Preliminary Researches On The Functional Mechanism Of Stra8 During Spermatogenesis

Stimulated by retinoic acid gene 8(Stra8)is specifically expressed in mammalian germ cells.Stra8-knockout males are infertile,with spermatogenesis blocked in the stage of meiosis.The functional mechanism of Stra8 during spermatogenesis is still not clear yet.In order to explore the molecular function of Stra8,we carry out the following three aspects studies.Part Ⅰ.Preliminary studies of genes and networks regulated by Stra8 during spermatogenesisObjectives:To explore the functional mechanism of Stra8 during spermatogenesis,find potentially related downstream genes and determine its downstream regulating networks.Methods:Stra8-knockout homozygotic type(Stra8-/-)and wild type(Stra8+/+)mice from 5 to 12 days after birth were collected.Parafin sections of different days testes were prepared and than stained by Hematoxylin-eosin and immunofluorescence.DEAD(Asp-Glu-Ala-Asp)box polypeptide 4(Ddx4),a spermatogenic cell marker,was used for immunofluorescence.The Ddx4 positive spermatogenic cells in seminiferous tubule were counted and analysed by t-test.Testes of 11 days after birth Stra8-/-and Stra8+/+ mice were used for RNA Sequncing.The expression level of differential expressed RNAs in Stra8-/-and Stra8+/+ mice were verified by qRT-PCR.Results:From 5 to 11 days after birth,testis histological structure and the number of spermatogenic cell per tubule had no significant statistical difference between Stra8-/-and Stra8+/+ mice.While from 12 days after birth,there were more primary spermatocytes in Stra8+/+mice than in Stra8-/-mice.Besides,the number of spermatogenic cell per tubule in Stra8+/+ mice is significantly different from Stra8-/-mice.RNA-Sequnce results showed that there were 96 RNA differently expression in Stra8-/-mice when compared with Stra8+/+ mice.Among these RNAs,40 of them showed down-regulation whereas the other 56 RNA showed up-regulation.qRT-PCR results verified the expression pattern of 21 selected mRNAs in Stra8-/-and Stra8+/+mice.Conclutions:We found 96 differentially expressed RNAs between Stra8-/-and Stra8+/+mice with the method of RNA-Sequnce.Among these RNA,21 mRNA may be involved in three regulatory networks:spermatocyte meiosis,spermatagonia self-renewal and spermatagonia differentiation.We predicted that Stra8 might target these mRNAs and then regulate the process of spermatogenesis.Part Ⅱ.Preparation of rabbit anti mouse Setd8 polyclonal antibodyObjectives:To obtain mouse setd8 protein and prepare rabbit anti-mouse Setd8 polyclonal antibody.Methods:The recombinant plasmid pET-30a-Setd8 was constructed by double enzyme digestion and linkage,and then transformed into E.coli BL21.The expression of the target protein was induced by IPTG and the expression product was purified by Ni-NTA affinity chromatograph.The purified protein was used to immunize New Zealand white rabbits for producing polyclonal antibody.ELISA,Western blot and immunohistochemistry were applied to identify the titer and specificity of the antibody.Results:The prokaryotic expression vector pET-30a-Setd8 was successfully constructed.After inducing by IPTG,recombinaned Setd8 protein expressed effectively in E.coli BL21.ELISA showed that the titer of rabbit anti-Setd8 antiserum was 1:1 000 000.Western blotting demonstrated that the polyclonal antibody could recognize the native mouse Setd8 protein.Immunohistochemistry revealed that Setd8 protein recognized by the polyclonal antibody mainly distributed in the nucleus of spermatogonia in adult mouse testis.Conclusions:Using the prokaryotic expression vector pET-30a-Setd8,we have successfully prepared the polyclonal antibody with high affinity and specificity.Part Ⅲ.Purification of spermatogonia and spermatocytes from adult mouse testisObjectives:To develop a convenient,reliable and easily operated method by which spermatogonia and spermatocytes can be isolated from adult mouse testis.Methods:Single cell suspension was prepared from adult mice testes by using the combined enzyme digestion method.We use a modified discontinuous Percoll gradient(17%,22%,25%,28%,31%,34%,37%,40%and 90%)centrifugation method to isolate spermatogonia and spermatocytes from the cellular suspension.Isolated spermatogonia and spermatocytes were determined by Wright-Giemsa staining.Results:After centrifuged,single-cell suspension formed nine fractions.The 22%fraction contained mainly spermatids.While in the 31%,34%and 37%fractions,most cells were spermatogonia and spermatocytes.Conclusions:By using combined enzymatic digestion and discontinuous percoll gradient centrifugation,purified spermatogonia and spermatocytes could be isolated from adult mice testes.