SARS-CoV-2 Enters Human Leydig Cells and Affects Testosterone Production In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a SARS-like coronavirus, continues to produce mounting infections and fatalities all over the world. Recent data point to SARS-CoV-2 viral infections in the human testis. As low testosterone levels are associated with SARS-CoV-2 viral infections in males and human Leydig cells are the main source of testosterone, we hypothesized that SARS-CoV-2 could infect human Leydig cells and impair their function. We successfully detected SARS-CoV-2 nucleocapsid in testicular Leydig cells of SARS-CoV-2-infected hamsters, providing evidence that Leydig cells can be infected with SARS-CoV-2. We then employed human Leydig-like cells (hLLCs) to show that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 is highly expressed in hLLCs. Using a cell binding assay and a SARS-CoV-2 spike-pseudotyped viral vector (SARS-CoV-2 spike pseudovector), we showed that SARS-CoV-2 could enter hLLCs and increase testosterone production by hLLCs. We further combined the SARS-CoV-2 spike pseudovector system with pseudovector-based inhibition assays to show that SARS-CoV-2 enters hLLCs through pathways distinct from those of monkey kidney Vero E6 cells, a typical model used to study SARS-CoV-2 entry mechanisms. We finally revealed that neuropilin-1 and cathepsin B/L are expressed in hLLCs and human testes, raising the possibility that SARS-CoV-2 may enter hLLCs through these receptors or proteases. In conclusion, our study shows that SARS-CoV-2 can enter hLLCs through a distinct pathway and alter testosterone production.

Alterations of gene profiles in Leydig-cell-regenerating adult rat testis after ethane dimethane sulfonate-treatment.

Only occupying about 1%-5% of total testicular cells, the adult Leydig cell (ALC) is a unique endocrine cell that produces androgens. Rat Leydig cells regenerate after these cells in the testis are eliminated with ethane dimethane sulfonate (EDS). In this study, we have characterized Leydig cell regeneration and messenger ribonucleic acids (mRNA) profiles of EDS treated rat testes. Serum testosterone, testicular gene profiling and some steroidogenesis-related proteins were analyzed at 7, 21, 35 and 90 days after EDS treatment. Testicular testosterone levels declined to undetectable levels until 7 days after treatment and then started to recover. Seven days after treatment, 81 mRNAs were down-regulated greater than or equal to two-fold, with 48 becoming undetectable. These genes increased their expression 21 days and completely returned to normal levels 90 days after treatment. The undetectable genes include steroidogenic pathway proteins: steroidogenic acute regulatory protein, Scarb1, Cyp11a1, Cyp17a1, Hsd3b1, Cyp1b1 and Cyp2a1. Seven days after treatment, there were 89 mRNAs up-regulated two-fold or more including Pkib. These up-regulated mRNAs returned to normal 90 days after treatment. Cyp2a1 did not start to recover until 35 days after treatment, indicating that this gene is only expressed in ALCs not in the precursor cells. Quantitative polymerase chain reaction, western blotting and semi-quantitative immunohistochemical staining using tissue array confirmed the changes of several randomly picked genes and their proteins.

Identification, proliferation, and differentiation of adult Leydig stem cells.

Leydig cells, the testosterone-producing cells of the adult testis, rarely turn over. However, their elimination with ethane dimethanesulfonate (EDS) is followed by the appearance of new, fully functional adult Leydig cells. The cells that give rise to the new Leydig cells have not been well characterized, and little is known about the mechanism by which they are regulated. We isolated cells expressing platelet-derived growth factor receptor-α, but not 3ß-hydroxysteroid dehydrogenase (3ß-HSD(neg)) from the testes of EDS-treated adult rats. Depending on conditions, these cells proliferated indefinitely or differentiated and produced testosterone. To localize these cells and to determine the effect of the testicular environment on their function, the seminiferous tubules and testicular interstitium were physically separated and cultured. During the first 72 h in culture, 3ß-HSD(neg) cells on the tubule surfaces underwent divisions. Some of these cells later expressed 3ß-HSD and produced testosterone. Removal of the newly formed 3ß-HSD(pos) cells from the tubule surfaces with EDS, followed by further culture of the stripped tubules, resulted in the reappearance of testosterone-producing cells. These results, taken together, suggest that the precursors for newly formed Leydig cells are stem cells, with many if not all situated on the surfaces of the seminiferous tubules. Although normally quiescent, the stem cells are capable of self-renewal and differentiation. The development of the tubule culture system should provide a valuable in vitro approach to assess the role(s) of niche components on the function of adult Leydig stem cells despite their residing in a complex mammalian tissue.

Mono-(2-ethylhexyl) phthalate affects the steroidogenesis in rat Leydig cells through provoking ROS perturbation.

Di-2-ethylhexyl phthalate (DEHP), one of the most widely used plasticizers in a number of day-life products, exerts both short-term and long-lasting effects on testicular steroidogenesis during in utero exposure. These actions might be caused by its primary metabolite, mono-(2-ethylhexyl) phthalate (MEHP). In the present study, we investigated the effects of MEHP on steroidogenesis of different stages of rat Leydig cells, progenitor (PLCs), immature (ILCs) and adult (ALCs). Results showed that MEHP affected reactive oxygen species (ROS) generation as well as androgen production in ALCs, but not in PLCs and ILCs, which coincided with hydrogen peroxide (H(2)O(2)). Low concentrations of MEHP (20-200µM) provoked ROS perturbation and caused the stimulation of steroidogenic acute regulatory (StAR), cytochrome P450 side-chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenases (3ß-HSD) and 17ß-hydroxysteroid dehydrogenases (17ß-HSD) activities which elevated T production of ALCs. Contrast to the effect in low doses, high levels of MEHP (2000µM and over) induced overloaded oxidative stress and inhibited steroidogenesis by reducing the activities of these enzymes in ALCs. These results indicated that oxidative stress and subsequent steroidogenic enzymes changes in ALCs were the potential underlying mechanism of the biphasic effects of DEHP on androgen production.

Effect of brominated flame retardant BDE-47 on androgen production of adult rat Leydig cells.

As one of the most abundant polybrominated diphenylethers (PBDEs) detected in adipose tissue and breast milk of humans, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is considered as a potential endocrine disruptor. The objective of this study is to explore whether environment-related level of BDE-47 could affect the androgen production in rat Leydig cells. Rat adult Leydig cells (ALCs) were treated with 10(-8) to 10(-4)M BDE-47 in vitro, the production of testosterone (T) and steroidogenic acute regulatory (StAR) protein level were determined. BDE-47 significantly increased basal T production and steroidogenic acute regulatory protein (StAR) level of ALCs after treatment with 10(-4)M BED-47. Overall, LH (0.1ng/ml) stimulated T production in ALCs by 6 folds, however it did not increase T production in BDE-47-treated ALCs when compared to untreated ALC. Both 8-Br-cAMP (for cAMP signaling) and 22R-hydroxycholesterol (22-diol, for P450 cholesterol side chain cleavage enzyme P450scc activity) significantly increased T production in ALCs treated with BDE-47 from 10(-7) to 10(-5)M. The results of this study indicate that environment-related level of BDE-47 in vitro increased T production in a dose-dependent manner. The stimulated effects of BDE-47 on StAR and P450scc might play key roles in BDE-47-mediated stimulation of T production.

Role of 11ß-OH-C(19) and C(21) steroids in the coupling of 11ß-HSD1 and 17ß-HSD3 in regulation of testosterone biosynthesis in rat Leydig cells.

Here we describe further experiments to support our hypothesis that bidirectional 11ß-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17ß-HSD3, incubation of Leydig cells with corticosterone (B) or several C(19)- and C(21)-11ß-OH-steroids, in the presence of [(3)H]-11-dehydro-corticosterone (A), stimulated 11ß-HSD1-reductase activity. However, in presence of 30 µM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30 min and concomitantly inhibited 11ß-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p<0.05) to 224-267 picomole/25,000 cells/30 min when 10 µM 11ß-OH-steroids (in addition to 30 µM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5 µM), and B or A (500 nM). Incubations of 0.3-6.0 µM of corticosterone (plus or minus 30 µM AD) were then performed to test the effectiveness of 17ß-HSD3 as a possible NADP(+) regenerating system. In the absence of AD, increasing amounts (3-44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 µM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3-5-fold producing 4-210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11ß-HSD1 is enzymatically coupled to 17ß-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.

Inhibition of LH-stimulated androgen production in rat immature Leydig cells: Effects on nuclear receptor steroidogenic factor 1 by FGF2.

Both fibroblast growth factor 2 (FGF2) and luteinizing hormone (LH) have been reported to regulate androgen production in Leydig cells in progenitor Leydig cells. The objective of the present study is to examine the regulation of androgen production in rat immature Leydig cells (ILCs). ILCs were isolated from 35-day-old rat testes and cultured in DMEM/F12 medium with LH (1 ng/ml) or FGF2 (10 ng/ml). 5alpha-Androstane-3alpha, 17beta-diol (3alpha-DIOL), the primary androgen in ILCs, and testosterone (T) were measured by Radioimmuno assay. The results showed the LH stimulated androgen production in ILCs, and FGF2 did not. However, FGF2 decreased the LH-stimulated androgen production. Real-time PCR and enzyme assay showed that FGF2 decreased levels of several steroidogenic enzymes, inhibited the expressions of steroidogenic acute regulatory (StAR) protein and steroidogenic factor 1 (Nr5a1) in LH-stimulated ILCs. FGF2-mediated inhibition of Nr5a1gene expression may be the mechanism through which FGF2 inhibits LH-stimulated androgen production.

Normal responses to restraint stress in mice lacking the gene for neuronal nitric oxide synthase.

The hormonal changes associated with immobilization stress (IMO) include a swift increase in corticosterone (CORT) concentration and a decrease in circulating testosterone (T) levels. There is evidence that the production of the short-lived neuromodulator nitric oxide (NO) is increased during stress in various tissues, including the brain. NO also suppresses the biosynthesis of T. Both the inducible and the neuronal isoforms of NO synthase (iNOS and nNOS, respectively) have been implicated in this suppression, but the evidence has not been conclusive. We used adult wild-type (WT) and nNOS knockout male mice (nNOS-/-) to assess the respective roles of CORT and nNOS-derived NO in stress mediated inhibition of T production. Animals were assigned to either basal control or 3-hour IMO groups. No difference in basal plasma and testicular T levels were observed between WT and nNOS-/-, although testicular weights of mutant mice were slightly lower compared to WT animals. The plasma contents of luteinizing hormone (LH) and CORT in unstressed mice of both genotypes were similar. Exposure to 3 hours of IMO increased plasma CORT and decreased T concentrations in mice of both genotypes. However, comparable levels of plasma LH and testicular nitrite and nitrate (NOx), NO stable metabolites, were detected in control and stressed WT and nNOS-/- mice. Adrenal concentrations of NOx declined after IMO, but the reduction was not statistically significant. These findings implicate CORT rather than NO generated by nNOS in the rapid stress-induced suppression of circulating T.

Increased proliferation but decreased steroidogenic capacity in Leydig cells from mice lacking cyclin-dependent kinase inhibitor 1B.

Proliferating cells express cyclins, cell cycle regulatory proteins that regulate the activity of cyclin-dependent kinases (CDKs). The actions of CDKs are regulated by specific inhibitors, the CDK inhibitors (CDKIs), which are comprised of the Cip/Kip and INK4 families. Expression of the Cip/Kip CDKI 1B (Cdkn1b, encoding protein CDKN1B, also called p27(kip1)) in developing Leydig cells (LCs) has been reported, but the function of CDKN1B in LCs is unclear. The goal of the present study was to determine the effects of CDKN1B on LC proliferation and steroidogenesis by examining these parameters in Cdkn1b knockout (Cdkn1b(-/-)) mice. LC proliferation was measured by bromodeoxyuridine incorporation. Testicular testosterone levels, mRNA levels, and enzyme activities of steroidogenic enzymes were compared in Cdkn1b(-/-) and Cdkn1b(+/+) mice. The labeling index of LCs in Cdkn1b(-/-) mice was 1.5% +/- 0.2%, almost 7-fold higher than 0.2% +/- 0.08% (P < 0.001) in the Cdkn1b(+/+) control mice. LC number per testis in Cdkn1b(-/-) mice was 2-fold that seen in the Cdkn1b(+/+) control mice. However, testicular testosterone levels, mRNA levels of steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), and 3beta-hydroxtsteroid dehydrogenase 6 (Hsd3b6), and their respective proteins, were significantly lower in Cdkn1b(-/-) mice. We conclude that deficiency of CDKN1B increased LC proliferation, but decreased steroidogenesis. Thus, CDKN1B is an important regulator of LC development and function.

In utero and lactational exposures to diethylhexyl-phthalate affect two populations of Leydig cells in male Long-Evans rats.

Diethylhexylphthalate (DEHP) has been classified as an antiandrogen. However, whether in utero and lactational exposures of DEHP affect Leydig cells has not been well established. In the present study, the effects of DEHP exposures on fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) were assessed. Pregnant dams of Long-Evans rats were treated with 0, 10, and 750 mg/kg body weight DEHP from Gestational Day 12.5 to Postnatal Day (PND) 21.5. Fetal Leydig cell clustering and FLC-specific gene expression were examined. Anogenital distances (AGDs) of male pups were assessed at PND 2. Serum testosterone levels of male pups and mRNA levels of ALC-specific genes were measured at PNDs 21 and 49. The AGDs of male pups were significantly shorter in the group treated with 750 mg/kg DEHP (mean +/- SEM, 3.68 +/- 0.16 mm) compared with control (4.62 +/- 0.13 mm). The FLCs were aggregated after 10 and 750 mg/kg DEHP exposures. Several FLC-specific genes, including luteinizing hormone receptor (Lhcgr) and steroidogenic enzyme genes, were downregulated at both doses. Serum testosterone levels were significantly lower compared with control at PND 21 after treatment of 10 or 750 mg/kg DEHP, and continued to be lower even up to 49 days postpartum at the higher dose. The mRNA levels for Lhcgr and steroidogenic enzyme genes were significantly lower at both doses of DEHP at PND 21, whereas there were no significant differences for these genes at PND 49. In conclusion, in utero and continued lactational exposures to DEHP exert long-term disruption of steroidogenesis of ALCs.

Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero.

Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long-Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2-20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6-30 cells increased in all treatment groups, with 29 +/- 2% in control vs. 37 +/- 3, 35 +/- 3, and 56 +/- 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.

Inhibition of 11beta-hydroxysteroid dehydrogenase enzymatic activities by glycyrrhetinic acid in vivo supports direct glucocorticoid-mediated suppression of steroidogenesis in Leydig cells.

Previous studies have suggested that glucocorticoid (GC) can directly affect testicular testosterone (T) biosynthesis by Leydig cells through a receptor-mediated mechanism. Interconversion of corticosterone (CORT), the active form in rodents, and 11-dehydroCORT, the biologically inert 11-keto form, is catalyzed by 11betaHSD1. This enzyme thus controls the intracellular concentration of active GC. We have postulated that elevated CORT levels resulting from stress exceed the Leydig cell's capacity for metabolic inactivation of CORT, resulting in suppressed T production. The present study tested whether inhibition of 11betaHSD1 in vivo, by the administration of glycyrrhetinic acid (GA), increases intracellular active GC concentration and thereby affects serum T concentration and Leydig cell T production. Adult Sprague-Dawley rats were treated with vehicle (corn oil), CORT, GA, or GA + CORT. Serum luteinizing hormone (LH), CORT, and T levels were measured, as were the steroidogenic capacities of purified Leydig cells. Twofold elevations of CORT were achieved by the administration of either CORT or GA alone, but in both cases there was no effect on serum T levels. However, when CORT and GA were administered in combination, serum CORT levels increased 3.5-fold (to 420 +/- 34 ng/mL) and serum T levels were reduced significantly (to 0.72 +/- 0.07 ng/mL; control, 2.12 +/- 0.23 ng/mL). Serum levels of LH were not affected by CORT, GA, or GA + CORT. Consistent with the reduced serum T levels following GA + CORT, steroidogenic enzyme expression and capacities were significantly reduced compared to control. These data support a role for 11betaHSD1 in modulating intracellular CORT concentrations and, in turn, for a direct effect of GC on Leydig cells in response to stress.

Development of a cryopreservation protocol for Leydig cells.

BACKGROUND: In the present study, we describe a procedure to cryopreserve the postnatal members of the Leydig cell lineage, including progenitor (PLC), immature (ILC) and adult (ALC) Leydig cells from, respectively 21-, 35- and 90-day-old rats. METHODS: The cells were resuspended in a culture medium supplemented with 1% bovine serum albumin (Dulbecco's Modified Eagle's Medium [DMEM]/F12) to a final concentration of 2 x 10(6)cells/ml and the effects of varying concentrations of dimethylsulfoxide (DMSO) (5, 10, 15 or 20%) were assessed after freezing at -70 degrees C and then storing in liquid nitrogen. After 12 months of frozen storage, these cells were thawed rapidly at 37 degrees C and Trypan Blue exclusion staining and attachment to culture dishes were assessed as measures of viability. RESULTS: The trypan blue exclusion and attachment rates for Leydig cell stages were around 85% in the presence of 15% DMSO. After frozen storage, Leydig cell steroidogenic capacity in response to a range of LH doses, (0.01-100 ng/ml) was unchanged compared with freshly isolated control cells. Furthermore, the steady-state mRNA levels for Leydig cell specific transcripts were maintained. CONCLUSIONS: This study demonstrates that purified rat Leydig cells at a range of developmental stages can be frozen and that the cryopreserved cells retain normal function.

Biphasic effects of postnatal exposure to diethylhexylphthalate on the timing of puberty in male rats.

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP), which are commonly found in cosmetics and in flexible plastics distributed by the food, construction, and medical products industries, have been classified as anti-androgens. High-dose DEHP exposure in utero is associated with decreased androgen levels. However, when administered after birth, low doses of DEHP (eg, 10 mg/kg body weight) may stimulate androgen production. In the present study, the potential of phthalate exposure to advance or delay the timing of puberty was assessed. Male Long-Evans rat pups were chronically subjected to low or high doses of DEHP, with the androgen-driven process of preputial separation serving as an index of pubertal timing. Rats were treated with 0, 10, 500, or 750 mg/kg body weight DEHP for 28 days starting at day 21 postpartum. The average age at which the animals completed preputial separation was measured in each group. The age of preputial separation was 41.5 +/- 0.1 days postpartum in controls (vehicle). The 10 mg/kg DEHP dose advanced pubertal onset significantly to 39.7 +/- 0.1 days postpartum, whereas the 750 mg/kg DEHP dose delayed pubertal onset to 46.3 +/- 0.1 days postpartum. The 10 mg/kg DEHP dose also significantly increased serum testosterone (T) levels (3.13 +/- 0.37 ng/mL) and seminal vesicle weights (0.33 +/- 0.02 g) compared with control serum T (1.98 +/- 0.20 ng/mL) and seminal vesicle weight (0.26 +/- 0.02 g), while the 750 mg/kg dose decreased serum T (1.18 +/- 0.18 ng/mL) as well as testes and body weights. Direct action of the DEHP metabolite, monoethylhexylphthalate (MEHP), on Leydig cell steroidogenic capacity was investigated in vitro. MEHP treatment at a low concentration (100 microM) increased luteinizing hormone-stimulated T production, whereas 10 mM concentrations were inhibitory. In conclusion, data from the present study indicate that DEHP has a biphasic effect on Leydig cell function, with low-dose exposure advancing the onset of puberty. High doses of DEHP, which are anti-androgenic, may also be outside the range of real environmental exposure levels.

Testosterone production in mice lacking inducible nitric oxide synthase expression is sensitive to restraint stress.

Immobilization stress (IMO) induces a rapid increase in glucocorticoid secretion [in rodents, corticosterone CORT)] and this is associated with decreased circulating testosterone (T) levels. Nitric oxide (NO), a reactive free radical and neurotransmitter, has been reported to be produced at higher rates in tissues such as brain during stress. The biosynthesis of T is also known to be dramatically suppressed by NO. Specifically, the inducible isoform of nitric oxide synthase (iNOS) was directly implicated in this suppression. To assess the respective roles of CORT and NO in stress-mediated inhibition of T production, adult wild-type (WT) and inducible nitric oxide synthase knockout (iNOS(-/-)) male mice were evaluated. Animals of each genotype were assigned to either basal control or 3-h IMO groups. Basal plasma and testicular T levels were equivalent in both genotypes, whereas testicular weights of mutant mice were significantly higher compared with WT animals. Exposure to 3-h IMO increased plasma CORT and decreased T concentrations in mice of both genotypes. Testicular T levels were also affected by stress in WT and mutant males, being sharply reduced in both genotypes. However, the concentrations of nitrite and nitrate, the stable metabolites of NO measured in testicular extracts, did not differ between control and stressed WT and iNOS(-/-) mice. These results support the hypothesis that CORT, but not NO, is a plausible candidate to mediate rapid stress-induced suppression of Leydig cell steroidogenesis.

Gene expression during development of fetal and adult Leydig cells.

In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

In search of rat stem Leydig cells: identification, isolation, and lineage-specific development.

Leydig cells (LCs) are thought to differentiate from spindle-shaped precursor cells that exhibit some aspects of differentiated function, including 3beta-hydroxysteroid dehydrogenase (3betaHSD) activity. The precursor cells ultimately derive from undifferentiated stem LCs (SLCs), which are postulated to be present in testes before the onset of precursor cell differentiation. We searched for cells in the neonatal rat testis with the abilities to: (i) proliferate and expand indefinitely in vitro (self renew); (ii) differentiate (i.e., 3betaHSD and ultimately synthesize testosterone); and (iii) when transplanted into host rat testes, colonize the interstitium and subsequently differentiate in vivo. At 1 week postpartum, spindle-shaped cells were seen in the testicular interstitium that differed from the precursor cells in that they were 3betaHSD-negative, luteinizing hormone (LH) receptor (LHR)-negative, and platelet-derived growth factor receptor alpha (PDGFR alpha)-positive. These cells were purified from the testes of 1-week-old rats. The cells contained proteins known to be involved in LC development, including GATA4, c-kit receptor, and leukemia inhibitory factor receptor. The putative SLCs expanded over the course of 6 months while remaining undifferentiated. When treated in media that contained thyroid hormone, insulin-like growth factor I, and LH, 40% of the putative SLCs came to express 3betaHSD and to synthesize testosterone. When transplanted into host rat testes from which LCs had been eliminated, the putative SLCs colonized the interstitium and subsequently expressed 3betaHSD, demonstrating their ability to differentiate in vivo. We conclude that these cells are likely to be the sought-after SLCs.

Stimulation of testosterone production in rat Leydig cells by aldosterone is mineralocorticoid receptor mediated.

The testis is known to be a site of corticosterone action, and testosterone production in Leydig cells is directly inhibited by glucocorticoids. Glucocorticoids bind to both glucocorticoid receptors (GRs) and to mineralocorticoid receptors (MRs). In Leydig cells, selective mineralocorticoid binding could result from oxidative inactivation of glucocorticoid by type 1 and/or 2 11beta-hydroxysteroid dehydrogenase (11betaHSD), as both isoforms are expressed. However, it remains unclear whether Leydig cells express MRs and respond directly to mineralocorticoid action. Therefore, the aims of the present study were to ascertain: (1) whether MR mRNA, protein and receptor binding are present in Leydig cells; and (2) if the mineralocorticoid modulates testosterone production. The mRNA encoding MR, as well as protein, and binding activity were each observed in adult rat Leydig cells. MR-ligand binding specificity within isolated Leydig cells was evaluated further by measuring displacement of MR binding to aldosterone by corticosterone in the presence and absence of carbenoxolone, an inhibitor of 11betaHSD1 and 2 that decreases conversion to biologically inert 11-dehydrocorticosterone. Carbenoxolone inhibited 11betaHSD oxidative activity, and reduced corticosterone-binding by 50%. Mineralocorticoid effects on steroidogenesis were assessed in the presence of aldosterone (0.01-10 nM) with or without the MR antagonist, RU28318. Aldosterone induced dose-dependent increases in both basal and luteinizing hormone-stimulated testosterone production. RU28318 eliminated the increase, indicating that these effects of aldosterone were mediated by the MR. The effects of aldosterone and luteinizing hormone (0.1 ng/ml) on testosterone production were synergistic, suggesting that the two hormones increased steroidogenesis through separate pathways. We conclude that Leydig cells express MRs and that testosterone production is subject to regulation by aldosterone.

Paracrine modulation of androgen synthesis in rat leydig cells by nitric oxide.

The free radical nitric oxide (NO), generated through the oxidation of L-arginine to L-citrulline by NO synthases (NOSs), has been shown to inhibit steroidogenic pathways. NOS isoforms are known to be present in rat and human testes. Our study examined the sensitivity of Leydig cells to NO and determined whether NOS activity resides in Leydig cells or in another cell type such as the testicular macrophage. The results showed a low level of L-[14C]arginine conversion in purified rat Leydig cell homogenates. Administration of the NOS inhibitor L-N(G)-nitro-arginine methyl ester (L-NAME), or the calcium chelator ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA), had no effect on L-[14C]citrulline accumulation. Increased intracellular Ca2+ concentrations that were induced by a calcium ionophore, or the addition of luteinizing hormone (LH), failed to affect NO formation in intact cells that were cultured in vitro. Introduction of a high concentration of the NO precursor L-arginine did not decrease testosterone (T) production, and NOS inhibitors did not increase T biosynthesis. However, exposing Leydig cells to low concentrations of the NO donor S-nitrosoglutathione (GSNO) induced a dramatic blockade of T production under basal and LH-stimulated conditions. DNA array assays showed a low level of expression of endothelial NOS (eNOS), while the neuronal and inducible isoforms of NOS (nNOS and iNOS) were below detection levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses confirmed these findings and demonstrated the presence of high iNOS messenger RNA (mRNA) levels in activated testicular macrophages that produced large amounts of NO. These data suggest that, while T production in rat Leydig cells is highly sensitive to NO and an endogenous NO-generating system is not present in these cells, NOS activity is more likely to reside in activated testicular macrophages.

11{beta}-Hydroxysteroid dehydrogenase 2 in rat leydig cells: its role in blunting glucocorticoid action at physiological levels of substrate.

Corticosterone (CORT) suppresses Leydig cell steroidogenesis by inhibiting the expression of proteins involved in testosterone biosynthesis including steroidogenic acute regulatory protein and steroidogenic enzymes. In most cells, intracellular glucocorticoid levels are controlled by either or both of the two known isoforms of 11beta-hydroxysteroid dehydrogenase (11beta HSD): the nicotinamide adenine dinucleotide phosphate reduced-dependent low-affinity type I 11beta HSD (11beta HSD1) oxidoreductase and the nicotinamide adenine dinucleotide-dependent 11beta HSD2 high-affinity unidirectional oxidase. In Leydig cells, 11beta HSD1 alone may not be sufficient to prevent glucocorticoid-mediated suppression due to its low affinity for CORT at basal concentrations. The high-affinity unidirectional 11beta HSD2, if also present, may be critical for lowering intracellular CORT levels. In the present study, we showed that 11beta HSD2 is present in rat Leydig cells by PCR amplification, immunohistochemical staining, enzyme histochemistry, immunoprecipitation, and Western blotting. Real-time PCR showed a 6-fold enrichment of 11beta HSD2 mRNA in these cells, compared with whole testis and that the amount of 11beta HSD2 message was about 1000-fold lower, compared with 11beta HSD1. Diffuse immunofluorescent staining of 11beta HSD2 protein in the Leydig cell cytoplasm was consistent with its localization in the smooth endoplasm reticulum. 11beta HSD1 or 11beta HSD2 activities were selectively inhibited using antisense methodology: inhibition of 11beta HSD1 lowered reductase activity by 60% and oxidation by 25%, whereas inhibition of 11beta HSD2 alone suppressed oxidase activity by 50%. This shows that the high-affinity, low-capacity 11beta HSD2 isoform, present at only one thousandth the level of the low-affinity isoform may significantly affect the level of CORT. The inhibition of either 11beta HSD1 or 11beta HSD2 significantly lowered testosterone production in the presence of CORT. These data suggest that both types I and II 11beta HSD in Leydig cells play a protective role, opposing the adverse effects of excessive CORT on testosterone production.