Abstract:

The aim of this study was to investigate the effect of heat stress on m⁶A methylation modification in Hu sheep ovarian granulosa cells, so as to lay the foundation for revealing the regulatory mechanism of m⁶A methylation modification on the development and function of Hu sheep ovarian granulosa cells under heat stress. In this study, fresh ovaries of 2-year-old Hu ewes were collected, and granulosa cells in 3-5 mm follicles on the surface of ovaries were collected. They were randomly divided into two groups: control group (37 ℃) and heat stress group (42 ℃, 2 h per day for 3 consecutive days). The m⁶A peak was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the gene expression data were obtained and analyzed. There were 135 significantly different m⁶A peaks in MeRIP-seq between the control group and the heat stress group, which were mapped to 130 differentially methylated genes, mainly enriched in the PI3K-Akt signaling pathway, glutathione metabolism and Wnt signaling pathway. RNA-seq identified 359 significantly differentially expressed genes, which were mainly enriched in steroid synthesis, cholesterol biosynthesis and other signaling pathways. The combined analysis of MeRIP-seq and RNA-seq data showed that there were significant differences in m⁶A modification and gene expression levels of five genes, DTX3L, MAGEF1, MAN1C1, CCDC77 and RAD51, which were mainly enriched in the Notch signaling pathway. This study results shows that heat stress can change the mRNA m⁶A modification level of Hu sheep ovarian granulosa cells, which may eventually affect cell proliferation, apoptosis, estrogen synthesis and other related pathways, providing a theoretical basis for further exploring the specific mechanism of m⁶A modification under heat stress.

Key words: heat stress, ovarian granulosa cells, Hu sheep, m⁶A methylation modification, MeRIP-seq