Use of in vitro methodology to investigate phthalate effects on the differentiation of seminiferous tubule-associated stem cells to form Leydig cells and on the Leydig cells derived from the stem cells.

Introduction: Leydig cells isolated from the testis are able to sustain high levels of testosterone production in vitro, but only for up to 3 days. Such cells are valuable for addressing the acute effects of chemicals on steroidogenic function, but not for repeated or chronic effects. Methodology is now available by which adult Leydig cells can be derived in vitro from seminiferous tubule-associated stem cells. In contrast to isolated Leydig cells, the Leydig cells derived in this way can synthesize and secrete high levels of testosterone for months. Herein, we asked whether this system might be used to address the effect of mono-(2-ethylhexyl) phthalate (MEHP) exposure on the formation of Leydig cells from tubule-associated stem cells, and on the Leydig cells after their formation. Methods: Adult Brown Norway rats received an intraperitoneal injection of ethane dimethanesulfonate (EDS) to eliminate the existing Leydig cells. Seminiferous tubules then were isolated and cultured in medium containing Insulin-Transferrin- Selenium (ITS), Smoothened Agonist (SAG), and luteinizing hormone (LH). Results: Culture of the tubules for 8 weeks resulted in the formation of cells on the surfaces of the tubules that stained for CYP11A1 and STAR and produced high levels of testosterone. When the tubules were cultured in medium containing increasing concentrations of MEHP, concentration-dependent effects on Leydig cell formation occurred. To determine the effect of MEHP on newly produced Leydig cells, tubules were cultured for 8 weeks in the absence of MEHP, resulting in the formation of adult Leydig cells, and then in medium containing increasing concentrations of MEHP. Concentration-dependent decreases in testosterone production by the adult Leydig cells were seen, and these decreases proved to be reversible. Discussion: The use of this new system should make it possible to determine the mechanisms by which acute, repeated, or chronic exposures to increasing concentrations of MEHP and/or exposure to other chemicals affect the formation of Leydig cells from stem cells, as well as the steroidogenic function of adult Leydig cells.

Mitochondrial dynamics, Leydig cell function, and age-related testosterone deficiency.

The mitochondrial translocator protein (18 kDa; TSPO) is a high-affinity cholesterol-binding protein that is an integral component of the cholesterol trafficking scaffold responsible for determining the rate of cholesterol import into the mitochondria for steroid biosynthesis. Previous studies have shown that TSPO declines in aging Leydig cells (LCs) and that its decline is associated with depressed circulating testosterone levels in aging rats. However, TSPO's role in the mechanistic decline in LC function is not fully understood. To address the role of TSPO depletion in LC function, we first examined mitochondrial quality in Tspo knockout mouse tumor MA-10 nG1 LCs compared to wild-type MA-10 cells. Tspo deletion caused a disruption in mitochondrial function and membrane dynamics. Increasing mitochondrial fusion via treatment with the mitochondrial fusion promoter M1 or by optic atrophy 1 (OPA1) overexpression resulted in the restoration of mitochondrial function and mitochondrial morphology as well as in steroid formation in TSPO-depleted nG1 LCs. LCs isolated from aged rats form less testosterone than LCs isolated from young rats. Treatment of aging LCs with M1 improved mitochondrial function and increased androgen formation, suggesting that aging LC dysfunction may stem from compromised mitochondrial dynamics caused by the age-dependent LC TSPO decline. These results, taken together, suggest that maintaining or enhancing mitochondrial fusion may provide therapeutic strategies to maintain or restore testosterone levels with aging.

Single-cell RNA sequencing of adult rat testes after Leydig cell elimination and restoration.

Spermatogenesis is an efficient, complex, and highly organized proliferation and differentiation process that relies on multiple factors including testosterone produced by the Leydig cells. Although the critical role played by testosterone in spermatogenesis is well recognized, the mechanism by which it works is still not completely understood, partially due to the inability to specifically and precisely monitor testosterone-dependent changes within developing germ cells. Here we present single-cell RNA sequencing data from10,983 adult rat testicular cells after the rats were treated with ethanedimethanesulfonate, which temporarily eliminates Leydig cells. The elimination and recovery of Leydig cells represented a complete testosterone depletion and restoration cycle. The dataset, which includes all developing germ cells from spermatogonia to spermatozoa, should prove useful for characterizing developing germ cells, their regulatory networks, and novel cell-specific markers. The dataset should be particularly useful for exploring the effects of the androgen environment on the regulation of spermatogenesis. As this is the first single-cell RNA-Seq dataset for rat testes, it can also serve as a reference for future studies.

Identification of Rat Testicular Leydig Precursor Cells by Single-Cell-RNA-Sequence Analysis.

Stem Leydig cells (SLCs) play a critical role in the development and maintenance of the adult Leydig cell (ALC) population. SLCs also are present in the adult testis. Their identification, characteristics, and regulation in the adult testis remain uncertain. Using single-cell RNA-seq, we found that the mesenchymal stromal population may be involved in ALC regeneration. Upon ALC elimination, a fraction of stromal cells begins to proliferate while a different fraction begins to differentiate to ALCs. Transcriptomic analysis identified five stromal clusters that can be classified into two major groups representing proliferation and differentiation populations. The proliferating group represents stem cells expressing high levels of CD90, Nes, Lum, Fn and Gap43. The differentiating group represents a progenitor stage that is ready to form ALCs, and specifically expresses Vtn, Rasl11a, Id1 and Egr2. The observation that the actively dividing cells after ALC loss were not those that formed ALCs suggests that stem cell proliferation and differentiation are regulated separately, and that the maintenance of the stromal stem cell pool occurs at the population level. The study also identified specific markers for the major interstitial cell groups and potential paracrine factors involved in the regulation of SLCs. Our data suggest a new theory about SLC identity, proliferation, differentiation, and regulation.

MEHP induces alteration of mitochondrial function and inhibition of steroid biosynthesis in MA-10 mouse tumor Leydig cells.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in manufacturing. Previous studies have shown that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, has inhibitory effects on luteinizing hormone (LH)-stimulated steroid biosynthesis by Leydig cells. The molecular mechanisms underlying its effects, however, remain unclear. In the present study, we examined the effects of MEHP on changes in mitochondrial function in relationship to reduced progesterone formation by MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with MEHP (0-300 µM for 24 h) resulted in dose-dependent inhibition of LH-stimulated progesterone biosynthesis. Biochemical analysis data revealed that the levels of the mature steroidogenic acute regulatory protein (STAR), a protein that works at the outer mitochondrial membrane to facilitate the translocation of cholesterol for steroid formation, was significantly reduced in response to MEHP exposures. MEHP also caused reductions in MA-10 cell mitochondrial membrane potential (ΔΨm) and mitochondrial respiration as evidenced by decreases in the ability of the mitochondria to consume molecular oxygen. Additionally, significant increases in the generation of mitochondrial superoxide were observed. Taken together, these results indicate that MEHP inhibits steroid formation in MA-10 cells at least in part by its effects on mitochondrial function.

Sirt1 and Nrf2: regulation of Leydig cell oxidant/antioxidant intracellular environment and steroid formation†.

Previous studies reported that, with aging, Leydig cell intracellular antioxidants are reduced in concentration and intracellular ROS levels increase, suggesting that oxidant/antioxidant imbalance may contribute to the reduced testosterone production that characterizes the aging cells. As yet, little is known about how the Leydig cell oxidant/antioxidant environment is regulated. Sirt1, an enzyme that deacetylates transcription factors, and the transcription factor Nrf2, have been shown to be associated with cellular response to oxidative stress. We hypothesized that Sirt1 and/or Nrf2 might be involved in regulating the oxidant/antioxidant environment of Leydig cells, and therefore, the testosterone production. We found that Sirt1 and Nrf2 are present in the Leydig cells of Brown Norway rats, though reduced in aged cells. In MA-10 cells in which Sirt1 or Nrf2 were suppressed by nicotinamide (NAM) or ML385, respectively, or in which siRNAs were used for knockdown of Sirt1 or Nrf2, increased ROS levels and decreased progesterone production occurred. In rat Leydig cells, inhibition of Sirt1 by culturing the cells with NAM resulted in increased ROS and reduced testosterone production, and subsequent removal of NAM from the culture medium resulted in increased testosterone production. Activation of rat Leydig cells Sirt1 with honokiol or of Nrf2 with sulforaphane resulted in the maintenance of testosterone production despite the exposure of the cells to oxidizing agent. These results, taken together, suggest that Sirt1 and Nrf2 are involved in maintaining the Leydig cell oxidant/antioxidant environment, and thus in maintaining steroid production.

TSPO ligand FGIN-1-27 controls priapism in sickle cell mice via endogenous testosterone production.

Priapism, a prolonged penile erection in the absence of sexual arousal, is common among patients with sickle cell disease (SCD). Hypogonadism is also common in patients with SCD. While the administration of exogenous testosterone reverses hypogonadism, it is contraceptive. We hypothesized that the stimulation of endogenous testosterone production decreases priapism by normalizing molecular signaling involved in penile erection without decreasing intratesticular testosterone production, which would affect fertility. Treatment of SCD mice with FGIN-1-27, a ligand for translocator protein (TSPO) that mobilizes cholesterol to the inner mitochondrial membrane, resulted in eugonadal levels of serum testosterone without decreasing intratesticular testosterone production. Normalized testosterone levels, in turn, decreased priapism. At the molecular level, TSPO restored phosphodiesterase 5 activity and decreased NADPH oxidase-mediated oxidative stress in the penis, which are major molecular signaling molecules involved in penile erection and are dysregulated in SCD. These results indicate that pharmacologic activation of TSPO could be a novel, targetable pathway for treating hypogonadal men, particularly patients with SCD, without adverse effects on fertility.

Amhr2-Cre-Mediated Global Tspo Knockout.

Although the role of translocator protein (TSPO) in cholesterol transport in steroid-synthesizing cells has been studied extensively, recent studies of TSPO genetic depletion have questioned its role. Amhr2-Cre mice have been used to generate Leydig cell-specific Tspo conditional knockout (cKO) mice. Using the same Cre line, we were unable to generate Tspo cKO mice possibly because of genetic linkage between Tspo and Amhr2 and coexpression of Amhr2-Cre and Tspo in early embryonic development. We found that Amhr2-Cre is expressed during preimplantation stages, resulting in global heterozygous mice (gHE; Amhr2-Cre+/-,Tspo -/+). Two gHE mice were crossed, generating Amhr2-Cre-mediated Tspo global knockout (gKO; Tspo -/-) mice. We found that 33.3% of blastocysts at E3.5 to E4.5 showed normal morphology, whereas 66.7% showed delayed development, which correlates with the expected Mendelian proportions of Tspo +/+ (25%), Tspo -/- (25%), and Tspo +/- (50%) genotypes from crossing 2 Tspo -/+ mice. Adult Tspo gKO mice exhibited disturbances in neutral lipid homeostasis and reduced intratesticular and circulating testosterone levels, but no change in circulating basal corticosterone levels. RNA-sequencing data from mouse adrenal glands and lungs revealed transcriptome changes in response to the loss of TSPO, including changes in several cholesterol-binding and transfer proteins. This study demonstrates that Amhr2-Cre can be used to produce Tspo gKO mice instead of cKO, and can serve as a new global "Cre deleter." Moreover, our results show that Tspo deletion causes delayed preimplantation embryonic development, alters neutral lipid storage and steroidogenesis, and leads to transcriptome changes that may reflect compensatory mechanisms in response to the loss of function of TSPO.

Celebrating the Silver Anniversary of the North American Testis Workshop.

OBJECTIVE: To provide an overview of the history of the North American Testis Workshop (NATW), of its relationship to the American Society of Andrology (ASA), and of the publications that resulted from the first 25 workshops. METHODS: The collection of volumes and journal articles that relate to the NATW was searched. DISCUSSION AND CONCLUSION: During the first twenty-five meetings of the NATW, a remarkable number of breakthroughs regarding every aspect of the testis were presented. We anticipate that with the acceleration of new genetic, epigenetic, and molecular knowledge of the functions of testicular cells, we will continue to learn about the discovery of new and clinically important aspects of testicular function during the next twenty-five NATWs.

Effects of pharmacologically induced Leydig cell testosterone production on intratesticular testosterone and spermatogenesis†.

The Leydig cells of the mammalian testis produce testosterone (T) in response to luteinizing hormone (LH). In rats and men with reduced serum T levels, T replacement therapy (TRT) will raise T levels, but typically with suppressive effects on sperm formation. The rate-determining step in T formation is the translocation of cholesterol to the inner mitochondrial membrane, mediated by protein-protein interactions of cytosolic and outer mitochondrial membrane proteins. Among the involved proteins is cholesterol-binding translocator protein (TSPO) (18 kDa TSPO). We hypothesized that in contrast to TRT, the administration of the TSPO agonist N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27), by stimulating the ability of the Leydig cells to produce T, would result in the elevation of serum T levels while maintaining intratesticular T concentration and therefore without suppression of spermatogenesis. Age-related reductions in both serum and intratesticular T levels were seen in old Brown Norway rats. Both exogenous T and FGIN-1-27 increased serum T levels. With exogenous T, serum LH and Leydig cell T formation were suppressed, and intratesticular T was reduced to below the concentration required to maintain spermatogenesis quantitatively. In contrast, FGIN-1-27 stimulated Leydig cell T formation, resulting in increased serum T without reductions in intratesticular T concentrations or in testicular sperm numbers. FGIN-1-27 also significantly increased serum and intratesticular T levels in rats made LH-deficient by treatment with the gonadotropin-releasing hormone antagonist cetrorelix. These results point to a possible approach to increasing serum T without negative effects on spermatogenesis, based upon stimulating T production by the Leydig cells themselves rather than administering T exogenously.

Stem Leydig Cells in the Adult Testis: Characterization, Regulation and Potential Applications.

Androgen deficiency (hypogonadism) affects males of all ages. Testosterone replacement therapy (TRT) is effective in restoring serum testosterone and relieving symptoms. TRT, however, is reported to have possible adverse effects in part because administered testosterone is not produced in response to the hypothalamic-pituitary-gonadal (HPG) axis. Progress in stem cell biology offers potential alternatives for treating hypogonadism. Adult Leydig cells (ALCs) are generated by stem Leydig cells (SLCs) during puberty. SLCs persist in the adult testis. Considerable progress has been made in the identification, isolation, expansion and differentiation of SLCs in vitro. In addition to forming ALCs, SLCs are multipotent, with the ability to give rise to all 3 major cell lineages of typical mesenchymal stem cells, including osteoblasts, adipocytes, and chondrocytes. Several regulatory factors, including Desert hedgehog and platelet-derived growth factor, have been reported to play key roles in the proliferation and differentiation of SLCs into the Leydig lineage. In addition, stem cells from several nonsteroidogenic sources, including embryonic stem cells, induced pluripotent stem cells, mature fibroblasts, and mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord have been transdifferentiated into Leydig-like cells under a variety of induction protocols. ALCs generated from SLCs in vitro, as well as Leydig-like cells, have been successfully transplanted into ALC-depleted animals, restoring serum testosterone levels under HPG control. However, important questions remain, including: How long will the transplanted cells continue to function? Which induction protocol is safest and most effective? For translational purposes, more work is needed with primate cells, especially human.

Cholesterol accumulation, lipid droplet formation, and steroid production in Leydig cells: Role of translocator protein (18-kDa).

BACKGROUND: Cholesterol import into the mitochondria of steroid-producing cells is the rate-determining step in steroidogenesis. Numerous studies have provided evidence that the cholesterol-binding translocator protein (18 kDa TSPO) plays an important role in cholesterol translocation into mitochondria and that it also might act on cholesterol homeostasis. Several TSPO-specific ligands have been shown to increase steroid production in vitro and in vivo. OBJECTIVES: The present study assessed the effects of the TSPO drug ligand FGIN-1-27 on cholesterol accumulation and lipid droplet formation in relationship to steroid formation. MATERIALS AND METHODS: Using MA-10 and primary Leydig cells, immunocytochemical and molecular methods were used to examine cholesterol accumulation, the formation of lipid droplets, and steroid formation in response to LH and FGIN-1-27. Additionally, we determined the effects of Tspo knockout by CRISPR/Cas9, and of siRNA knockdowns of Tspo and Plin2 (Perilipin 2; also known as adipose differentiation-related protein, ADFP) on LH- and FGIN-1-27-induced steroidogenesis. RESULTS: In response to LH and FGIN-1-27, cultured MA-10 cells and primary Leydig cells increased steroid formation, cholesterol accumulation, and lipid droplet formation. Cholesterol accumulation in the lipid droplets also was increased in Tspo knockout cells. Knockout of Tspo or its knockdown in MA-10 cells resulted in reduced progesterone formation in response to both LH and FGIN-1-27, as did knockdown of Plin2. Steroid production also was inhibited by the cholesteryl ester hydrolase inhibitor diethylumbelliferyl phosphate. DISCUSSION AND CONCLUSION: These results support the conclusion that FGIN-1-27 stimulates steroid formation by increasing TSPO-mediated cholesterol translocation into the inner mitochondria for steroidogenesis, as well as into the cytosol for lipid droplet formation. FGIN-1-27 also increased steroid formation at least in part by inducing the conversion of cholesteryl ester located in lipid droplets to cholesterol, thus making available more substrate for steroid formation.

Testosterone treatment of aged male mice improves some but not all aspects of age-associated increases in influenza severity.

The severity of influenza increases with age, with worse disease in aged males than females. Testosterone concentrations decline with age in males, which may impact influenza pathogenesis. Aged male mice were treated with testosterone or placebo and outcomes during influenza A virus (IAV) infection were compared with adult male mice. Aged males experienced greater morbidity and mortality than adult males, which was partially improved by testosterone treatment of aged males. Aged males cleared IAV from lungs slower than adult males, regardless of testosterone treatment. As compared with adult males, aged males experienced pulmonary, but not systemic, cytokine dysregulation, and delayed influx and contraction of IAV-specific CD8+ T cells in the lungs. Testosterone treatment in aged males partially restored pulmonary cytokine responses to levels consistent with adult males but did not alter the age-associated changes in IAV-specific CD8+ T cells. Testosterone only modestly improves outcomes of influenza in aged males.

Redox regulation of hormone sensitive lipase: Potential role in the mechanism of MEHP-induced stimulation of basal steroid synthesis in MA-10 Leydig cells.

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a plasticizer with endocrine disruptor activity that has been shown to stimulate basal steroid biosynthesis in Leydig cells. The mechanism by which it does so is unknown. Using MA-10 mouse tumor Leydig cells, we assessed the effects of MEHP on reactive oxygen species (ROS) levels, and on the signal transduction pathways that mobilize cholesterol. Exposure to 0-300 µM MEHP stimulated basal progesterone production in a dose-dependent manner. Progesterone stimulation was correlated with increases in the phosphorylation of hormone-sensitive lipase (HSL; aka cholesteryl ester hydrolase), which is involved in the production of free cholesterol, and of steroidogenic acute regulatory (STAR) protein expression. Co-treating MA-10 cells with MEHP and the ROS scavenger N-acetyl cysteine (NAC) blocked the activation of HSL, blunted MEHP-induced STAR, and reduced basal progesterone formation. These observations suggest that ROS generation by MEHP leads to activation of HSL and increase in STAR which, together, result in increased free-cholesterol bioavailability and progesterone formation.

Effects of spermatogenic cycle on Stem Leydig cell proliferation and differentiation.

We reported previously that stem Leydig cells (SLC) on the surfaces of rat testicular seminiferous tubules are able to differentiate into Leydig cells. The proliferation and differentiation of SLCs seem likely to be regulated by niche cells, including nearby germ and Sertoli cells. Due to the cyclical nature of spermatogenesis, we hypothesized that the changes in the germ cell composition of the seminiferous tubules as spermatogenesis proceeds may affect tubule-associated SLC functions. To test this hypothesis, we compared the ability of SLCs associated with tubules at different stages of the cycle to differentiate into Leydig cells in vitro. SLCs associated with stages IX-XI were more active in proliferation and differentiation than SLCs associated with stages VII-VIII. However, when the SLCs were isolated from each of the two groups of tubules and cultured in vitro, no differences were seen in their ability to proliferate or differentiate. These results suggested that the stage-dependent local factors, not the SLCs themselves, explain the stage-dependent differences in SLC function. TGFB, produced in stage-specific fashion by Sertoli cells, is among the factors shown in previous studies to affect SLC function in vitro. When TGFB inhibitors were included in the cultures of stages IX-XI and VII-VIII tubules, stage-dependent differences in SLC development were reduced, suggesting that TGFB may be among the paracrine factors involved in the stage-dependent differences in SLC function. Taken together, the findings suggest that there is dynamic interaction between SLCs and germ/Sertoli cells within the seminiferous tubules that may affect SLC proliferation and differentiation.

Acute effects of the translocator protein drug ligand FGIN-1-27 on serum testosterone and luteinizing hormone levels in male Sprague-Dawley rats†.

We reported that FGIN-1-27 (N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide, FGIN), a synthetic ligand for translocator protein (TSPO, 18 kDa), increased serum testosterone levels in young and aged Brown Norway rats after its administration daily for 10 days. It is not known, however, how soon after treatment with FGIN serum testosterone rises, how long levels remain elevated after cessation of treatment, or whether the drug acts solely through TSPO. Adult Sprague-Dawley male rats received a single ip dose of FGIN (1 mg/kg BW). Serial blood samples were collected, and serum testosterone and luteinizing hormone (LH) were assessed hourly throughout 24 h. Testosterone concentration was maximal by 3 h, remained significantly higher than the controls at 10 h, and returned to the control level by 24 h. Consistent with the in vivo study, culturing isolated Leydig cells with either FGIN (40 µM) or LH (0.1 ng/ml) resulted in significantly increased testosterone production by 30 min, and the stimulatory effects persisted through 48 h. At a very early (15 min) treatment time, however, FGIN significantly increased testosterone production but LH had not yet done so. Surprisingly, in vivo treatment with FGIN not only increased serum testosterone but also serum LH concentration, raising the possibility that FGIN may increase serum testosterone concentration by dual mechanisms.

Androgen action in prostate function and disease.

Benign prostatic hyperplasia (BPH) is an enlargement of the prostate gland that is frequently found in aging men. Androgens are essential for the development and differentiated function of the prostate, as well as for proliferation and survival of prostatic cells. In man, dog and rodent, there are age-related decreases in serum testosterone. Despite the lower serum testosterone levels, benign prostatic hyperplasia increases with age in men and dogs, while age-dependent prostatic hyperplasia develops in the dorsal and lateral lobes of the rat prostate. The possible mechanisms that lead to prostate hyperplasia have been extensively studied over many years. It is clear that androgens, estrogens and growth factors contribute to the condition, but the exact etiology remains unknown. Prostate cancer (CaP) represents a significant cause of death among males worldwide. As is the case of BPH, it is clear that androgens (testosterone and dihydrotestosterone) and their metabolites play important roles in the disease, but cause-effect relationships have not been established. Androgen deprivation therapy has been used for decades, primarily in the metastatic stage, to inhibit androgen-dependent prostate cancer cell growth. Androgen deprivation, which can be achieved by targeting hormone biosynthesis or androgen receptor activation, results in symptom amelioration. However, most patients will develop hormone refractory cancer or castration-resistant prostate cancer (CRPC). Prostatic epithelial cells demonstrate enormous plasticity in response to androgen ablation. This characteristic of prostatic epithelial cells may give rise to different populations of cells, some of which may not be dependent on androgen. Consequently, androgen receptor positive and negative cells might co-exist within CRPC. A clear understanding of this possible cellular heterogeneity and plasticity of prostate epithelial cells is necessary to develop an optimal strategy to treat or prevent CRPC.

Long-term maintenance of luteinizing hormone-responsive testosterone formation by primary rat Leydig cells in vitro.

The inability of cultured primary Leydig cells to maintain luteinizing hormone (LH)-responsive testosterone formation in vitro for more than 3-5 days has presented a major challenge in testing trophic effects of regulatory factors or environmental toxicants. Our primary objective was to establish culture conditions sufficient to maintain LH-responsive testosterone formation by Leydig cells for at least a month. When isolated rat adult Leydig cells were cultured in DMEM/F12 and M199 culture medium containing insulin (10µg/ml), PDGFAA (10 ng/ml), lipoprotein (0.25 mg/ml), horse serum (1%) and a submaximal concentration of LH (0.2 ng/ml), the cells retained the ability to produce testosterone in vitro for at least 4 weeks. By using the longer-term culture conditions of this system, we were able to detect suppressive effects on testosterone production by low levels of the toxicant MEHP (mono-(2-ethylhexyl) phthalate), an active metabolite of the plasticizer DEHP, that were not detected by short-term culture.