The Research On Key Molecular Mining Of Spermatogenesis And Sex-sorted Semen In Dairy Goats

The dairy goat industry is an important part of dairy industry in China.At present,there are currently insufficient numbers of high-quality dairy goats.How to improve breeds has become a major challenge and demand for the dairy goat industry.Giving full play to the fecundity of excellent goat bucks has a significant genetic improvement effect on the dairy goat population.The fertility of male mammal depends on spermatogenesis.This process takes place in the seminiferous tubules of the testes,including spermatogonia mitosis,spermatocyte meiosis,and round spermatids deformation,which are subject to extremely strict and precise molecular regulation.The development of the current high-throughput sequencing technologies has led to the recognition of the great complexity of the spermatogenesis,which carries thousands of molecules.The precise expression of these key molecules is essential for maintaining spermatogenesis.However,the molecular mechanism of spermatogenesis is still unclear about dairy goats.In addition,using artificial insemination with sex-sorted semen is an effective way to rapidly multiply high-quality ewes.However,sex-sorted semen in dairy goats still needs further research.Therefore,this study analyzed the development of male germ cells and Sertoli cells by immunohistochemical staining,and explored the key molecular characterization of spermatogenesis in Guanzhong dairy goats based on transcriptomics,preliminarily established a novel method of sex-sorted semen of dairy goats.The main results of this study were as follows:1.The testicular tissues of dairy goats at 8 different ages(15,30,45,60,75,90,180,and 240-day-old)were collected,and the developmental characteristics of male germ cells and Sertoli cells were explored by immunohistochemical staining and histomorphological observation.The results were as follows:(1)The diameter of the seminiferous tubules of gradually increased with the development of the dairy goat testes,and the increased faster at75 to 90-day-old.The lumen of the seminiferous tubules,round spermatids and few sperm were observed at 90-day-old,which indicated that dairy goat bucks had entered to puberty.At 180-day-old,sperm was observed in seminiferous tubules,which indicated that dairy goat bucks had entered to sexual maturation.(2)Gonocytes were completely transformed to undifferentiated spermatogonia before 75-day-old.Undifferentiated spermatogonia began to differentiate into spermatogonia at 30-day-old,and the proliferation of undifferentiated spermatogonia was active between 75 to 90-day-old.(3)SOX9 and AMH could be used as markers of Sertoli cells,AMH was only expressed in immature Sertoli cells.Sertoli cells matured at 90-day-old,and the proliferation were active at 45-day-old.2.The lnc RNA and m RNA expression profiling of eight dairy goat testes at 45-day-old birth and 240-day-old were comprehensively analyzed by RNA-seq.The results were as follows:(1)Based on the comparison between 45 and 240-day-old seminiferous tubules samples,11,612 lnc RNAs and 18,217 m RNAs were identified,in which 7594 lnc RNAs and11,986 m RNAs were significantly differentially expressed(DE)between two stages.(2)229DE genes were identified related to spermatogenesis,such as KIT,DMRT1 and SOX9 were down-regulated,DDX4,SYCP1 and SYCP3 were up-regulated after sexual maturity.Furthermore,such as PI3K/Akt,MAPK,AMPK,Ras and Rap1 signaling pathways could regulate of spermatogenesis.(3)The lnc RNA-m RNA interaction networks were constructed,which found that TCONS＿00029023,TCONS＿00035144,TCONS＿00017872,TCONS＿00051085,TCONS＿00033882,and their target genes GSKIP,KAT8,MBD3L1,NANOS3,PRM3 might play important roles during spermatogenesis.3.Single-cell RNA sequencing(sc RNA-seq)technology was used to sequence single cell suspensions of seminiferous tubules at 45,90,and 180-day-old dairy goats,respectively.The results were as follows:(1)25,373 cells from three different ages of seminiferous tubules were captured.Cluster analysis of seminiferous tubule cells from neonatal,immature,and sexual maturation dairy goat testes revealed several cell types,including cell populations with characteristics of spermatogonia,spermatocytes,round spermatids,Sertoli cells,Leydig cells,macrophages,and endothelial cells.The proportion of testicular cells in the seminiferous tubules of dairy goats at different developmental stages had been clarified for the first time.(2)Several potential marker genes for germ and somatic cells were identified,such as SOHLH1,PAGE4 and DMRT1 for spermatogonia,YBX2,HSPB9 and LYPD4 for spermatocytes,PRM2,SPEM1 and PRSS37 for round spermatid,AMH,INHA and EGR1 for Sertoli cells,INSL3,ACTA2 and STAR for Leydig cells.Furthermore,such as testosterone,retinoic acid,PDGF,FGF,WNT signaling pathways were analyzed and found molecular characterization between testicular somatic cells and germ cells.(3)The cell trajectory analysis of male germ cells was determined by Monocles,and the unsupervised pseudo-time exhibited the developmental trajectory of male germ cells in dairy goat.The molecular characterization occurred from spermatogonia to spermatids were revealed,such as DMRT1,SYCP2 and TNP1,and constructed the single-cell transcriptional map of spermatogenesis in dairy goats.4.The coding genes on the sex chromosomes of dairy goats were revealed by RNA-seq data and conducted sex-sorted semen research.The results were as follows:(1)500 genes were identified as expressed genes from X chromosome in dairy goats,such as ABCB7,ABCD1 and ACE2.Nine genes were identified as expressed genes from Y chromosome,such as SRY,USP9 Y and UTY.Nineteen encode receptors genes were identified in X chromosome,such as TLR7 and TLR8.(2)TLR7/8 was expressed in approximately 50% of round spermatids in the testes and sperm,as measured from cross-sections of the epididymis and ejaculate,respectively.(3)TLR7/8 was located at the tail of the X-sperm.Upon TLR7/8activation,phosphorylated forms of glycogen synthase kinase α/β(GSK3 α/β)was detected in the X-sperm,causing reduced mitochondrial activity,ATP levels,and sperm motility.However,Y-sperm was not affected.(4)Following in vitro fertilization using the TLR7/8-activated X-sperm by sorted,80.52% ± 6.75% of the embryos were females,and 19.48% ±6.75% of the embryos were males.In conclusion,this study revealed the development of male germ cells and Sertoli cells from seminiferous tubules.Then,using RNA-seq and sc RNA-seq technologies to identify the key genes and lnc RNA related to spermatogenesis,identified several cell types from the seminiferous tubules,and constructed a single-cell transcription map of spermatogenesis.In addition,using RNA-seq sequencing data to reveal the key genes encoded by the X and Y chromosomes,and preliminarily established a novel method of sex-sorted semen of dairy goats.This study would provide a theoretical basis for the molecular breeding and sex-sorted semen of dairy goats,and also enriched understanding for mammalian spermatogenesis and male fertility.