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.

Rapid glucocorticoid mediation of suppressed testosterone biosynthesis in male mice subjected to immobilization stress.

Physical and psychosocial stress challenge homeostasis, increasing glucocorticoid secretion (in rodents, corticosterone [CORT]) while decreasing testosterone (T) levels. The dynamics of stress-induced changes in T, CORT, and luteinizing hormone (LH) concentrations in mice have not been investigated previously. In particular, it remains to be established whether there is a rapid effect of CORT that is directly mediated by glucocorticoid receptors (GRs) in the testis. Therefore, serum and intratesticular T, serum CORT, and LH levels were measured during acute immobilization (IMO) stress, using the C57BL/6 strain of mice. The effects of testicular GR blockade were investigated by administration of the GR antagonist, RU486, via intratesticular (IT) or intraperitoneal (IP) injection. CORT levels increased in stressed males starting at 15 minutes, reaching a fivefold higher plateau by 1 hour compared with controls (P < .01). Conversely, starting from 30 minutes on, both serum and intratesticular T levels decreased in stressed males to 30% and 8% of control values, respectively, by 6 hours (P < .01). In contrast, LH was unchanged by IMO stress for up to 6 hours. Intratesticular treatment with RU486 partially prevented the IMO-induced decline in T levels. CORT treatment reduced intracellular cyclic adenosine monophosphate (cAMP) content in Leydig cells by 15 minutes and T production by 30 minutes in vitro. We conclude that 1) the rapid changes in T suggest a suppression of T biosynthesis by glucocorticoid through a nongenomic mechanism, lowering the production of cytoplasmic cAMP; 2) changes in gonadotropic stimulation of Leydig cells are unlikely to explain the suppression of T levels during acute stress; and 3) the results are consistent with a direct inhibitory action of CORT on Leydig cells.

Inhibition of recovery of spermatogenesis in irradiated rats by different androgens.

We previously showed that exogenous testosterone (T) inhibited GnRH-antagonist-stimulated spermatogenic recovery in irradiated rats through an androgen-receptor-mediated action. In the present study, we tested whether the inhibition is attributable to T, a specific androgenic metabolite of T, or a general property of androgens in this system. In addition, we also tested whether estradiol-17beta (E2), a metabolite of T, is similarly inhibitory. Rats irradiated with 5 Gy were treated with a GnRH antagonist during wk 3-7. Neither irradiation nor GnRH-antagonist treatment produced biologically significant changes in the relative intratesticular levels of several androgenic metabolites. Next, groups of rats, irradiated and treated with GnRH antagonist as above, were given various doses of one of the following androgens: T, 5alpha-dihydrotestosterone, 7alpha-methyl-19-nortestosterone, methyltrienolone, or E2. The percentage of tubules showing differentiation (tubule differentiation index) was increased to 68% by the GnRH antagonist, from a value of 0.1% in irradiated-only rats at 13 wk after irradiation. All of the added androgens inhibited spermatogenic recovery, lowering the tubule differentiation index to between 0.4-36%, but no inhibition was observed with the addition of E2. Of all the androgen treatments tested, T (given as daily injections of T propionate) minimally inhibited spermatogenic recovery while maintaining androgen-responsive tissue weights, and might be most useful in clinical studies. Hormonal measurements in androgen-treated rats were most consistent with the androgen inhibition of spermatogenic recovery in irradiated rats being a combined result of a direct inhibitory effect of all androgens on the testis and an indirect effect through the pituitary by raising levels of FSH, which seems to add to the inhibition of spermatogenic recovery.