SSCLCs Differentiated From PGCs In Chicken And Initiation Mechanism Of Meiosis In Vitro

Primordial Germ Cells(PGCs)are progenitor cells of gametogenesis,which involves a multi-step complex process,however,the process and mechanism of poultry sperm generation are still unclear.In recent years,the problem of continuous decline of breeding chickens’ capacity has always plagued the development of the poultry industry.Studies have shown that 5-12%of breeding roosters are eliminated each year due-to the poor spermatogenic capacity,and the emergence rate of breeding chickens has declined,showing a "upside down" trend,which is closely related to the spermatogenesis of breeding roosters.Secondly,transgenic chickens have great potential as bioreactors and can be used to produce high-value medicinal proteins.It’s one of the main ways to produce transgenic chickens and bioreactors that PGCs introduced exogenous genes using the method of artificial modification and then produced mature sperm in vitro induction.Therefore,the study of in vitro induction system can reproduce the precise process of PGCs differentiation into spermatozoa during spermatogenesis in poultry,which could establish an important platform for the study of spermatogenesis mechanism in poultry.In this paper,chicken PGCs cultured in vitro were induced to differentiate into Spermatogonial Stem Cells Like Cells(SSCLCs),and the meiosis process was initiated to explore the dynamic map of gene expression changes during the induction,which laid a foundation for further in-depth analysis of the process of avian Spermatogonial Stem Cells and the production of transgenic chicken.The results are as follows:1.In order to obtain chicken PGCs cultured in vitro,4.5-5.5 days of chicken embryo gonads were digested using trypsin,and successfully isolated and cultured chicken PGCs in feeder mediun;then in order to improve the purity and proliferation efficiency in vitro,the PGCs were cultured in the feeder-free medium which suitable for stem cell without feeder culture.The results showed that PGCs aggregated in small groups after 4 days of continuous cultivation in the optimized feeder-free medium,and a large number of PGCs aggregated after 7 days of cultivation.this aggregation phenomenon is more obvious than that in feeder culture systems,which was more conducive to the short-term rapid proliferation and renewals(25 d)and higher purity of PGCs(over 40%);besides,the feeder-free medium with N2,B27 and CS was more conducive to the proliferation and renewal of PGCs in vitro;the cellular immunofluorescence staining analysis proved that the PGCs cultured in feeder medium and in the feeder-free medium still maintained the characteristics of sternness and reproduction and had not occurred differentiation in vitro,so as to prepare for the subsequent induced differentiation of PGCs in vitro.2.In order to explore the induction effect of cytokines bFGF and GDNF on chicken PGCs in vitro,bFGF and GDNF were added to the induction medium to induce chicken PGCs in vitro.Flow cytometry analysis showed that compared with the group added with bFGF and GDNF,the proportion of double-positive SSCLCs in the group without bFGF/GDNF and the group with bFGF or GDNF added alone was decreased significantly(P<0.05);the mRNA level of CVH and ITGA6 gene expression were significantly higher than the other two groups after the addition of bFGF and GDNF factors(P<0.05),and meiosis initiation gene STRA8 was highly expressed,those results indicated that the cytokines bFGF and GDNF had an important role in the induction and differentiation of PGCs.Base on the above experimental results,the induction system of chicken PGCs in vitro was determined that the medium containing cytokines bFGF and GDNF is beneficial to differentiate into SSCLCs and enter meiosis.3.In order to clarify the dynamre map of the change of doaole positive SSCLCs ratio and gene expression during the induction of chicken PGCs in vitro,the PGCs cultured for 7 days in feeder medium were induced in vitro firstly by adding the induction system in vitro constructed above.The results found that the integrin-α6 positive SSCLCs appeared after induction for 24 h,the proportion of Integrin-β1 and SSEA-1 double positive SSCLCs reached 27.9%after 48 h of induction using flow cytometry;besides,the quantitative PCR analysis found that the expression of stem cell factor OCT4 gene was significantly decreased after 48 hours of induction(P<0.05),and the expression levels of reproductive marker genes DAZL and ITGB1 increased significantly after 72 h of induction(P<0.05),indicating that the cells gradually exited the pluripotent and self-renewal state after induction,while the high reproductive marker genes expression means the PGCs differentiation towards reproduction.At the same time,the meiotic initiation gene STRA8 increased significantly after 48 h of induction(P<0.05),and showed a downward trend after 72 hours of induction,and the expression of the recombination complex gene SYCP3 decreased at 48 h,but the difference was not significant,and the expression level at 72 h was significantly increased(P<0.05),indicating that the cells have started meiotic activity,and were already in meiosis I phase,homologous recombination and other important biological events during the induction process.Similarly,during the process of differentiation in virto induced for the PGCs cultured without feeding layer medium,the cells gradually withdrew from the pluripotent state,while the reproductive marker genes CVH and ITGB1 were highly expressed,and the meiosis marker genes STRA8 and SYCP3 were also highly expressed,marking cells undergo meiosis;and the feeder-free culture system has better induction effect in vitro under this experimental induction system(efficiency increased 24%).4.In order to study the effect of different gender PGCs on the efficiency of SSCLCs formation due to mutual interference in the induction of differentiation,the mixed male and female PGCs were induced for the PGCs cultured with feeder-free medium.The results found that the proportion of SSCLCs in both groups increased with the induction time,the proportion of double positive SSCLCs in the mixed male and female PGCs was 13.8%higher than that in the male PGCs group after 72 hours of induction,showing that female PGCs can promote the differentiation of male PGCs to form SSCLCs in vitro under this experimental induction system.In summary,chicken PGCs cultured in an induction medium with bFGF and GDNF can differentiate into SSCLCs in vitro and the induction differentiation system of PGCs into SSCLCs in vitro was successfully established,which can promote the induced cells into meiosis.In addition,the feeder-free medium is more conducive to the proliferation and renewal of PGCs,and has more efficient in the differentiation into SSCLCs,moreover,female PGCs can promote the differentiation of male PGCs to form SSCLCs in vitro under this experimental induction system.