BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals

Objectives BCL2-associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24-skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis. Materials and Methods CRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high-testosterone mice model. H&E staining, qRT-PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis. Results Bag6 exon24 knockout mice show sub-fertility along with partially impaired blood-testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24-deficient testes and sterol regulatory element-binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up-regulated, which causes a high serum testosterone level. Additionally, serine/arginine-rich splicing factor 1 down-regulates BAG6 exon24-skipped transcripts in porcine Sertoli cells by binding to 35-51 nt on BAG6 exon24 via its N-terminal RNA-recognition domain. Conclusions Our findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.