Autoimmune Regulator Enhanced the Expression of Caspase-3 and Did Not Induce Massive Germ Cell Apoptosis in GC1-Spg Cells

Abstract

Background/Aims: AIRE is known for its involvement in autoreactive T-cell deletion in thymic epithelium. Though extrathymic expression of AIRE is well documented, the functional relevance of AIRE in non-thymus tissues is emerging. AIRE is expressed in neonatal and adult testis, and has been implicated in sporadic germ cell apoptosis in developing testis. In this study we examined whether AIRE has any role in inducing apoptosis in cultured spermatogonial cells. Methods: We over-expressed AIRE or CARD domain of AIRE in GC1-spg cells and evaluated its impact on cell cycle using fluorescence activated cell sorting following Hoechst 33342 staining. Apoptosis was assayed using Annexin-V staining. Caspase-3 cleavage was assessed on western blots and caspase-3 expression was quantitated using realtime PCR. Results: We report that C18-4 cells which are derived from Type A spermatogonia expressed AIRE, while GC1-spg which is closer to Type B spermatogonia was negative for AIRE expression. Overexpression of AIRE or CARD domain of AIRE induced Caspase-3 expression in GC1-spg cells. Silencing of AIRE in C18-4 cells inhibited Caspase-3 expression. When overexpressed, AIRE and CARD brought about a very negligible increase in germ cell death and resulted in altered cell cycle pattern with a reduction in G1 phase. This was not associated with any increase in activation of Caspase-3. Conclusion: We conclude that the CARD domain of AIRE enhances caspase-3 expression through possible direct DNA binding and triggers non-apoptotic downstream signaling in cultured spermatogonial cells.