Overexpression Of STRA8,BOULE And DAZL Genes Promote Goat Bone Mesenchymal Stem Cells Trans-differentiation Toward Putative Male Germ Cells In Vitro

Livestock pass their desirable traits through germ cells.Therefore,it’s critical to understand the mechanism of gametogenesis and development for cultivating high-quality livestock population.However,the gametogenesis is a complex process and it’s hard to label in vivo which have impeded further study of gametogenesis.On the other hand,due to its self-renewal ability and multilineage differentiation potential,stem cell have been proved to be an effective and efficient tool in investigations of germ cell development.Bone mesenchymal stem cells(BMSCs)is a kind of adult stem cells from bone marrow,which are convenient and pluripotent similar to embryonic stem cell,and are research hotspot in induced germ cells in vitro.Recent researches showed that it has already promoted human and mouse BMSCs trans-differentiate toward germ cell-like cells by RA induction or other treatments,but hardly got the haploid cells.The process of gametogenesis in vivo is controlled by a series of germ cell related genes.Stimulated by retinoic acid gene 8(STRA8)mainly expresses during the period from mitosis into meiosis which is a significant marker gene in germ cells.BOULE and DAZL are important members of DAZ(deleted in azoospermia)family.And DAZL plays a role in germ cell differentiation,meiosis and etc.As for BOULE,it expresses during the late stage of gametogenesis which participates in the process of the meiosis of spermatocyte and spermiotelcosis.Stem cell can effectively trans-differentiate into specific cell line by controlled the expression of some key genes in cell differentiation.Therefore,this research tries to promote the differentiation of goat BMSCs(gBMSCs)to germ cells by overexpression of germ cells specific genes STRA8,BOULE and DAZL through transfection of plasmid DNA and in vitro transcription mRNA and create a non-integration multi-gene overexpression inducing system which also provides a model for investigating the mechanism of gametogenesis.The main results were as follows:1.The expression of goat STRA8 gene and construction of the eukaryotic expression vector.To study the expression pattern of STRA8 during goat testicular development,immunohistochemistry and qRT-PCR were used in goat testes of different develop stages(10 days postnatal,10 dpp;4 month-old,4M;8 month-old,8M).The result showed that STRA8 expressed in all of these three develop stages,and was mainly observed in the nucleus of spermatogonia and primary spermatocytes,and had a gradually increased expression pattern along testis maturation and development from 10 dpp to 8 M,reaching the highest expression level in 8 M than other groups(P<0.05).Then a PCR method was used in cloning 1104 bp goat STRA8 gene coding regions,which was subcloned into plasmid pEX-4-STRA8 and confirmed by PCR and restriction enzyme digestion.2.The expression of goat BOULE and DAZL genes and construction of the eukaryotic expression vectors.To study the expression patterns of BOULE and DAZL during goat testicular development,immunohistochemistry and qRT-PCR were used in goat testes of different develop stages(10 dpp,4 M,8 M).The result showed that BOULE was expressed in cytoplasm of spermatocytes and spermatids,no staining of spermatogonia was observed.Meanwhile,DAZL was presented in the cytoplasm of spermatogonia,primary spermatocytes,some secondary spermatocytes and a small number of round spermatids.Both of them have a significant higher expression levels in 8 M goat testis than 10 dpp and 4 M groups(P<0.05).Then PCR method was used to clone an 816 bp goat BOULE gene coding regions and a 966 bp goat DAZL gene coding regions respectively,for constructed the recombinant plasmids pEX-4-BOULE and pEGFP-DAZL,and were confirmed by PCR and restriction enzyme digestion.3.Research on multi-gene overexpression promote gBMSCs in vitro trans-differentiation toward putative male germ cells by DNA plasmid transfection.The aim of this study was to develop a plasmid DNA-basis method of multi-gene overexpression induction system.Promoting gBMSCs trans-differentiate toward germ cell by overexpression STRA8,BOULE and DAZL genes.Transfected pEX-4-STRA8(iSTRA8)or pEX-4-BOULE(iBOULE)or pEGFP-DAZL(iDAZL)or their combination(iSBD)into gBMSCs by liposome,and to study whether three genes would function independently or synergistically during germ cells induction in vitro.The result showed that,PGCs specification genes STELLA,C-KIT and MVH,male germ cell special genes DAZL,PIWIL2 and RNF17,meiosis gene SCP3 were expressed in some of the induction groups.In addition,the meiotic marker SCP3 protein significantly increased in iSTRA8,iDAZL and iSBD group,compared with iBOULE group and control(P<0.05).Indicated that this plasmid DNA-basis method of multi-gene overexpression induction system can promote gBMSCs trans-differentiate toward germ cell,and suggested that the genes network between STRA8,BOULE and DAZL may be important for the gametogenesis.4.Research on multi-gene overexpression promote gBMSCs in vitro trans-differentiation toward putative male germ cells by mRNA transfection.The present study tried to overexpression of STRA8,BOULE and DAZL genes in gBMSCs by transfected the in vitro transcription mRNA.There were two kinds of induction scheme in this study,one was three-time co-transfected the mRNA combination of three genes in 8 days induction period(mi-SBD),another was three-time mRNA transfected in STRA8,STRA8+BOULE+DAZL,BOULE+DAZL order during 8 days induction period(mi-S+BD),to simulation of STRA8,BOULE and DAZL genes time-order expression patterns in vivo.The result showed that,firstly,the expression levels of germ cell markers all up-regulated in these two group cells after 8-day transfection period,such as OCT4、STELLA、C-KIT、MVH、PIWIL2、RNF17、SCP3.Then,DAZL protein widely expressed in cell cytoplasm of mi-SBD and mi-S+BD groups after induction;there were SCP3 positive cells presented in both of the two induction groups,and its expression levels both were higher than control(P<0.05);the expression levels of MVH protein were increased in both two groups(P<0.05),and mi-S+BD was higher than mi-SBD group(P<0.05).In addition,the H19 locus were hypomethylated in cells of mi-SBD and mi-S+BD groups(P<0.05),and mi-S+BD groups was significantly lower than control(P<0.01).Indicated that gBMSCs have already trans-differentiated toward male germ cell by multi-gene mRNA transfected induction.And time-order transfected induction method simulate the genes time-order expression patterns in vivo,which increased the expression levels of germ cell special marker,promoted the meiosis,and down-regulated the methylation level in gBMSCs.Suggested that the multi-genes time-order transfected induction method was more effective and efficient.