Selective GPER activation decreases proliferation and activates apoptosis in tumor Leydig cells.

We have previously shown that estrogens binding to estrogen receptor (ER) α increase proliferation of Leydig tumor cells. Estrogens can also bind to G protein-coupled ER (GPER) and activation of this receptor can either increase or decrease cell proliferation of several tumor types. The aim of this study was to investigate GPER expression in R2C rat tumor Leydig cells, evaluate effects of its activation on Leydig tumor cell proliferation and define the molecular mechanisms triggered in response to its activation. R2C cells express GPER and its activation, using the specific ligand G-1, is associated with decreased cell proliferation and initiation of apoptosis. Apoptosis after G-1 treatment was asserted by appearance of DNA condensation and fragmentation, decrease in Bcl-2 and increase in Bax expression, cytochrome c release, caspase and poly (ADP-ribose) polymerase-1 (PARP-1) activation. These effects were dependent on GPER activation because after silencing of the gene, using a specific small interfering RNA, cyt c release, PARP-1 activation and decrease in cell proliferation were abrogated. These events required a rapid, however, sustained extracellular regulated kinase 1/2 activation. G-1 was able to decrease the growth of R2C xenograft tumors in CD1 nude mice while increasing the number of apoptotic cells. In addition, in vivo administration of G-1 to male CD1 mice did not cause any alteration in testicular morphology, while cisplatin, the cytotoxic drug currently used for the therapy of Leydig tumors, severely damaged testicular structure, an event associated with infertility in cisplatin-treated patients. These observations indicate that GPER targeting for the therapy of Leydig cell tumor may represent a good alternative to cisplatin to preserve fertility in Leydig tumor patients.

Expression of cyclooxygenase-1 (COX-1) and COX-2 in human male gametes from normal patients, and those with varicocele and diabetes: a potential molecular marker for diagnosing male infertility disorders.

Rising rates of varicocele and diabetes mellitus (DM) pose a significant problem to human fertility. Recent studies have pointed out the impact of cyclooxygenase (COX) in the regulation of testicular function and male fertility. Prominent COX-2 expression has been described recently in the testes of infertile patients, but little is known about the role and identity of COX isoforms in human sperm under certain disease states such as varicocele and DM. We therefore examined the expression profile and ultrastructural localization of COX-1 and COX-2 concomitantly in semen samples from healthy donors, and patients with varicocele and DM. Using Western blotting assay, 'varicocele' and 'diabetic' sperm showed enhanced COX isoforms expression with respect to the 'healthy' sperm. Immunogold labeling revealed human sperm anatomical regions containing COX-1 and COX-2, confirming their increased expression in pathological samples. Our data demonstrate that both COX isoforms are upregulated in the spermatozoa of varicocele and diabetic patients, suggesting the harmful effect of the diseases also at the sperm molecular level, going beyond the abnormal morphology described to date. In conclusion, COX enzymes may possess a biological relevance in the pathogenesis and/or maintenance of male factor infertility associated with varicocele and DM, and may be considered additional molecular markers for the diagnosis of male infertility disorders.

Conventional progesterone receptors (PR) B and PRA are expressed in human spermatozoa and may be involved in the pathophysiology of varicocoele: a role for progesterone in metabolism.

The physiological roles of intracellular progesterone (PRG) receptors (PRs) have been studied intensively in female mammals, while their functions in male are scarce. Conventional PRs were evidenced in our study by Western blotting, concomitantly in healthy spermatozoa and in oligoasthenoteratozoospermic samples without and with varicocoele. Transmission electron microscopy revealed the presence of the PRs on the membrane as well as in the nucleus, mitochondria and flagellum. A reduced expression of the PRs was observed only in varicocoele spermatozoa. Responses to PRG treatment on cholesterol efflux, tyrosine phosphorylation, src and Akt activities, acrosin activity and acrosome reaction in varicocoele spermatozoa were reduced or absent. To further investigate PRG significance in human male gamete, we focused its action on lipid and glucose metabolism. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities suggests that PRG through the PRs exerts a lipolytic effect on human spermatozoa. An increase in glucose-6-phosphate dehydrogenase activity was also obtained, evidencing a role for PRG on glucose metabolism. In 'varicocoele' spermatozoa, the PRG did not induce energy consumption. The action of PRs on sperm metabolism is a novel finding that renews the importance of PRG in male fertility. Our results showed that varicocoele may lead to male factor infertility by a mechanism involving a decreased PR expression in human spermatozoa that evidences a detrimental effect on spermatozoa at the molecular level, going beyond the abnormal sperm morphology described to date.