BCAS2 Is Involved In Alternative MRNA Splicing During Spermatogenesis

Spermatogenesis is a complex and dunamic process involving mitotic cell division,meiosis and spermiogenesis to give rise to haploid spermatozoa.The expression of functional genes should be accurately adjusted and controlled in each stage of spermatogenesis.Alternative splicing is critical for post-transcriptional regulation of gene expression and significantly expands the forms and functions of the proteins in highly complex organisms and tissues.Substantial evidences have suggested that alternative splicing regulates the expression and function of many genes important for spermatogenesis.In addition,a number of splicing factors are primarily or exclusively expressed in mouse testis,suggesting that alternative splicing might play important roles during spermatogenesis.BCAS2 is a core component of the Prpl9 complex and plays important roles in several processes including pre-mRNA splicing.Previously,we found that BCAS2 exhibits specific expression patterns in male germ cells.In this study,based on the mouse model in which BCAS2 is specifically depleted in spermatogonia,we investigated whether BCAS2 is involved in alternative splicing during spermatogenesis.Through immunostaining of BCAS2 during testes development and isolating spermatogenic cells,we found that BCAS2 is specifically enriched in spermatogonia of mouse testes.Mice with conditionally disrupted Bcas2 in male germ cells using Vasa-Cre(Bcas2F/-;Vasa-Cre)developed grossly normal,but were infertile when mated with normal fertile females,although copulatory plugs could be observed.BCAS2 depletion in spermatogonia impaired spermatogenesis with the reduction of germ cells and absence of meiosis events(recombination and synapsis)in meiosis prophase I.Further analysis of the number,location,proliferation and apoptosis of spermatogonia in Bcas2F/-;Vasa-Cre testes revealed that spermatogonia appeared grossly normal,but the spermatocytes in meiosis prophase I were rarely observed and the expression of STRA8 was significantly decreased in BCAS2-depleted mouse testis.These data indicate that BCAS2 is required for the initiation of meiosis during spermatogenesis.To investigate the molecular mechanisms of BCAS2 depletion in germ cells,we isolated mRNA from control and Bcas2F/-;Vasa-Cre testes at P9 and perform RNA sequencing.We did not observed large-scale changes on the transcription level in Bcas2F/-;Vasa-Cre testes.However,the splicing of tubulin,a splicing target gene was disturbed in the absence of BCAS2,indicating that BCAS2 might function through regulating pre-mRNA splicing.To explore the function of BCAS2 in pre-mRNA splicing,we further analyzed the alteration of alternative splicing patterns in Bcas2F/-;Vasa-Cre testes.The result revealed that 245 genes are altered in alternative splicing forms in BCAS2 depleted testes,including 6 functional genes(Dazl,Ehmt2,Hmgal,Cit,Hsfl and Bcl2111)during spermatogenesis.Then,using RT-PCR and real-time RT-PCR analysis,we succesfully verified the abrrant alternative splicing patterns of three spermatogenesis-related genes(Dazl,Ehmt2 and Hmga1).In addition,disruption of Bcas2 resulted in a significant decrease of the full-length form and an increase of the short form(lacking exon 8)of DAZL protein.Moreover,the total level of DAZL protein was dramatically decreased in BCAS2 depleted testes.Taking the advantage of this model,we demonstrated an important role of BCAS2 in pre-mRNA splicing in the mitosis-to-meiosis transition of mouse spermatogonia.The evidences from this study for the first time illustrate that alternative splicing machinery regulates meiosis initiation in germ cells during mouse spermatogenesis,providing novel insights to the molecular basis for meiosis initiation and the physiological fuctions of alternative splicing.