF could exert negative autocrine modulation on the stimulatory action of luteinizing hormone on Leydig cell function.     

Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells

Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2.     

In vitro interactions between Sertoli cells and steroidogenic cells

Both the cell and the species specificities of the steroidogenic potentiating activity (SPA) of Sertoli cells on Leydig cells were studied using a coculture system. Coculture of purified pig Leydig cells with rat or pig Sertoli cells in the presence of FSH led in both cases, to a significant increase in hCG receptor number and in hCG-stimulated testosterone production. Similarly, coculture of bovine adrenal cells with rat or pig Sertoli cells enhanced the steroidogenic response of adrenal cells to ACTH and angiotensin II. Such effects were not observed when pig Leydig cells or bovine adrenal cells were cocultured with bovine aortic endothelial cells. Coculture of Sertoli and Leydig cells in the presence of hCG, resulted in a significant increase in FSH receptor number and in FSH-induced plasminogen activator activity. Such effects did not occur when Sertoli cells were cocultured with either adrenal or aortic endothelial cells.     

Percoll-gradient separation of Leydig cells from postnatal rat testes

The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data.     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paracrine effect of seminiferous tubule factors on rat Leydig cell testosterone production: Role of cytoskeleton

In Percoll purified Leydig cells from mature rat we have demonstrated that the basal testosterone production (9.5 ng/10⁶ Leydig cells/24 h) is increased 10-fold in presence of a saturating amount of hCG (1 IU/mL) and diminished in a dose-related manner when larger concentrations of gonadotropin are used to reach 14 ng/10⁶ Leydig cells for 50 IU of hCG. If 40% (v/v) seminiferous tubule medium (STM) is added together with hCG (1 IU/mL) to the incubation medium, a further increase (62%) of testosterone output is noticed. Obviously, when the testosterone production is low as a consequence of a higher dose of hCG (50 IU/mL), the STM (80%) improves the steroid synthesis five-fold (67.4 ng). Concerning the cytoskeletal components (microtubules, intermediate filaments and microfilaments) which have been examined in presence or absence of hCG and STM, we have found a rearrangement of cytoskeletal elements as well as cell-shape changes in relation with hormonal activity of the cells. The most prominent alterations of cytoskeletal elements have been observed after 24 h of incubation with 1 IU/mL of hCG added together with 80% of STM. The obtained results suggest that paracrine factor(s) presents in STM and acting in synergy with LH/hCG generate(s) the rearrangement of cytoskeletal structures which, in turn, facilitates the availability of cholesterol for the mitochondria and finally enhances the testosterone production in the rat Leydig cells.     

Paracrine regulation of testicular function

Data from several experimental approaches have been reviewed and the findings clearly indicate the existence of multiple interactions between testicular cells and the potential role of these interactions in the paracrine control of testicular functions. Both testicular interstitial fluid and spent media from cultured Sertoli cells had an acute steroidogenic effect on Leydig cells, and this effect is not species specific. The secretion of this steroidogenic factor(s), which is probably a protein, is enhanced by previous FSH treatment of Sertoli cells. Coculture for 2-3 days of pig Leydig cells with homologous or heterologous Sertoli cells enhances Leydig cell specific functions (hCG receptor number and hCG responsiveness) and induces Leydig cell hypertrophy. A similar but less pronounced trophic effect is seen when Leydig cells are cultured with spent media from Sertoli cells cultured in the presence of FSH and high concentrations of insulin, but the spent media from Sertoli cells cultured in the absence of these two hormones inhibits Leydig cell specific functions. Somatomedin-C might play an important role in the positive trophic effect of Sertoli cells on Leydig cells, since this peptide is secreted by Sertoli cells and it has trophic effects on the specific function of Leydig cells. Moreover, Sertoli cells, probably through a diffusible factor and cell-to-cell contacts, control the multiplication, meiotic reduction and maturation of germ cells. In turn, the activity of Sertoli cells is modulated by the stage of neighbouring germ cells. Thus, if a normal Sertoli cell function (which depends not only on FSH but also on Leydig and myoid cell secretory products) is an absolute requirement for germ cell multiplication and maturation, these cells, in turn, cyclically regulate Sertoli cell function and through these cells the size and probably the function of Leydig cells.     

LH contamination may explain FSH effects on rat Leydig cells

Treatment of immature, hypophysectomized male rats with 50 micrograms ovine FSH (NIH-FSH-S12) twice a day for 5 days stimulated the maximum quantity of 17 beta-hydroxyandrogen produced by isolated Leydig cells in response to hCG. Pretreatment of the FSH preparation with an LH antiserum in one study markedly reduced and in another study completely abolished this stimulatory effect of FSH, but only slightly impaired the capacity of the hormone to stimulate the Sertoli cell in vivo (epididymal androgen-binding protein). Administration of another highly potent FSH preparation (LER-1881) had no discernible effects on the dose-response characteristics of the Leydig cells but was superior to the NIH-FSH-S12 in its capacity for stimulating the Sertoli cell. When all hormone preparations were tested for their ability to stimulate steroid secretion from normal Leydig cells in vitro, a close correlation was obtained between their Leydig cell-stimulating activity (a measure of LH contamination) and their capacity to alter Leydig cell responsiveness after in-vivo treatment. FSH treatment had no effects on specific LH binding per 10(6) Leydig cells. It is concluded that the stimulatory influence of FSH on rat Leydig cells may to some extent be a result of the LH contaminating the hormone preparation.     

A transplantable rat Leydig cell tumor--5. Cellular localization of beta-adrenergic and prostaglandin receptors and their effects on testosterone production

The cellular localization of beta-adrenergic and prostaglandin (PG) receptors and their effects on adenylate cyclase activity (AC) and testosterone production in vitro were investigated in a transplantable rat Leydig cell tumor (H-540). Separation of the tumor cells in Percoll gradients revealed that the specific binding of [3H]PGE1 and [125I]Cyanopindolol was found in the same fraction as that of [125I]LH. This fraction--judged by light microscopy of smears--consisted of tumor Leydig cells. In addition, [125I]cyanopindolol was found specifically bound in the red blood cell fraction. In the Leydig tumor cells, approx 25% of the beta-adrenergic receptors was identified as beta 1-receptors, whereas approx 75% of the receptors were of the beta 2-subtype. The AC in Percoll purified Leydig tumor cells was stimulated by hCG (6-fold), PGE1 (2-fold), PGE2 (1.5-fold), PGI1 (2-fold) and isoproterenol (2-fold). The AC in the red blood cell fraction was stimulated by isoproterenol whereas the PGs and hCG had little or no effect. hCG, isoproterenol and PGE1 were able to stimulate testosterone production in vitro. At 44 h incubation, PGE1 was the most potent stimulator of testosterone production. In conclusion, tumor Leydig cells possess hCG, PGE1, PGI2 and beta-adrenergic receptors coupled to the AC. PGE1 and beta-adrenergic agonists stimulate testosterone production after prolonged incubation in vitro.     

Interleukin-1 alpha as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of the adult Leydig cell population in the postnatal testis

Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor alpha (TGFalpha) is a mitogen for mesenchymal precursor cells. Moreover, both TGFalpha and TGFbeta (to a lesser extent than TGFalpha) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Rat Leydig cells use apolipoprotein E depleted high density lipoprotein to regulate testosterone production

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 g/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 g protein HDL/ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.     

Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone

Testosterone (T) is an absolute requirement for spermatogenesis and is supplied by mature Leydig cells stimulated by LH. We previously showed in gonadotropin-deficient hpg mice that T alone initiates qualitatively complete spermatogenesis bypassing LH-dependent Leydig cell maturation and steroidogenesis. However, because maximal T effects do not restore testis weight or germ cell number to wild-type control levels, additional Leydig cell factors may be involved. We therefore examined 1). whether chronic hCG administration to restore Leydig cell maturation and steroidogenesis can restore quantitatively normal spermatogenesis and testis development and 2). whether nonandrogenic Leydig cell products are required to initiate spermatogenesis. Weanling hpg mice were administered hCG (0.1-100 IU i.p. injection three times weekly) or T (1-cm subdermal Silastic implant) for 6 weeks, after which stereological estimates of germinal cell populations, serum and testicular T content, and testis weight were evaluated. Human CG stimulated Leydig cell maturation and normalized testicular T content compared with T treatment where Leydig cells remained immature and inactive. The maximal hCG-induced increases in testis weight and serum T concentrations were similar to those for T treatment and produced complete spermatogenesis characterized by mature, basally located Sertoli cells (SCs) with tripartite nucleoli, condensed haploid sperm, and lumen development. Compared with T treatment, hCG increased spermatogonial numbers, but both hCG and T had similar effects on numbers of spermatocytes and round and elongated spermatids per testis as well as per SC. Nevertheless, testis weight and germ cell numbers per testis and per SC remained well below phenotypically normal controls, confirming the involvement of non-Leydig cell factors such as FSH for quantitative normalization of spermatogenesis. We conclude that hCG stimulation of Leydig cell maturation and steroidogenesis is not required, and that T alone mostly replicates the effects of hCG, to initiate spermatogenesis. Because T is both necessary and sufficient for initiation of spermatogenesis, it is likely that T is the main Leydig cell secretory product involved and that additional LH-dependent Leydig cell factors are not essential for induction of murine spermatogenesis.     

An early steroidogenic defect in hormone-induced Leydig cell desensitization

To define the nature of the lesion of the early steroidogenic pathway (prior to pregnenolone formation) in gonadotropin-induced desensitization of rat testicular Leydig cells, we evaluated cholesterol side-chain cleavage activity in isolated mitochondria by measurement of pregnenolone synthesis and [14C]isocaproic acid formation from [26-14C]cholesterol. The enzyme activity was shown to be reduced after in vivo treatment with 10 micrograms hCG when compared to that of mitochondria from control animals only when measured in the presence of limiting NADPH concentrations (100 microM). Sonication of mitochondria from control and hCG-treated rats caused complete loss of cholesterol side-chain cleavage activity. When acetone-powdered adrenal cell mitochondria were employed as the source of the enzyme, the addition of sonicated Leydig cell mitochondria from control and hCG-treated animals caused the same differences as those observed with intact Leydig cell mitochondria in the presence of low concentration of NADPH. The Km value of the adrenal enzyme for NADPH incubated with Leydig cell mitochondria increased from 0.111 mM in control to 0.37 mM after hCG, with no changes in Vmax. Moreover, cholesterol side-chain cleavage activity of adrenal mitochondria assayed in the presence of 100 microM cholesterol was progressively inhibited by increasing amounts of acetone powder from Leydig cell mitochondria of control and hCG-treated rats, with ID50 of 500 and 280 micrograms protein, respectively. The inhibiting factor was not a lipid or steroid but a heat-labile protein, with an approximate Stokes radius of 4.8 nm and an isoelectric point of 5.05 +/- 0.23 SD (n = 8). The inhibitory effect was confined to the Leydig cell mitochondrial membrane, and was not related to changes in oxidative phosphorylation. NADPH was not directly oxidized or immobilized by the mitochondrial factor, and this inhibiting substance was not adsorbed on 2',5' ADP-Sepharose 4B. These results have demonstrated that a heat-labile inhibiting protein factor is present in mitochondria from normal Leydig cells and is markedly activated or increased by hCG treatment. This substance that competitively modulates cholesterol side-chain cleavage activity could contribute to the early steroidogenic lesion, and also serve as an endogenous modulator of steroid hormone biosynthesis.     

Monoclonal antibodies against rat Leydig cell surface antigens

Monoclonal antibodies (MAbs) directed against the Leydig cell surface may be used to identify this cell in testicular preparations. Collagenase-dispersed adult rat interstitial cells were fractionated on Percoll density gradients, and Leydig cell-enriched fractions were used to prepare MAbs. Hybridomas were screened by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF) on isolated testicular cells and immunocytochemical localization on paraffin sections of adult testes. In density gradient fractions, immunoglobulin (Ig) M MAbs (LC-1C6 and LC-6H6) labeled the surface of cells possessing the morphological characteristics of Leydig cells. The density gradient profiles of MAb-binding activity observed by IIF and ELISA were parallel with the Leydig cell distribution as determined by [125I]-human chorionic gonadotropin (hCG) binding, testosterone response to hCG in vitro, 3 beta-hydroxysteroid dehydrogenase histochemistry and electron microscopy. The MAbs prominently labeled most interstitial cells in sections, but there was little or no labeling of connective tissue, endothelial or seminiferous tubule cells. Both MAbs recognized components of Mr 58,000 in Western blots of Leydig cell-enriched extracts. The results indicate that LC-1C6 and LC-6H6 recognize antigens on the Leydig cell surface that are not present on other isolated testicular cells from the adult rat. These MAbs are specific markers of the Leydig cell in situ and in vitro.     

Effect of serum and serum lipoproteins on testosterone production by adult rat Leydig cells in vitro.

The effect of serum factors other than luteinizing hormone on Leydig cell testosterone secretion was examined using an in vitro bioassay system based on the stimulation of purified adult rat Leydig cells during a 20 h incubation in the presence of a maximal dose of human chorionic gonadotrophin (hCG). Charcoal-extracted serum and testicular interstitial fluid (IF) from normal adult male rats were separated into lipoprotein and lipoprotein-deficient fractions by density ultracentrifugation. Stimulatory bioactivity was found in the lipoprotein fraction of both serum and IF, although the levels of lipoprotein and corresponding bioactivity recovered from IF were significantly lower (25%) than those of serum. There was no difference between the effects of serum lipoproteins on Leydig cell testosterone production stimulated by either hCG or dibutyryl cAMP. In time-course studies, the serum lipoprotein fraction had no effect on hCG-stimulated testosterone production in vitro at 3.0 or 6.0 h, but partially prevented the normal decline in hCG-stimulated testosterone production after 6.0 h. In contrast, unfractionated serum was stimulatory at all time-points. In the absence of hCG, the lipoprotein fraction was stimulatory at both 6.0 and 20 h, although not at 3.0 h. The lipoprotein-deficient protein fraction of serum had no effect on hCG-stimulated testosterone production alone, but significantly enhanced the bioactivity of the lipoprotein fraction, and caused a dose-dependent stimulation of testosterone production in the presence of a constant concentration of serum lipoproteins. Both a stimulatory peak of activity (apparent MW 40-80 kDa), and a large MW (> 100 kDa) inhibitor of testosterone production were identified in serum after fractionation by gel filtration (Sephadex G-100). The data indicate that (i) the stimulatory effect of serum on short-term hCG-stimulated Leydig cell testosterone production in vitro is predominantly due to the serum lipoprotein fraction, possibly by providing additional precursors for testosterone synthesis, (ii) the biological activity of the lipoproteins is influenced by both stimulatory and inhibitory serum proteins in addition to luteinizing hormone, and (iii) that serum lipoproteins may be involved in supporting Leydig cell steroidogenesis in vivo.     

In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen.

In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S-F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors-the characteristic of fetal Leydig cells.