The Analysis Of Heterogeneity In Porcine Spermatogonia Based On The Single Cell Sequencing

In mammalian testis,spermatogenesis orgniated from spermatogonia,which undergo a series of proferation and differentiation to form spermatozoa.Moreover,spermatogonia can be divided into undifferentiated spermatogonia and differentiating spermatogonia according to their monology and ablity of proliferation.The differentiation and markers of mouse spermatogonial cells have been studied in depth,but the heterogeneity of porcine spermatogonial cells remains unclear.Therefore,based on single cell RNA sequencing,this study analyzed the heterogeneity of pig spermatogonia,and further analyzed the classification of pig spermatogonia subpopulations.To further explore the surface molecular markers of undifferentiated and differentiating spermatogonia,we analyzed the different expression genes of each spermatogonia clusters.The surface molecular markers of porcine spermatogonia were identified by immunofluorescence staining.The main results are as follows:Firstly,In this study,we first profiled single-cell transcriptomes from the porcine testis at 150 days of age.The cells were clustered into 20 clusters which identified as undifferentiated spermatogonia,differentiating spermatogonia,leptene/zygotene spermatocytes,pachtene spermatocytes,diplotene spermatocytes,MII spermatocytes,round spermatids,elongated spermatids,sperm,myoid,Sertoli and Leydig cell types.In addition,we delineated four distinct spermatogonial states in porcine testis,including undifferentiated spermatogonia,termed Un-diff,which displays high properties in maintaining stemness.And the other three states with high proliferative activity were defined as differentiating spermatogonia(Diff-1,Diff-2 and Diff-3).To further illustrate the accuracy of the identified marker genes,we performed immunostaining for CD99,CDH1 and PODXL2 to validated our single-cell measurements.In sum,CDH1 and CD99 served as marker genes for undifferentiated spermatogonia,and the results manifested that PODXL2 marks the differentiating soermatogonal population in porcine spermatogenesis.Secondly,the specificity of CDH1 was confirmed by immunofluorescence,q-PCR and immunohistochemical.The result showed 80% overlapped with DBA and 90% overlapped with UCHL1.Meanwhile,CDH1 positive cells have high proliferative activity and were almost no apoptosis cells.Thirdly,the expression and location of CD99 showed more similarities with UCHL1 at different developmental stages in the porcine testicular tissue.CD99 positive cells could co-localized with UCHL1 with 90%,which suggested CD99 positive cells are a rare population in undifferentiated spermatogonia.Finally,with age,almost all PODXL2 positive cells were UCHL1 negative.Meanwhile,PODXL2 positive cells could co-localize with KIT at the same age at 90 days.Our results identified that PODXL2 could serve as marker gene for differentiating spermatogonia.Taken together,in this study,we confirmed CDH1 and CD99 positive cells are a rare population in undifferentiated spermatogonia.Meanwhile,PODXL2 could mark the differentiating spermatogonia.Our results revealed the transcriptome heterogeneity of porcine spermatogonia,while also providing multiple insights into the development of porcine spermatogonia.