Histological Analysis and Identification of Spermatogenesis-related Genes in 2-, 6-, and 12-month-old Sheep Testes

Testis development and spermatogenesis are vital factors that influence male animal fertility. In order to identify spermatogenesis-related genes and further provide a theory basis for finding biomarkers related to male sheep fertility, 2-, 6-, and 12-month-old Small Tail Han Sheep testes were selected to investigate the dynamic changes of sheep testis development. Hematoxylin-eosin routine staining and RNA-Seq technique were used to perform histological and transcriptome analysis for these testes. The results showed that 630, 102, and 322 differentially expressed genes (DEGs) were identified in 2- vs 6-month-old, 6- vs 12-month-old, and 2- vs 12-month-old testes, respectively. GO and KEGG analysis showed the following: DEGs in 2- vs 6-month-old testes were mainly related to the GO terms of sexual maturation and the pathways of multiple metabolism and biosynthesis; in 6- vs 12-month-old testes, most of the GO terms that DEGs involved in were related to metabolism and translation processes; the most significantly enriched pathway is the ribosome pathway. The union of DEGs in 2- vs 6-month-old, 6- vs 12-month-old, and 2- vs 12-month-old testes was categorized into eight profiles by series cluster. Subsequently, the eight profiles were classified into four model profiles and four co-expression networks were constructed based on the DEGs in these model profiles. Finally, 29 key regulatory genes related to spermatogenesis were identified in the four co-expression networks. The expression of 13 DEGs (CA3, APOH, MYOC, CATSPER4, SYT6, SERPINA10, DAZL, ADIPOR2, RAB13, CEP41, SPAG4, ODF1, and FRG1) was validated by RT-PCR.