# Effect of Alternative Splicing Euchromatic Histone Lysine Methyltransferase 2 (EHMT2/G9A) on Spermatogenesis in Mongolian Horses

## TL;DR

This study explores how alternative splicing of the EHMT2 gene affects spermatogenesis in Mongolian horses by examining gene expression and cell activity in Sertoli cells.

## Contribution

The study reveals that alternative splicing of EHMT2 influences spermatogenesis-related gene expression in Mongolian horse Sertoli cells.

## Key findings

- EHMT2 is localized in the nuclei of Sertoli cells in Mongolian horses.
- Alternative splicing of EHMT2 increases the expression of several spermatogenesis-related genes compared to the control group.
- Lentiviral overexpression of AS and non-AS EHMT2 variants alters gene expression patterns in Sertoli cells.

## Abstract

In this study, the localization of the EHMT2/G9A gene in Sertoli cells was examined, the higher-level structures of the two splice variants of EHMT2 were predicted, two lentiviral vectors to infect Sertoli cells were constructed, proliferation and activity were evaluated, and the differential expression of sperm-related genes was measured.

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.