Hormonal Regulation of the Tubulobulbar Complex in the Testis.

Abstract

Spermatogenesis is absolutely dependent on follicle stimulating hormone (FSH) and androgens; acute suppression of these hormones inhibits germ cell development and thus sperm production. The removal of intercellular junctions and release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to acute hormone suppression. Retention of spermatids following hormone suppression is characterised by the persistence of intercellular junctions between the Sertoli cell and the adjacent elongated spermatid. Recent reports have suggested that the tubulobulbar complex (TBC) and the related endocytotic pathways may be important mediators of intercellular junction internalisation by the Sertoli cell, and hence sperm release. We hypothesised that the TBC is regulated by FSH and androgens in the testis. The aim of this study was to characterise the hormonal dependence of TBC formation and function by the Sertoli cell. Using a previously characterised in vivo model of acute hormone suppression in rats, we assessed the expression of known and predicted TBC components by array analysis and immunohistochemistry. Following hormone suppression, known components including clathrin, Wasl and the Arp2/3 complex, and previously uncharacterised components including Eps15 and Picalm appear mislocalised or absent, suggesting that the TBC may fail to form appropriately in the absence of hormone action in vivo. Eps15 and Picalm were also suppressed following acute hormone suppression in primary Sertoli cells in vitro by Western blot analysis. In order to assess the impact of FSH and androgens upon clathrin-mediated endocytosis, we measured the kinetics of fluorescently labelled transferrin uptake by Sertoli cells using flow cytometry as a marker of endocytotic function. Using this approach, we confirmed that endocytosis is hormonally stimulated in the Sertoli cell.We have previously demonstrated that FSH and androgen regulate a subset of Sertoli cell miRNAs. It was of particular interest that miR-23b, which is stimulated following hormone suppression, was predicted to repress several TBC associated proteins. To assess this, we cloned the 3′untranslated regions of each gene into a dual luciferase vector, and validated novel inhibitory actions of miR-23b on Eps15, Picalm, Wasl and Dynamin 3. This inhibition provides a facile mechanism by which hormones can act to regulate TBC formation in the testis.In conclusion, we have demonstrated that hormonal suppression in the testis disrupts the localisation of known and predicted components of the TBC. Disrupted removal of intercellular junctions may contribute to the failure of sperm release following hormone suppression, and identifies a novel mechanism by which hormones control male fertility. Research supported by the National Health and Medical Research Council of Australia, Program Grant #494802 and CDF fellowship to CAH #1013533. (platform)