Regulatory Roles Of Rybp In Mouse Spermatogenesis

Part 1 Generation of Male Germline Conditional Knockout Mouse Model of Rybp with Stra8 cre Transgenic Mice[Purpose] Generating a male germline conditional knockout（cKO）mouse model of Rybp to study the function of Rybp in spermatogenesis.[Methods] Firstly,we generated a Rybp conditional knockout mouse model（Rybpfl/+）employing the gene editing technology.Secondly,we caged Rybpfl/+ mice with stra8 cre transgenic mice to breed the male germline conditional knockout mice of Rybp.Then,we picked out aimed mice(Stra8^cre;Rybpfl/fl)using mouse genotyping.Finally,we verified the knockout efficiency of Rybp by q RT-PCR and Western Blot.[Results] We successfully generated the male germline conditional knockout mouse model of Rybp（Rybpfl/fl;Stra8Cre）by crossing Rybpfl/+ mice with Stra8 Cre transgenic mice.Successful breeding of Rybp cKO mice was confirmed by genotyping analysis.q RT–PCR showed that the m RNA level of Rybp in cKO testes decreased significantly compared to that of controls.In line with the experimental result of q RT –PCR,Western Blot indicated that the protein level of Rybp in cKO testes decreased significantly compared to that of controls.[Conclusions] We successfully generated the male germline conditional knockout mouse model of Rybp,and Rybp was inactivated in male germ cells efficiently.Part 2 Role of Rybp in Spermatogenesis and Its Possible Mechanisms[Purpose] Studying the role of Rybp in spermatogenesis.[Methods] We performed morphological experiment to study the influence of Rybp deletion in male germline on spermatogenesis.We performed Meiotic Chromosome Spreads and TUNEL analysis to study the influence of Rybp deletion in male germline on meiosis and apoptosis.We used q RT-PCR and immunofluorescence to detect changes of gene expression and histone modifications on Rybp deletion in male germline respectively.[Results] Testes of Rybp cKO mice were significantly smaller than that of controls.PAS staining of testis sections showed that seminiferous tubules of testes from cKO mice exhibited reduced diameter and contained lower cell numbers per tubule.Meiotic Chromosome Spreads indicated that testes from Rybp cKO mice had a smaller portion of pachytene spermatocytes and a larger portion of diplotene spermatocytes.TUNEL assay indicated that apoptotic cells in Rybp cKO testes were significantly increased,and a part of leptotene to zygotene spermatocytes underwent apoptosis.Immunofluorescence indicated that the levels of histone modifications H3K9me3 and H3K27me3 were downregulated in Rybp cKO testes.q RT-PCR indicated that expression levels of retrotransposons,cleavage-stage genes and pachytene pi RNA precursors were not affected on Rybp knockout in male germline.[Conclusions] Deletion of Rybp in male germline impaired meiosis of spermatocyte.Knockout of Rybp in male germline reduced the levels of histone modifications H3K9me3 and H3K27me3,which might result in apoptosis of a part of leptotene to zygotene spermatocytes.Part 3 Deletion of Rybp in Male Germline Reduced Sperm Motility[Purpose] Studying the influence of Rybp knockout in male germline on sperm count,sperm motility and sperm morphology.[Methods] We performed semen analysis to detect sperm count,sperm motility and sperm morphology abnormities in Rybp cKO mice.[Results] Semen analysis indicated that sperm count and sperm morphology of Rybp cKO mice were similar to that of controls.However,loss of sperm motility was observed in Rybp cKO mice,with increase of immotile sperm population and decrease of progressive sperm population.[Conclusions] Knockout of Rybp in male germline resulted in loss of sperm motility.Part 4 Conditional Knockout of Rybp in Oocyte Has No Influence on Oogenesis and Preimplantation Embryo Development[Purpose] Studying the function of Rybp in oogenesis and preimplantation embryo development.[Methods] Firstly,we generated a conditional knockout mouse model of Rybp in oocyte（Rybp M-cKO）with Zp3 Cre transgenic mice.Then we utilized superovulation and in vitro mouse embryo culture to study the influence of conditional knockout of Rybp in oocyte on oogenesis,fertilization and preimplantation embryo development.[Results] Oocytes from Rybp M-cKO mice could develop to MII stage and fertilize WT sperm,and these zygotes could develop to blastocysts.Notably,zygotes produced by Rybp depleted Oocytes and sperm could develop to blastocysts.[Conclusions] Conditional knockout of Rybp in oocyte has no influence on oogenesis and preimplantation embryo development.