The epigenetic regulation of gene expression through the covalent modification of histones is crucial for developing germline cells. To study the regulatory role of alternative splicing (AS) of euchromatic histone lysine methyltransferase 2 (EHMT2/G9A) in spermatogenesis in Mongolian horses, this study first examines the localization of the EHMT2 gene in testicular support cells and then predicts the higher-order structures of sequences with and without AS. Two types of lentiviral vectors for overexpression were subsequently constructed for the EHMT2 gene, one with AS and one without, to infect support cells. The proliferation and activity of infected cells were measured using CCK8, and the differential expression of spermatogenesis-related genes in the two types of support cells was analyzed via qRT–PCR. We analyzed the expression of EHMT2 by immunofluorescence staining. EHMT2 was expressed in the nuclei of Sertoli cells. The expression of spermatogenesis-related genes was measured in the two types of cells. The results reveal that the expression levels of the FSH, Stra8, CCNB2, CDC27, NRG1, PPP2R5C, CCNB2, and YWHAZ genes in the AS group were greater than those in the control group. These results indicate that AS events in EHMT2 affect gene expression and thus affect spermatogenesis.

Background: Normal spermatogenesis in Mongolian horses depends on the mitotic division of spermatogonia, two successive meiotic divisions, and the morphological transformation of spermatids into mature spermatozoa. The MEI1 gene is involved in the meiosis cycle and is required for normal chromosome association during meiosis. Previous studies have shown that alternative splicing of MEI1 may promote spermatogenesis in Mongolian horses. In this paper, the regulatory effects of different MEI1 alternative splicing events on Mongolian horse spermatogenesis are investigated. Results: In this study, two overexpressed lentiviral vectors with mutually exclusive exon (MXE) and skipped exon (SE) events of MEI1 were constructed and successfully used to infect Sertoli cells. After 72 h of viral infection, the expression of MEI1 was higher in the SE event than in the MXE event (p < 0.001), as shown by fluorescence quantification; transcriptomics and metabolomics were then used to screen and annotate the differential genes and metabolites, and 193 differentially expressed genes (comprising 109 genes, such as MEI1, and 84 genes with upregulated and downregulated expression, respectively) and 11,360 differentially expressed metabolites (comprising 7494 and 3866 metabolites with upregulated and downregulated expression, respectively) were screened. Differential genes and metabolites were mainly enriched in several metabolic pathways related to spermatogenesis. Differential genes such as IL31RA, ATP2B3, and CASQ2 were highly expressed in SE events, while IL11, PRLR, and CCR7 were highly expressed in MXE events. Metabolites such as folic acid and spermine were highly expressed during SE events, while citric acid and glutathione were highly expressed during MXE events. This suggests that both MXE and SE events of the MEI1 gene can promote the activity of the spermatogenesis signaling pathway. Conclusions: The MXE and SE splicing events of the MEI1 gene may influence spermatogenesis by regulating the expression of spermatogenesis-related genes and metabolites. These findings provide a theoretical foundation for further investigations into the regulatory mechanisms of different alternative splicing events in Mongolian horse spermatogenesis.