Study On Candidate Genes Promoting The Differentiation Of Male Mouse Primordial Germ Cells Into Spermatogonium

Objective: Primordial Germ Cell(PGC)is the first type of germ cells formed during embryonic development.It is the direct precursor of oogonium and spermatogonium,which is of great significance to heredity and reproduction.The gonad(also known as the genital ridge)refers to a pair of organs that have not yet differentiated into ovaries or testes in mice.They are composed of somatic cells and PGCs,and have dimorphism that can differentiate into both ovaries and testes.This differentiation process is called gonadal sex determination.PGCs in both sexual genital ridge are also dimorphic before differentiation,and their differentiation into gonium is regulated by somatic cells in the genital ridge,that is,PGCs in the female genital ridge differentiate into oogonium,while PGCs in the male genital ridge differentiate into spermatogonium.However,there are few studies on the acting factors and their mechanisms of corresponding differentiation pathway in PGCs.In this study,we screen the genes that specifically expressed in PGCs of male genital ridges at the 13.5th day of embryonic stage(E13.5d)by microcellular amplified transcriptome sequencing,and predict the transcription factors that regulate them,so as to explore the molecular regulatory mechanism of PGCs differentiation into spermatogonium.Methods: 1.PGCs of E13.5d C57BL/6J mouse embryos were isolated by cell sorting experiment with magnetic beads,then constructed single cell transcription library,the transcription profile of E13.5d PGCs was obtained by microcellular amplified transcriptome sequencing;2.Screen out male-specific expression genes in PGCs through bioinformatics analysis;3.Verified the expression of male-specific genes Etd and Acbd7 in PGCs at E13.5d by immunohistochemistry,real-time fluorescence quantitative PCR and fluorescence in situ hybridization;4.Screen transcription factors that may transcription regulate Etd and Acbd7 through bioinformatics analysis,and verified the expression of candidate transcription factors Yy1,Ets-1,Jun B,Jun D,Hoxd9 and Hoxd10 in genital ridge at E13.5d by real-time fluorescence quantitative PCR.Results: 1.PGCs was separated by cell sorting experiment with magnetic beads,and alkaline phosphatase staining verified the positive rate of PGCs>95%.2.Immunohistochemistry,real-time fluorescence quantitative PCR and fluorescence in situ hybridization confirmed the specific expression of Etd and Acbd7 in PGCs of male genital ridge at E13.5d.3.Real-time fluorescence quantitative PCR verified the expression of transcription factors Yy1,Ets-1,Jun B,Jun D,Hoxd9 and Hoxd10 in PGCs of male genital ridge at E13.5d.Conclusion: Acbd7 and Etd genes are specifically expressed in PGCs of male C57BL/6J mice at E13.5d,and may be the important regulate genes that promote PGCs differentiation into spermatogonium.