Novel expression of spermatogenesis-associated protein 3 gene in mouse spermatogenic cells and its influence upon apoptosis and autophagy in HEK 293T cells

doi:10.16098/j.issn.0529-1356.2018.01.007

Abstract

Abstract:

Objective To detect the expression of spermatogenesis-associated protein 3(spata3) gene in mouse spermatogenic cells and to analyze its probable function on apoptosis and autophagy in HEK 293T cells over-expressed spata3. The overall aim is to explore the possible significance of spata3 during mouse spermatogenesis. Methods Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of spata3 mRNA and its encoded protein in a variety of mouse tissues, respectively. Immunohistochemical staining and indirect immunofluorescent staining were used to locate the SPATA3 protein in spermatogenic cells. The recombinant eukaryotic expression plasmid Plv-EGFP-2 (a) puro-spata3 were transiently transfected into HEK 293T cells using liposome and the expression level of apoptosis-related proteins including cleaved-Caspase3, poly ADP-ribose polymerase(PARP), BAX, Bcl-2 and autophagy-related protein LC3A/B were investigated by Western blotting. Results Spata3 mRNA and its encoded product were specifically expressed in mouse testis. SPATA3 positive signals were mainly located in the cytoplasm and the nucleus of both the round spermatids and the pachytene spermatocytes. Moreover, SPATA3 stainning signals were detected in the cytoplasm of elongating spermatids. The expression levels of cleaved-Caspase-3 and cleaved-PARP were not significant difference between HEK 293T cells over-expressed spata3 and control groups. The content of BAX in 293T cells overexpressed spata3 0.815±0.020 was higher than that in 293T cells 0.469±0.012 and in 293T cells transfected with Plv-EGFP-2(a) puro 0.588±0.018, while the expression of Bcl-2 in HEK 293T cells over-expressed spata3 0.214±0.020 was lower than that in HEK 293T cells 0.507±0.021 and in HEK 293T cells transfected with Plv-EGFP.2(a) puro 0.545±0.024. The amount of LC3A/B-Ⅱin HEK 293T cells over-expressed spata3 0.741±0.037 were detected higher than that in HEK 293T cells 0.136±0.011 and in HEK 293T cells transfected with Plv-EGFP-2(a) puro 0.169±0.012. Conclusion Spata3 is specifically expressed in mouse spermatogenic cells and its over-expression in HEK 293T cells has no significant effect upon apoptosis, but could promote autophagy.

Key words: Spermatogensis-associated protein 3gene, HEK 293T cell, Cell transfection, Autophagy, Western blotting, Mosue

WANG Yu-jing WEN Li-min BAI Xin-yan CAO Rui WANG Hai-long GUO Rui. Novel expression of spermatogenesis-associated protein 3 gene in mouse spermatogenic cells and its influence upon apoptosis and autophagy in HEK 293T cells[J]. Acta Anatomica Sinica, 2018, 49(1): 41-48.

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