Decreased abundance of GDNF mRNA transcript in the immature Sertoli cells of cattle in response to protein kinase inhibitor staurosporine

Abstract

Sertoli cells (SC) have important functions in spermatogenesis by regulating development of spermatogenic cells. Glial cell line-derived neurotrophic factor (GDNF) are produced by SC. Although the effects of GDNF on spermatogenesis have been well studied, the understanding of how GDNF is synthesized is still limited, especially in food animal producing species. Because protein kinase (PK) has varied functions in multiple cellular processes and the PK pathway modulates SC functions, the objective of the present study was to determine whether PK modulates the abundance of GDNF protein in SC of cattle. To conduct this study, immature SC were enriched from cryopreserved testicular tissues of 1-day-old bulls. These cells had a marked proliferation capacity. Results from immunostaining analysis indicated that there was a sustained abundance of SC mRNA marker protein transcripts and marker proteins: androgen bind protein (ABP), GATA4 and VIMENTIN. There was subsequent characterization of SC treated with the PK inhibitor staurosporine for 0, 1 or 2 h. Results from real-time-PCR and Western blot analyses indicated the treatment (2 h) resulted in a decrease in Gdnf mRNA transcript and GDNF protein. Additionally, the staurosporine treatment resulted in an increase in the abundance of anti-apoptosis Bcl2 and decrease in pro-apoptosis Bax mRNA transcripts. Furthermore, results of the TUNEL assay indicated there was a decrease in apoptosis in the staurosprine-treated SC. Collectively, results indicate the PK signaling is involved in regulation of GDNF protein abundance in the immature SC and the survival of these cells in cattle.