Cross-Species Testicular Single Cell Atlase And Testicular Mesenchymal Cell Heterogeneity Studies

Male infertility does not only occur in humans,but also in economically farmed animals such as cattle,sheep and pig,which affects the normal life of men and family happiness,and also endangers the livestock farming industry to a great extent.The testis is a key organ for spermatogenesis and androgen synthesis,and lesions of the testis itself due to genetic or acquired defects,called primary testicular dysfunction,are an important cause of male infertility.Single-cell sequencing technology has multiple roles such as measuring the gene expression of each cell individually and capturing the diversity between different cell types.Studying the testis from a single-cell perspective provides insight into its function and cellular composition.Single-cell profiles of testes from a variety of mammalian species[1,2],including primates,rodents and even-toed ungulates,already exist,but comparative analyses across species are missing.Although the development of mammalian testis cells is a highly conserved process throughout evolution,mammalian differences in testis cell type composition,function,and marker genes are largely unknown.Constructing cross-species single-cell maps to better understand testis conserved and heterogeneous from an evolutionary perspective can track changes in gene expression profiles of the same cell type during development and differentiation,leading to a more in-depth understanding of male sterility.Moreover,most of the attention on male infertility has been focused on spermatogonia,spermatocytes,and supporting cells,etc.Testicular mesenchymal cells are generally considered to have only the function of synthesizing and secreting androgens,and their relationship with male infertility has rarely been studied.In this paper,cross-species analysis of transcriptome profiles of single cells from sexually mature human,mouse,pig and lake sheep testes reveals cell types and functions that are conserved and differ between species.Single-cell data were obtained by isolating and purifying rat and mouse testicular mesenchymal cells to go into the subtypes of mesenchymal cells and their relevance to sperm development.The main findings are briefly described as follows:1.certain marker genes were found to be consistently present and reliable in mammalian testis,such as sox9 and wt1 in supporting cells,myh11 and acta2 in peritubular myeloid cells,and dmrt1 and uchl1 in spermatogonia,and piwil1 and sycp1 in spermatocytes are also differentially expressed.Several previous studies found that the typical marker genes of spermatocytes are prm1 and tnp1,and according to the process of cell annotation in multiple species,it was found that spermatocytes could indeed be specifically identified in human and mouse testis tissues,but prm1,tnp1 and tssk6 were commonly found in different cell types of pig,sheep and buffalo testes,and were not better able to distinguish spermatocytes.2.The gene expression patterns of sexually mature mammalian testis tissues were basically similar,and correlation analysis revealed that interspecies differences were greater than intraspecies differences,with pig testis tissues being the closest to human,while mice were more different from human.Analysis of conserved and differential between different cell types revealed that SPG,SPC and MC are conserved,while most somatic cells such as: EC,SLC/ILC,ALC and SC differ more between species.3.The association of ALC with sperm cells was detected from human testicular cell trajectory analysis,and single cell sequencing was performed using rat and mouse testicular mesenchymal cells to further explore LCs.cell trajectory analysis found the differentiation nodes of two ALC subpopulations,and analysis of node gene enrichment revealed that ALC-I gene was enriched in lipid catabolic process and fatty acid metabolic process in rats,while ALC-II gene was enriched in sperm differentiation and germ cell development.In mice,one ALC subpopulation was enriched to immune-related biological processes and the other was enriched to biological processes such as sperm development,sperm differentiation and germ cell development.The finding that a new subpopulation of ALC is indeed associated with sperm development may open up a new idea for the study of azoospermia.4.To investigate the molecular mechanism of differentiation of SLC to ALC-II,we found that the core genes that tend to differentiate to ALC-II are pgk2 and odf1;the transcription factors active in ALC-II are Nr5a1 and Ppara.