Letrozole protects against cadmium-induced inhibition of spermatogenesis via LHCGR and Hsd3b6 to activate testosterone synthesis in mice.

The heavy metal cadmium is proposed to be one of the environmental endocrine disruptors of spermatogenesis. Cadmium-induced inhibition of spermatogenesis is associated with a hormone secretion disorder. Letrozole is an aromatase inhibitor that increases peripheral androgen levels and stimulates spermatogenesis. However, the potential protective effects of letrozole on cadmium-induced reproductive toxicity remain to be elucidated. In this study, male mice were administered CdCl2 (4 mg/kg BW) orally by gavage alone or in combination with letrozole (0.25 mg/kg BW) for 30 days. Cd exposure caused a significant decreases in body weight, sperm count, motility, vitality, and plasma testosterone levels. Histopathological changes revealed extensive vacuolization and decreased spermatozoa in the lumen. However, in the Cd + letrozole group, letrozole treatment compensated for deficits in sperm parameters (count, motility, and vitality) induced by Cd. Letrozole treatment significantly increased serum testosterone levels, which were reduced by Cd. Histopathological studies revealed a systematic array of all germ cells, a preserved basement membrane and relatively less vacuolization. For a mechanistic examination, RNA-seq was used to profile alterations in gene expression in response to letrozole. Compared with that in the Cd-treated group, RNA-Seq analysis showed that 214 genes were differentially expressed in the presence of letrozole. Gene ontology (GO) enrichment analysis and KEGG signaling pathway analysis showed that steroid biosynthetic processes were the processes most affected by letrozole treatment. Furthermore, we found that the expression of the testosterone synthesis-related genes LHCGR (luteinizing hormone/choriogonadotropin receptor) and Hsd3b6 (3 beta- and steroid delta-isomerase 6) was significantly downregulated in Cd-treated testes, but these genes maintained similar expression levels in letrozole-treated testes as those in the control group. However, the transcription levels of inflammatory cytokines, such as IL-1ß and IL-6, and oxidative stress-related genes (Nrf2, Nqo1, and Ho-1) showed no changes. The present study suggests that the potential protective effect of letrozole on Cd-induced reproductive toxicity might be mediated by the upregulation of LHCGR and Hsd3b6, which would beneficially increase testosterone synthesis to achieve optimum protection of sperm quality and spermatogenesis.

AR Signaling in Prostate Cancer Regulates a Feed-Forward Mechanism of Androgen Synthesis by Way of HSD3B1 Upregulation.

3ßHSD1 enzymatic activity is essential for synthesis of potent androgens from adrenal precursor steroids in prostate cancer. A germline variant in HSD3B1, the gene that encodes 3ßHSD1, encodes for a stable enzyme, regulates adrenal androgen dependence, and is a predictive biomarker of poor clinical outcomes after gonadal testosterone deprivation therapy. However, little is known about HSD3B1 transcriptional regulation. Generally, it is thought that intratumoral androgen synthesis is upregulated after gonadal testosterone deprivation, enabling development of castration-resistant prostate cancer. Given its critical role in extragonadal androgen synthesis, we sought to directly interrogate the transcriptional regulation of HSD3B1 in multiple metastatic prostate cancer cell models. Surprisingly, we found that VCaP, CWR22Rv1, LNCaP, and LAPC4 models demonstrate induction of HSD3B1 upon androgen stimulation for approximately 72 hours, followed by attenuation around 120 hours. 3ßHSD1 protein levels mirrored transcriptional changes in models harboring variant (LNCaP) and wild-type (LAPC4) HSD3B1, and in these models androgen induction of HSD3B1 is abrogated via enzalutamide treatment. Androgen treatment increased flux from [3H]-dehydroepiandrosterone to androstenedione and other downstream metabolites. HSD3B1 expression was reduced 72 hours after castration in the VCaP xenograft mouse model, suggesting androgen receptor (AR) regulation of HSD3B1 also occurs in vivo. Overall, these data suggest that HSD3B1 is unexpectedly positively regulated by androgens and ARs. These data may have implications for the development of treatment strategies tailored to HSD3B1 genotype status.

Multiple signaling pathways mediate interleukin-4-induced 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase type 1 gene expression in human breast cancer cells.

The 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) isoenzymes catalyze an essential step in the formation of all classes of active steroid hormones. We have recently shown that 3beta-HSD type 1 gene expression is specifically induced by interleukin (IL)-4 and IL-13 in breast human cancer cell lines and in normal human mammary epithelial cells in primary culture. There is evidence that IL-4 stimulates bifurcating signaling pathways in which the signal transducer and activator of transcription-6 (Stat6)-signal pathway is involved in differentiation and gene regulation, whereas insulin receptor substrate (IRS) proteins mediate the mitogenic action of IL-4. In fact, we have shown that Stat6 was activated by IL-4 in all cell lines studied where IL-4 induced 3beta-HSD expression, but not in those that failed to respond to IL-4. The present study was designed to investigate the potential contribution of IRS proteins and their downstream targets to IL-4-induced 3beta-HSD type 1 gene expression. IL-4 rapidly induced IRS-1 and IRS-2 phosphorylation in ZR-75-1 human breast cancer cell lines. Moreover, insulin-like growth factor (IGF)-I and insulin, which are well known to cause IRS-1 and IRS-2 phosphorylation, increased the stimulatory effect of IL-4 on 3beta-HSD activity. IRS-1 and IRS-2 are adapter molecules that provide docking sites for different SH2-domain-containing proteins such as the phosphatidylinositol (PI) 3-kinase. In this light, the inhibition of IL-4-induced 3beta-HSD expression by wortmannin and LY294002, two potent PI 3-kinase inhibitors, indicates the probable involvement of the PI 3-kinase signaling molecules in this response to IL-4. Furthermore, it has been suggested that the IRS proteins are part of the signaling complexes that lead to activation of the mitogen-activated protein (MAP) kinase by insulin; thus we investigated the potential role of the MAP kinase (MAPK) cascade in the IL-4 action. In ZR-75-1 cells, both the activation of MAPK by IL-4 and the IL-4-induced 3beta-HSD activity were completely blocked by PD98059, an inhibitor of MAPK activation. Wortmannin also blocked MAPK activation by IL-4, IGF-I, and insulin, suggesting that the MAPK cascade acts as a downstream effector of PI 3-kinases. To further understand the cross-talk between signaling pathways involved in IL-4 action, we investigated the possible involvement of protein kinase C (PKC). The potential role of PKC was suggested by the observation that the well known PKC activator phorbol-12-myristate-13-acetate (PMA) potentiated the IL-4-induced 3beta-HSD activity. Taken together, these findings suggest the existence of a novel mechanism of gene regulation by IL-4. This mechanism would involved the phosphorylation of IRS-1 and IRS-2, which transduce the IL-4 signal through a PI 3-kinase- and MAPK-dependent signaling pathway. The inability of IGF-I, insulin, and PMA to stimulate 3beta-HSD expression by themselves in the absence of IL-4 makes obvious the absolute requirement of an IL-4-specific signaling molecule. Our findings thus suggest that the multiple pathways downstream of IRS-1 and IRS-2 must act in cooperation with the IL-4-specific transcription factor Stat6 to mediate the induction of 31beta-HSD type 1 gene expression in ZR-75-1 human breast cancer cells.

Stat5-mediated regulation of the human type II 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene: activation by prolactin.

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD). A 9/9 match with the consensus Stat5 response element was identified at -110 to -118 in the human Type II 3beta-HSD promoter. 3beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated Stat5 element were tested for PRL activation. Expression vectors for Stat5 and the PRL receptor were cotransfected with a -300 --> +45 3beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected Stat5 expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative Stat5 response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, 32P-labeled promoter fragments containing the Stat5 element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to Stat5. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.

Induction of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase type 1 gene transcription in human breast cancer cell lines and in normal mammary epithelial cells by interleukin-4 and interleukin-13.

Sex steroids play a crucial role in the development and differentiation of normal mammary gland as well as in the regulation of breast cancer growth. Local intracrine formation of sex steroids from inactive precursors secreted by the adrenals, namely, dehydroepiandrosterone and its sulfate, may regulate growth and function of peripheral target tissues, including the breast. Both endocrine and paracrine influences on the proliferation of human breast cancer cells are well recognized. Breast tumors harbor tumor-associated macrophages and tumor-infiltrating lymphocytes that secrete a wide spectrum of cytokines. These factors may also contribute to neoplastic cell activity. The present study was designed to investigate the action of cytokines on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, which is an essential step in the biosynthesis of active estrogens and androgens in human breast cancer cell lines and in normal human mammary epithelial cells in primary culture. 3Beta-HSD activity was undetectable in ZR-75-1 and T-47D estrogen receptor-positive (ER)+ cells under basal growth conditions. This activity was markedly induced after exposure to picomolar concentrations of interleukin (IL)-4 or IL-13. The potent stimulatory effect of these cytokines on 3beta-HSD activity was also observed in the ER- MDA-MB-231 human breast cancer cell line and in normal human mammary epithelial cells (HMECs) in primary culture. The stimulation of 3beta-HSD activity by IL-4 and IL-13 results from a rapid increase in 3beta-HSD type 1 mRNA levels as measured by RT-PCR and Northern blot analyses. Such an induction of the 3beta-HSD activity may modulate androgenic and estrogenic biological responses as demonstrated using ZR-75-1 cells transfected with androgen- or estrogen-sensitive reporter constructs and treated with the adrenal steroid 5-androstene-3beta,17beta-diol. The DNA-binding activity of Stat6, a member of the signal transducers and activators of transcription gene family, is activated 30 min after exposure to IL-4 and IL-13 in human breast cancer cell lines as well as in HMECs in primary culture. In these cells, Stat6 activated by IL-4 or IL-13 binds to two regions of the 3beta-HSD type 1 gene promoter, containing Stat6 consensus sequences. IL-4 induction of 3beta-HSD mRNA and activity is sensitive to staurosporine. This protein kinase inhibitor also inhibits IL-4-induced Stat6 DNA-binding activity. Our data demonstrate for the first time that IL-4 and IL-13 induce 3beta-HSD type 1 gene expression, thus suggesting their involvement in the fine control of sex steroid biosynthesis from adrenal steroid precursors in normal and tumoral human mammary cells. Furthermore, aromatase and/or 5alpha-reductase(s) are expressed in the mammary gland and in a large proportion of human breast tumors. An increase in the formation of their substrates, namely, 4-androstenedione and testosterone, may well have a significant impact on the synthesis of active estrogens and androgens in these tissues.

Pharmacological analysis of sterol delta8-delta7 isomerase proteins with [3H]ifenprodil.

Sterol Delta8-Delta7 isomerases (SIs) catalyze the shift of the double bond from C8-9 to C7-8 in the B-ring of sterols. Surprisingly, the isoenzymes in fungi (ERG2p) and vertebrates [emopamil binding protein (EBP)] are structurally completely unrelated, whereas the sigma1 receptor, a mammalian protein of unknown function, bears significant similarity with the yeast ERG2p. Here, we compare the drug binding properties of SIs and related proteins with [3H]ifenprodil as a common high affinity radioligand (Kd = 1.4-19 nM), demonstrating an intimate pharmacological relationship among ERG2p, sigma1 receptor, and EBP. This renders SIs a remarkable example for structurally diverse enzymes with similar pharmacological profiles and the propensity to bind drugs from different chemical groups with high affinity. We identified a variety of experimental drugs with nanomolar affinity for the human EBP (Ki = 0.5-14 nM) such as MDL28815, AY9944, triparanol, and U18666A. These compounds, as well as the fungicide tridemorph and the clinically used drugs tamoxifen, clomiphene, amiodarone, and opipramol, inhibit the in vitro activity of the recombinant human EBP (IC50 = 0.015-54 microM). The high affinity of the human EBP for 3H-tamoxifen (Kd = 3 +/- 2 nM) implies that the EBP carries the previously described microsomal antiestrogen binding site. Interactions of the EBP with structurally diverse lipophilic amines suggest that novel compounds of related structure should be counterscreened for inhibition of the enzyme to avoid interference with sterol Delta8-Delta7 isomerization.

Both the immunosuppressant SR31747 and the antiestrogen tamoxifen bind to an emopamil-insensitive site of mammalian Delta8-Delta7 sterol isomerase.

SR31747 is a novel agent that elicits immunosuppressive and anti-inflammatory effects. This drug was shown to inhibit Delta8-Delta7 sterol isomerase in yeast. To test whether this enzyme could also be an SR31747 target in mammals, the binding, antiproliferative and sterol biosynthesis inhibitory properties of various drugs were studied in recombinant sterol isomerase-producing yeast cells. Our results clearly show that SR31747 is a high affinity ligand of recombinant mammalian sterol isomerase (Kd = 1 nM). Tridemorph, a sterol biosynthesis inhibitor that is widely used in agriculture as an antifungal agent, is also a powerful inhibitor of murine and human sterol isomerases (IC50 value in the nanomolar range). Some drugs, like cis-flupentixol, trifluoperazine, 7-ketocholestanol and tamoxifen, inhibit SR31747 binding only with the mammalian enzymes, whereas other drugs, like haloperidol and fenpropimorph, are much more effective with the yeast enzyme than with the mammalian ones. Emopamil, a high affinity ligand of human sterol isomerase, is inefficient in inhibiting SR31747 binding to its mammalian target, suggesting that the SR31747 and emopamil binding sites on mammalian sterol isomerase do not overlap. In contrast, SR31747 binding inhibition by tamoxifen is very efficient and competitive (IC50 value in the nanomolar range), indicating that mammalian sterol isomerase contains a so-called antiestrogen binding site. Tamoxifen is found to selectively inhibit sterol biosynthesis at the sterol isomerase step in the cells that are producing the mammalian enzyme in place of their own sterol isomerase. Finally, we also show that tridemorph, a sterol biosynthesis inhibitor widely used in agriculture as an antifungal agent, is not selective of yeast Delta8-Delta7 sterol isomerase but is also highly efficient against murine Delta8-Delta7 sterol isomerase or human Delta8-Delta7 sterol isomerase. This observation contrasts with our already published results showing that fenpropimorph, another sterol isomerase inhibitor used in agriculture, is only poorly efficient against the mammalian enzymes.

Regulation of cytochrome P450 cholesterol side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase type 1 and estradiol-17 beta-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells.

In human placenta the cytochrome P450 side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta-HSD-1) convert cholesterol and pregnenolone producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD-1) mediates the interconversion of estrone and estradiol. We have examined the effects of calcium on phorbol ester- and cAMP-induced P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs in human JEG-3 cells. A23187 increased in a dose-dependent fashion in the 1.3 kb 17 beta-HSD-1 mRNA whereas a weaker increase followed by a gradual depletion effect of A23187 was observed on 3 beta-HSD-1 mRNA. No significant effect of A23187 on P450scc mRNA was observed. Using 0.50 microM of A23187 the induction of 3 beta-HSD-1 and 17 beta-HSD-1 mRNAs was maximum within about 6 h whereas P450scc mRNA levels stayed unaffected throughout the time-course period. The action of A23187 was synergistic on cAMP-stimulated 17 beta-HSD-1 mRNA levels, while in a dose-dependent manner A23187 progressively depleted 3 beta-HSD-1 and P450scc mRNA abundance probably by activation of a calcium-/calmodulin-dependent phosphodiesterase. On the phorbol 12-myristate, 13-acetate (PMA)-stimulated 3 beta-HSD-1, 17beta-HSD-1 and P450scc mRNA levels only the lowest concentration of A23187 potentialized the PMA effect on the 17 beta-HSD-1 mRNA levels. Using thapsigargin (TG), a cell-permeable sesquiterpene lactone that releases calcium by inhibiting sarco/endoplasmic reticular calcium-ATPase, our data indicated the presence in JEG-3 cells of TG-sensitive and TG-insensitive calcium-ATPases regulating 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. These results emphasized the complexity of calcium contribution with the protein kinase A and C pathways in the regulation of P450scc, 3 beta-HSD-1 and 17 beta-HSD-1 mRNA levels. In addition, the different sensitivity of these genes to calcium suggest they could be activated by different subclasses of PKCs.

Testosterone decreases 3beta-hydroxysteroid dehydrogenase-isomerase messenger ribonucleic acid in cultured mouse Leydig cells by a strain-specific mechanism.

We previously reported a strain-related difference in basal 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) activity in response to testosterone in cultured Leydig cells. The data suggested that the response to testosterone was androgen receptor mediated and that testosterone was acting via a trans-acting factor distal to the androgen receptor to regulate Leydig cell basal 3betaHSD activity. This study was designed to determine whether the previous reported strain-related difference in basal 3betaHSD activity in response to testosterone was due to a difference at the 3betaHSD protein and/or at the mRNA level. In C57BL/6J Leydig cells, 2.0 microM testosterone significantly decreased basal 3betaHSD immunoreactive mass by day 6 in culture. Treatment with 2.0 microM testosterone and 2.0 microM hydroxyflutamide, an androgen receptor antagonist, negated the inhibitory effect of testosterone on C57BL/6J 3betaHSD immunoreactive mass. Treatment with 2.0 microM testosterone also significantly decreased 3betaHSD mRNA content in C57BL/6J Leydig cells, which was detectable on day 3 in culture. In contrast to Leydig cells from C57BL/6J mice, Leydig cells from C3H/HeJ mice were not susceptible to the inhibitory effect of testosterone on 3betaHSD. Treatment with 2.0 microM testosterone had no detectable effect on C3H/HeJ 3betaHSD immunoreactive mass or mRNA content at any time point in culture. These data indicate that the testosterone-induced loss of basal 3betaHSD activity in C57BL/6J Leydig cells can be accounted for by the loss of 3betaHSD immunoreactive mass, which is preceded by the loss of 3betaHSD mRNA, and that the strain-related difference in the regulation of 3betaHSD is present at all three levels. Thus, the putative trans-acting factor involved in the mechanism whereby testosterone decreases basal 3betaHSD is likely to regulate the amount of 3betaHSD mRNA.

[The effect of chemical damage to the adrenal cortex on its ontogenetic development]. / Vliianie khimicheskogo povrezhdeniia korkovogo veshchestva nadpochechnika na ego ontogeneticheskoe razvitie.

We studied adrenal gland of rats at the age of 1 month, which underwent injections of dioxin-preparations during a week. In 1, 6, 12 days; 1, 3, 5, 5, 7, 13 months adrenal gland mass, adrenal cortex size, adrenocorticocytes number, 3B-ol-steroid dehydrogenase and succinate dehydrogenase activities of the experimental animals differed greatly from that of the control. It was found that chemical damage of the gland at an early stage changes it greatly during the following ontogenic development.

Kinetics and stability of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni in the system of reverse micelles of aerosol OT in isooctane.

Partially purified delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni was studied kinetically after solubilization in reverse micelles of aerosol OT (AOT) in isooctane and water, as regards its application to biotechnology. With delta 5,10-estren-17 beta-ol-3-one as a substrate, KSI displays an enzyme activity in the micellar system but a low stability. In the presence of urea, the enzyme is, however, stable. Kinetic parameters of the stabilized enzyme are highly sensitive to both the hydration degree of the surfactant and its concentration. The hypothesis of the geometric correspondence of a non-spherical enzyme and spherical micellar matrix is considered.

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in two distinct density Leydig cells from immature rats. Differences in responsiveness to human chorionic gonadotropin or 8-bromoadenosine 3',5'-monophosphate.

The present studies examined the responsiveness to human chorionic gonadotropin (hCG) or 8-bromoadenosine 3',5'-monophosphate (8-Br-cAMP) of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature Band 2 (low density) or Band 3 (high density) Leydig cells isolated on Percoll gradients. Enzyme activity increased in relation to the dose of hCG or 8-Br-cAMP in both bands; however, activity in Band 2 cells increased about 200% above control, while activity in Band 3 cells increased only about 30-60% above control following 6 days of treatment. Maximal responses were observed 4-6 days following exposure to hCG or 8-Br-cAMP in both bands. Because elevated 5 alpha-reductase activity prevents testosterone accumulation in immature Leydig cells, Band 2 or Band 3 cells were cultured in the presence of 4-methyl-4-aza-3-oxo-pregnan-(20S)-carboxylate, a 5 alpha-reductase inhibitor, to assess the relationship between changes in delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity and testosterone formation. Although hCG or 8-Br-cAMP-stimulated delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity began to decline by day 8 or 10 of treatment, testosterone levels progressively increased for 10 days before declining in both bands. Thus, changes in enzyme activity did not strictly correlate with testosterone synthesizing capacity of cultured Leydig cells. Enzyme activity also was measured in cells cultured with the 5 alpha-reductase inhibitor to determine whether the responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of Band 2 or Band 3 cells was due, in part, to low testosterone levels. delta 5-3 beta-Hydroxysteroid dehydrogenase-isomerase activity in Band 2 or Band 3 cells cultured without or with the inhibitor was similar, suggesting that testosterone does not inhibit the enzyme in immature Leydig cells. The greater responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in Band 2 cells was not due to Leydig cell replication as neither total DNA or [3H]thymidine incorporation into DNA was altered by hCG or cAMP.