Single-cell RNA-sequencing reveals the dynamic process and novel markers in porcine spermatogenesis

Background:Spermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations.To date,the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported.Results:To characterize the atlas of porcine spermatogenesis,we profiled the transcriptomes of~16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing(scRNA-seq).The scRNA-seq analysis identified spermatogonia,spermatocytes,spermatids and three somatic cell types in porcine testes.The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis.The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq.Since we delineated four distinct spermatogonial subsets,we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia,respectively.Conclusions:The present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq.Four subsets of spermatogonia were identified and two novel cell surface markers were discovered,which would be helpful for studies on spermatogonial differentiation in pigs.The datasets offer valuable information on porcine spermatogenesis,and pave the way for identification of key molecular markers involved in development of male germ cells.

Transcriptome-wide Dynamics of m^(6)A mRNA Methylation During Porcine Spermatogenesis

Spermatogenesis is a continual process that occurs in the testes,in which diploid spermatogonial stem cells(SSCs)differentiate and generate haploid spermatozoa.This highly efficient and intricate process is orchestrated at multiple levels.N^(6)-methyladenosine(m^(6)A),an epigenetic modification prevalent in mRNAs,is implicated in the transcriptional regulation during spermatogenesis.However,the dynamics of m^(6)A modification in non-rodent mammalian species remains unclear.Here,we systematically investigated the profile and role of m^(6)A during spermatogenesis in pigs.By analyzing the transcriptomic distribution of m^(6)A in spermatogonia,spermatocytes,and round spermatids,we identified a globally conserved m^(6)A pattern between porcine and murine genes with spermatogenic function.We found that m^(6)A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators.In addition,transcriptomes in porcine male germ cells could be subjected to the m^(6)A modification.Our data show that m^(6)A plays the regulatory roles during spermatogenesis in pigs,which is similar to that in mice.Illustrations of this point are three genes(SETDB1,FOXO1,and FOXO3)that are crucial to the determination of the fate of SSCs.To the best of our knowledge,this study for the first time uncovers the expression profile and role of m^(6)A during spermatogenesis in large animals and provides insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.

Single-cell RNA-seq analysis of testicular somatic cell development in pigs

Spermatogenesis is the process by which diploid male germ cells propagate and differentiate into haploid flagellated spermatozoa.This highly complicated process is dependent on testicular somatic cells maturation.While the role of these somatic cells in spermatogenesis is relatively well established,knowledge about their development and maturation,particularly at the molecular level,is limited.In this study,we profiled the testicular single-cell transcriptomes of Guanzhong black pigs at the age of 7,30,60,90,and 150 days.Five types of Sertoli cells,five types of Leydig cells,and four types of peritubular myoid cells were identified.Histological analysis revealed the changes in proliferation levels and marker gene expressions,and the prion-like protein gene(PRND)was identified as a novel marker for Sertoli cells.Additionally,integrated analyses of porcine and human datasets revealed similarities between human and pig testicular somatic cells.Overall,the data obtained in this study contribute to the understanding of testicular development in pigs as a model species.

Ancient genomes reveal the genetic inheritance and recent introgression in Chinese indigenous pigs

Dear Editor,As one of the oldest domesticated livestock, pigs were independently domesticated in the Near East and East Asia,approximately 10,000 years ago (Larson et al., 2010).