Rodent Leydig Cells Research Articles

Crosstalk between androgen-ZIP9 signalling and key regulators of mitochondrial dynamics in rodent leydig cells

Crosstalk between androgen-ZIP9 signalling and key regulators of mitochondrial dynamics in rodent leydig cells

Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics

Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion in response to LH.

The ABC transporter Mrp4/Abcc4 is required for Leydig cell protection from chemotherapeutic drugs (844.1)

The thiopurines are widely used to treat a variety of diseases such as pediatric acute lymphoblastic leukemia, adult promyelocytic leukemia, and irritable bowel disease. In both human and rodents, one toxic consequence attributed to 6‐mercaptopurine (6MP) is detrimental reproductive effects related to impaired testosterone (T) production. The ABC transporter Mrp4 plays a protective role against 6MP toxicity as it exports 6MP nucleotide metabolites. As Mrp4 is highly expressed in human and rodent Leydig cells (LCs), we hypothesize it protects LCs against 6MP by extruding nucleotides from cells, promoting LC survival to maintain normal T synthesis. Administration of 6MP to Mrp4+/+ and Mrp4‐/‐ mice produced a strong reduction in LCs, testicular T levels, and increased LC death in Mrp4‐/‐ mice. However, as LCs are post‐mitotic, the mechanism of death is unrelated to 6MP nucleotide incorporation into DNA. We show that cultured Mrp4‐/‐ LCs are highly susceptible to 6MP, exhibiting cytotoxicity at doses 6‐fold lower than Mrp4+/+ LCs. Leydig cell death by 6MP appears to involve a mitochondrial pathway as a pan‐caspase inhibitor blocked 6MP‐mediated death. We will report on the mechanisms of this 6MP‐mediated mitochondrial death. An important implication of our study is humans harboring non‐functional Mrp4 alleles may be at risk for LC toxicity and pathophysiological consequences of reduced T concentrations.Grant Funding Source: This work is supported by grants from the National Institutes of Health (NIH) and ALSAC.

Inhibition of steroidogenesis in Leydig cells by Müllerian-inhibiting substance

Müllerian-inhibiting substance (MIS), a member of the transforming growth factor-β family of cytokines that signal through a heteromeric complex of single-transmembrane serine/threonine kinase receptors, is required for Müllerian duct regression and normal reproductive tract development in the male embryo. However, the continued expression of MIS at high levels in males until puberty and its induction in females after birth suggested other roles for MIS. Additionally, Leydig cell development and steroidogenic capacity and ovarian follicle recruitment were abnormal in MIS-knockout or MIS-overexpressing mice. We have shown that MIS inhibits the cAMP-induced expression of cytochrome P450 C17α-hydroxylase/C17-20 lyase (Cyp17) mRNA both in vitro and in vivo. Our current efforts are to understand the molecular mechanisms regulating both MIS type II receptor (MISRII) expression and its signaling in rodent Leydig cell lines. MISRII expression in R2C cells requires both steroidogenic factor-1 and an unknown protein to bind to its proximal promoter in the context of 1.6 kb 5′-flanking DNA. When bound by MIS, signaling by the receptor in MA-10 cells blocks the protein kinase A-mediated induction of Cyp17 expression by a cAMP regulatory element-binding protein independent mechanism. We continue to investigate the molecular mechanisms of MISRII expression and possible interactions between MIS-regulated SMAD activation and cAMP signaling. These studies will provide a better understanding of the role played by MIS during postnatal life.

Enhanced purification and production of Müllerian inhibiting substance for therapeutic applications

It is almost 60 years since Prof. Alfred Jost reported the seminal observations regarding Müllerian inhibiting substance (MIS). His experiments clearly showed that a testicular product other than testosterone, a Müllerian inhibitor, was responsible for Müllerian duct regression. Twenty-five years later Dr. Picon established an organ culture assay which paved the way for the initial studies into the biochemistry and biology of Müllerian inhibiting substance, also known as Anti-Müllerian hormone (AMH), undertaken first in Dr. Nathalie Josso’s Laboratory in Paris then in our own laboratory in Boston. Purification of MIS led to cloning the human gene and production of recombinant human (rhMIS). MIS is a 140 kDa glycoprotein homodimer which is activated by a biosynthetic protease, cleaving MIS into an aminoterminus (110 kDa) and a carboxyterminus (25 kDa). The latter domain is sufficient for biological activities. MIS functions by interacting with two receptors; a type II binds the hormone and at type I that initiates downstream signaling. The MIS type II receptor has been cloned and functionally confirmed as distinct from that of other members of the TGFβ superfamily. MIS can employ a number of type I receptors (ALK2, ALK3, ALK6) and BMP receptor specific SMADS 1, 5, and 8 in various tissue specific contexts. Cell lines derived from human ovarian, breast, and prostate tumors, and from rodent Leydig cell tumors, which respond to MIS in growth inhibition assays, all express the MIS type II receptor. A variety of signal transduction pathways are associated with the grown inhibition mediated by MIS. For example, breast and prostate cancer cell lines use a MIS-mediated NFκB pathway leading to G1 arrest and apoptosis. The ovarian cancer cell lines employ a pathway which enhances p16, modulates the E2Fs, and induces apoptosis. These signal transduction events can establish new rational treatment strategies to complement the growth inhibitory effects mediated by MIS. These combination strategies are being tested in vitro, and where appropriate will be tested in vivo using the highly purified MIS preparations, prior to use in early human clinical trials.

Ontogenesis of leptin receptor in rat Leydig cells.

There are still many controversies about the role of leptin in reproductive function and sexual development. We recently demonstrated that leptin receptors are expressed in rodent Leydig cells and that leptin has inhibitory effects on hCG-stimulated testosterone production by adult rat Leydig cells in culture. In this study, we evaluated the expression of leptin receptor (Ob-R) in rat testes from gestational to adult age in comparison with the pattern of expression of relaxin-like factor (RLF), a specific marker of Leydig cell differentiation status. Immunohistochemical analysis showed that, in prenatal life, Ob-R immunoreactivity was absent at early embryonic ages (E14.5) and appeared at a late embryonic age (E19.5); in postnatal life, immunoreactivity was evident only after sexual maturation (35-, 60-, and 90-days old), whereas it was absent in testes from sexually immature rats (7-, 14-, and 21-days old). Immunoreaction was always confined to Leydig cells and no signal of Ob-R was detected within the tubules. The pattern of expression of Ob-R during testicular development was similar with that of RLF immunoreactivity, which was present in mature fetal as well as adult-type Leydig cells. In contrast with the findings in the testis, in the hypothalamus, the immunohistochemical pattern of Ob-R was very similar between pre- and postpubertal life. Reverse transcription-polymerase chain reaction studies showed that Ob-R expression was present in embryonic, prepubertal, and adult rat testes; semiquantitative analysis showed that mRNA levels were much higher in late versus early embryonic testes, as well as in mature adults versus sexually immature testes, with a gradual increase from younger to older ages. Functional studies showed that, while leptin (150 ng/ml) significantly inhibited hCG-stimulated testosterone production in adult rat Leydig cells (46% reduction; P > 0.01), it did not modify prepubertal rat Leydig cells steroidogenic function in vitro. In conclusion, we showed that, in rat testis, Ob-R expression is characteristic of mature Leydig cells (fetal and adult type) and it is functional in adult but not prepubertal life.

Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells.

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of -282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

Expression of functional leptin receptors in rodent Leydig cells.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450-17alpha messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men.

Expression of Functional Leptin Receptors in Rodent Leydig Cells

Expression of Functional Leptin Receptors in Rodent Leydig Cells

Leptin and androgens in male obesity: evidence for leptin contribution to reduced androgen levels.

Leptin circulates in plasma at concentrations that parallel the amount of fat reserves. In obese males, androgen levels decline in proportion to the degree of obesity. Recently, we have shown that in rodent Leydig cells leptin inhibits hCG-stimulated testosterone (T) production via a functional leptin receptor isoform; others have found that leptin inhibits basal and hCG-induced T secretion by testis from adult rats. In this study, we further investigated the relationship linking leptin and androgens in men. Basal and hCG-stimulated leptin and sex hormone levels were studied in a large group of men ranging from normal weight to very obese (body mass index, 21.8-55.7). Initial cross-sectional studies showed that circulating leptin and fat mass (FM) were inversely related with total and free T (r = -0.51 and r = -0.38, P < 0.01 and P < 0.05, respectively). Multiple regression analysis indicated that the correlation between leptin or FM and T was not lost after controlling for SHBG and/or LH and/or estradiol (E2) levels and that leptin was the best hormonal predictor of the lower androgen levels in obesity. Dynamic studies showed that in obese men the area under the curve of T and free T to LH/hCG stimulation (5000 IU i.m.) was 30-40% lower than in controls and inversely correlated with leptin levels (r = -0.45 and r = -0.40, P < 0.01 and P < 0.05, respectively). Also, LH/hCG-stimulation caused higher increases in 17-OH-progesterone to T ratio in obese men than in controls, whereas no differences were observed between groups either in stimulated E2 levels or in the E2/T ratio. In all subjects, the percentage increases from baseline in the 17-OH-progesterone to T ratio were directly correlated with leptin levels or FM (r = 0.40 and r = 0.45, P < 0.01), but not with E2 or other hormonal variables. In conclusion, our studies, together with previous in vitro findings, indicate that excess of circulating leptin may be an important contributor to the development of reduced androgens in male obesity.

Müllerian-inhibiting substance regulates androgen synthesis at the transcriptional level.

Müllerian-inhibiting substance (MIS) is a hormone produced by Sertoli cells of the fetal testes that causes regression of the Müllerian ducts, the precursors to female reproductive tract structures that are present in the bipotential urogenital ridge. MIS is also produced in the adult gonads of both males and females, albeit at much lower levels than those measured during the fetal and perinatal periods. Adult transgenic mice chronically overexpressing MIS exhibit severe gonadal abnormalities and, in males, dramatically reduced levels of testosterone, which might lead to the incomplete virilization observed in some of the males. To understand the roles played by MIS in the adult gonad, we performed Northern analyses to show that the MIS type II receptor is expressed in purified Leydig cells and in two rodent Leydig cell lines, R2C and MA-10. Addition of purified recombinant human MIS to cultures of both R2C and MA-10 cells reduced steroid production. With MA-10 cells, the reduction of testosterone secretion into the medium was reduced to 1/10th of that in the control culture, which provided us with a means to study the molecular mechanisms underlying MIS-mediated suppression of testosterone synthesis. Northern analysis revealed that after stimulation with cAMP, the expression of messenger RNA for P450c17 hydroxylaselyase, the enzyme that catalyzes the conversion of progesterone to androstenedione, was reduced to background levels in the presence of MIS. Addition of cycloheximide, a protein synthesis inhibitor, did not prevent the effect of MIS, indicating a direct effect of MIS signal transduction on the expression of P450c17. Analysis of the transcriptional activity of Cyp17, the gene for murine P450c17, with Cyp17 promoter/luciferase reporter constructs shows that MIS regulates the transcription of Cyp17 in a concentration- and time-dependent manner. From our results, we conclude that MIS might play a physiological role in maintaining testosterone homeostasis. These findings will allow us in the future to use the transcriptional regulation of Cyp17 as a model to uncover the signal transduction pathways of MIS and the molecular mechanisms of its suppression of androgen synthesis.

Leydig cell hyperplasia and adenoma formation: Mechanisms and relevance to humans

Leydig cell adenomas are observed frequently in studies evaluating the chronic toxicity of chemical agents in laboratory animals. Doubts have been raised about the relevance of such responses for human risk assessment, but the question of relevance has not been evaluated and presented in a comprehensive manner by a broad group of experts. This article reports the consensus conclusions from a workshop on rodent Leydig cell adenomas and human relevance. Five aspects of Leydig cell biology and toxicology were discussed: 1) control of Leydig cell proliferation; 2) mechanisms of toxicant-induced Leydig cell hyperplasia and tumorigenesis; 3) pathology of Leydig cell adenomas; 4) epidemiology of Leydig cell adenomas; and 5) risk assessment for Leydig cell tumorigens. Important research needs also were identified. Uncertainty exists about the true incidence of Leydig cell adenomas in men, although apparent incidence is rare and restricted primarily to white males. Also, surveillance databases for specific therapeutic agents as well as nicotine and lactose that have induced Leydig cell hyperplasia or adenoma in test species have detected no increased incidence in humans. Because uncertainties exist about the true incidence in humans, induction of Leydig cell adenomas in test species may be of concern under some conditions. Occurrence of Leydig cell hyperplasia alone in test species after lifetime exposure to a chemical does not constitute a cause for concern in a risk assessment for carcinogenic potential, but early occurrence may indicate a need for additional testing. Occurrence of Leydig cell adenomas in test species is of potential concern as both a carcinogenic and reproductive effect if the mode of induction and potential exposures cannot be ruled out as relevant for humans. The workgroup focused on seven hormonal modes of induction of which two, GnRH agonism and dopamine agonism, were considered not relevant to humans. Androgen receptor antagonism, 5α-reductase inhibition, testosterone biosynthesis inhibition, aromatase inhibition, and estrogen agonism were considered to be relevant or potentially relevant, but quantitative differences may exist across species, with rodents being more sensitive. A margin of exposure (MOE; the ratio of the lowest exposure associated with toxicity to the human exposure level) approach should be used for compounds causing Leydig cell adenoma by a hormonal mode that is relevant to humans. For agents that are positive for mutagenicity, the decision regarding a MOE or linear extrapolation approach should be made on a case-by-case basis. In the absence of information about mode of induction, it is necessary to utilize default assumptions, including linear behavior below the observable range. All of the evidence should be weighed in the decision-making process.

Regulation of Leydig cell function by inhibins and activins

The role of inhibin related proteins is not confined to the regulation of pituitary FSH. These proteins are now recognised as growth and differentiation factors and in the testis they regulate both epithelial and interstitial cell functions. In species such as rodents and pigs, inhibin and activin are considered to be paracrine regulators of steroidogenesis whereby they modulate the endocrine LH signal. Porcine and rodent Leydig cells have been shown in vitro to respond to exogenously added activin A. We have previously proposed that activins have a significant role in Leydig cell development and in the fetal testis and at puberty, i.e. activin (like TGF β) holds Leydig cell growth in abeyance until differentiation or puberty. The roles of activins, inhibins and binding proteins for these ligands, such as follistatins, in the development of Leydig cells remains to be determined.

The proopiomelanocortin gene is expressed as both full-length and 5'truncated transcripts in rodent Leydig cells

A reverse transcription polymerase chain reaction (RT-PCR) assay was developed to distinguish between full-length transcripts and functionally aberrant, truncated transcripts of the proopiomelanocortin (POMC) gene. This assay was applied to RNA obtained from the testes of rats and mice, to Percoll-purified primary mouse Leydig cells, and to various Leydig-derived cell lines. The results indicate a very low level of expression for the full-length transcripts in whole rodent testis, in mouse Leydig cells and in the MA10 cell line. In Leydig cells, the major transcripts of the POMC gene were the shorter, truncated transcripts where the regions corresponding to exons 1 and 2 are missing.

Substance P- and neuron-specific enolase-like immunoreactivity of rodent Leydig cells in tissue section and cell culture

Comparative immunocytochemical studies concerning the presence of a neurotransmitter (substance P), and a marker of neuroendocrine cells (neuron-specific enolase), in the Leydig cells of 3 mammalian species (golden hamster, guinea pig, and rat) were carried out on tissue sections and cell cultures. Substance P(SP)-like immunoreactivity (-LI) was found to be present in both fetal and adult generation of Leydig cells in hamster and guinea pig, while neuron-specific enolase (NSE)-LI was detected in Leydig cells of the 3 species at all stages studied: fetal, neonatal and adult. In primary cultures of Leydig cells isolated from adult hamster testes, SP- and NSE-LI was also established. This result was considered as an indirect evidence for the synthesis of the substances under study by the steroidogenic cells of the testis. A comparison of these results with data obtained in vivo suggests that Leydig cells may be related to the APUD- or the diffuse neuroendocrine system.

Site-Directed Mutagenesis of the Human Chorionic Gonadotropin β-Subunit: Bioactivity of a Heterologous Hormone, Bovine α-Human des-(122–145)β

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.

Measurement of steroidogenesis in rodent Leydig cells: a comparison between pregnenolone and testosterone production

The efficiency and specificity of inhibition of pregnenolone metabolism in mature, immature rat Leydig cells, mouse and tumour Leydig cells by SU-10603, a 17α-hydroxylase inhibitor and epostane (WIN-32729), a 3β-hydroxysteroid dehydrogenase inhibitor, were studied. Metabolism of [ 14C]pregnenolone by mature rat Leydig cells was inhibited for more than 95% in the presence of 20 μM SU-10603 and 5 μM epostane. The sum of the different steroids produced by Leydig cells from immature rats incubated in the presence of a 5α-reductase inhibitor was only 80% of pregnenolone production in the presence of SU-10603 and epostane. Pregnenolone metabolism could also be inhibited in tumour Leydig cells but not in mouse Leydig cells. Pregnenolone and testosterone production by Leydig cells from mature rats were similar when steroidogenesis is maximally stimulated by luteinizing hormone (LH). However, in the presence of LH and bovine serum albumin (bSA), or 22 R-hydroxycholesterol and bSA, pregnenolone production was 1.7- and 6-fold higher, respectively, than testosterone production. The data show that for measuring the steroidogenic activity of Leydig cells estimation of pregnenolone production is more reliable than measuring testosterone production. At high activities of the cholesterol side-chain cleavage (CSCC) the conversion of pregnenolone into testosterone may become the rate-limiting step for testosterone production. Under all conditions the conversion of cholesterol into pregnenolone is the (hormonal regulated) rate-determining step for steroidogenesis.

Characterization of α-Adrenergic Binding Sites on Rodent Leydig Cells1

A radioligand binding technique was used to study beta-adrenergic binding sites on rodent Leydig cells. Beta-Adrenergic binding sites were found on Leydig cells in both the rat and mouse. Binding of [3H]CGP-12177 [4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazole-2-one] to purified rat Leydig cells was found to be saturable, temperature and time dependent, stereospecific, and readily reversible by the beta-adrenergic antagonist propranolol. Scatchard analysis revealed the presence of high-affinity sites with an apparent dissociation constant (Kd) of 0.79 +/- 0.22 nM and maximal binding capacity (Bmax) of 1716 +/- 245 sites per rat Leydig cell. Competition of various beta-adrenergic agonists and antagonists with [3H]CGP indicates an order of potency of L-isoproterenol greater than epinephrine = salbutamol greater than norepinephrine greater than D-isoproterenol and dl-propranolol = ICI 118,551 much greater than atenolol, respectively. These observations suggest that the binding sites are predominantly of the beta 2-receptor subtype. Incubation of freshly isolated rat Leydig cells with luteinizing hormone (100 ng/ml) caused consistent stimulation of androgen production, but only occasional stimulation by the beta-agonist isoproterenol (10 microM) was observed. However, these cells consistently responded to the beta-agonist after 3 h in primary cultures. These findings indicate that rodent Leydig cells possess beta-adrenergic binding sites and point out a possible dissociation between receptor recognition and physiologic response.