17ß-Estradiol regulates cyclin A1 and cyclin B1 gene expression in adult rat seminiferous tubules.

Spermatogenesis, which is the fundamental mechanism allowing male gamete production, is controlled by several factors, and among them, estrogens are likely concerned. In order to enlighten the potential role of estrogen in rat spermatogenesis, seminiferous tubules (ST) from two groups of seminiferous epithelium stages (II-VIII and IX-I) were treated with either 17ß-estradiol (E(2)) agonists or antagonists for estrogen receptors (ESRs). In this study, we show that cyclin A1 and cyclin B1 gene expression is controlled by E(2) at a concentration of 10(-9) M only in stages IX-I. This effect is mimicked by a treatment with the G-protein coupled estrogen receptor (GPER) agonist G1 and is abolished by treatment with the ESR antagonist ICI 182 780. Moreover, using letrozole, a drug that blocks estrogen synthesis, we demonstrate that these genes are under the control of E(2) within rat ST. Thus, germ cell differentiation may be regulated by E(2) which acts through ESRs and GPER, expressed in adult rat ST.

Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: ionic channels and gamma-glutamyl transpeptidase activity.

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.

Nongenomic and genomic effects of 1α,25(OH)2 vitamin D3 in rat testis.

The steroid hormone 1α,25(OH)(2)-vitamin D(3) (1,25D(3)) regulates gene transcription through a nuclear receptor (VDRnuc) and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). It has been described that successful mating and fertility rates are significantly decreased in vitamin D deficient male rats and a VDR null mutant rodent has decreased sperm count and motility and expresses rare spermatogenesis. Although the Sertoli cells are pointed as the major target of 1,25D(3) in the testis the mechanism of 1,25D(3) action, particularly in Sertoli cells, remains unclear. Several studies undertaken in the testicular cells showed that 1,25D(3) can produce both genomic and nongenotropic actions in those cells. 1,25D(3) can modulate kinase activities and ionic fluxes (Ca(2+) and Cl(-)) at the plasma membrane resulting in the regulation of secretory processes in Sertoli cells. The enormous complexity of the nongenomic actions of 1,25D(3) implies that specific receptor or specific ligand-binding sites located on the plasma membrane or in the nucleus are believed to initiate specific cell responses. Apparently the choice of the signaling pathways to be activated after the interaction of the hormone with cell surface receptors is directly related with the physiological action to be better accomplished. The demonstration that 1,25D(3) can regulate both Sertoli cell and sperm function may be useful for the study and development of new therapeutic strategies to the treatment of male reproductive disorders. This review summarizes recent research on the rapid actions of 1,25D(3) and identifies questions that remain to be answered in this area.

17 beta-estradiol activates rapid signaling pathways involved in rat pachytene spermatocytes apoptosis through GPR30 and ER alpha.

Aim of the present study was to investigate whether estrogens were able to directly activate rapid signaling pathways controlling spermatogenesis in rat pachytene spermatocytes (PS). Classically, estrogens act by binding to estrogen receptors (ERs) alpha and beta. Recently, it has been demonstrated that rapid estrogen action can also be activated through the G-protein-coupled receptor (GPR)-30. Herein, we demonstrated that rat PS express ER alpha, ER beta and GPR30. Treatment of PS with estradiol (E2), the selective GPR30 agonist G1 and the selective ER alpha agonist PPT determined activation of ERK1/2 which are part of GPR30 signaling cascade. ERK1/2 activation in response to E2 and G1 was correlated to an increased phosphorylation of c-Jun. All treatments failed to induce these responses in the presence of EGFR inhibitor AG1478, ERK inhibitor PD98059 and ER inhibitor ICI182780. mRNA expression of cell cycle regulators cyclin A1 and B1 was downregulated by E2 and G1 while an up-regulation of proapoptotic factor Bax was observed in the same conditions. These data demonstrate that E2, working through both ER alpha and/or GPR30, activates in PS the rapid EGFR/ERK/c-Jun pathway, modulating the expression of genes involved in the balance between cellular proliferation and apoptosis.

Aromatase expression in the normal human adult adrenal and in adrenocortical tumors: biochemical, immunohistochemical, and molecular studies.

OBJECTIVE: The aromatase enzyme catalyzes the final stage of estrogen biosynthesis pathway from androgens. Its expression in the adrenal is poorly studied except for the rare estrogen-producing adrenocortical tumors. In order to further characterize aromatase expression in the adrenal, we evaluated the aromatase enzyme activity, Cyp19a1 gene expression level, and promoter utilization in normal adrenal tissues and in adrenocortical secreting tumors. DESIGN AND METHODS: Six normal adult adrenals (NA), 2 feminizing adrenal tumors (FT), 10 cortisol-producing adenomas with overt (CS, n=4) or sub-clinical Cushing syndrome (SCS, n=6) and 3 aldosterone-producing adenomas (APA) were studied. Tissue aromatase activity was determined by the tritiated ((3)H)-water method. Total aromatase mRNA were measured by a competitive RT-PCR. Promoter regions PII and PI.4-derived transcripts were also studied in NA, FT, and other steroid-producing tumors by a semi-quantitative comparative RT-PCR. Immunofluorescence analysis was performed in normal human adrenal tissues. RESULTS: Aromatase activity was detected in NA tissues and in all tumor subtypes, at high levels in both FT. In NA, aromatase immunofluorescence was detected in the cytoplasm of steroidogenic cells, mainly from zona reticularis. Compared with NA, aromatase transcript levels were similar in CS and APA, lower in SCS and similar or higher in FT. Promoter analysis suggested predominant PII utilization in NA, APA, and SCS, but similar PII and PI.4 utilization in CS tumors. CONCLUSION: Aromatase is expressed at similar levels in normal adrenal and in adrenocortical tumors, but at variably high levels in FT. Different promoter utilization patterns are found among tumor subtypes.

Estrogens: a new player in spermatogenesis.

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them, estrogens are the end products obtained from the irreversible transformation of androgens by aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rat the aromatase activity is mainly located in Sertoli cells of immature animals and then in Leydig cells of adults. Moreover rat germ cells represent an additional source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes (PS) compared to gonocytes or round spermatids (RS); conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank vole, bear and monkey express also aromatase. In man besides Leydig cells, we have shown the presence of a biologically active aromatase and of estrogen receptors in ejaculated spermatozoa and in immature germ cells. Concerning aromatase, a 30% decrease of the amount of mRNA is observed in immotile compared to motile sperm fraction from the same sample; moreover the aromatase activity is also diminished of 34%. In asthenoteratozoospermic and teratozoospermic patients the aromatase gene expression is decreased by 67 and 52%, respectively when compared to normospermic controls. Statistical analyses between the sperm morphology and the aromatase/GAPDH ratio have revealed a high degree of correlation (r=-0.64) between the ratio and the percentage of abnormal spermatozoa (especially microcephaly and acrosmome malformations). Alterations of sperm number and motility have been described in men genetically deficient in aromatase, which together with our data, suggest a likely role for aromatase/estrogens in the acquisition of sperm motility. Therefore besides gonadotrophins and testosterone, estrogens produced locally should be considered as a physiologically relevant hormone involved in the regulation of spermatogenesis and spermiogenesis.