New Insights into the Mechanism of Insulin-like Peptide 3 (INSL3) Regulating the Growth and Development of Bovine Follicular Granulosa Cells

<i>Dabie Mountain cattle</i> are characterized by their ability to tolerate coarse feed, strong disease resistance, and delicious meat. Lower reproductive efficiency has become one of the key factors limiting its development. Therefore, this study investigated the developmental patterns of <i>Dabie Mountain cattle</i> follicles and screened key candidate genes for in vitro experimental validation. Research collected granulosa cells from small follicles (<5 mm), medium (5-8 mm), and big (>8 mm), followed by RNA extraction for transcriptomic sequencing. A total of 20,775 genes were identified, including 13,777 (66.3%) differentially expressed genes (DEGs). DEGs showing up-regulation and down-regulated in B vs. S, B vs. M, and M vs. S groups were collected. A total of 19 commonly up-regulated DEGs across the three groups were identified, including genes such as <i>DEFB</i>, <i>FAM124A</i>, and <i>RASSF10</i>. Additionally, 227 commonly down-regulated DEGs were identified, including genes such as <i>INSL3</i>, <i>GAS7</i>, and <i>PAQR7</i>. Protein interaction network analysis revealed an interaction between <i>INSL3</i> and <i>STAR</i>. <i>Bovine</i> ovarian granulosa cells (GCs) were collected to investigate the effect of the <i>INSL3</i> on GCs proliferation. The results revealed that <i>INSL3</i> expression was highest in small follicles and was almost absent in big follicles. Subsequently, the <i>INLS3</i> gene was knocked down in GCs using small interfering RNA. RT-qPCR results demonstrated that both si-INSL3 (239) and si-INSL3 (392) significantly knock down <i>INSL3</i> expression (<i>p</i> < 0.01), si-INSL3 (239) for follow-up research. CCK-8 was used to assess cell proliferation, revealing that <i>INSL3</i> knockdown significantly enhanced GCs viability and number at 24, 48, and 72 h (<i>p</i> < 0.05). Flow cytometry was used to detect cell cycle distribution. The results showed that knockdown of <i>INSL3</i> expression significantly decreased the proportion of G1 phase cells and significantly increased the number of S phase cells (<i>p</i> < 0.01). RT-qPCR was used to detect the expression of cell proliferation-related genes. The results showed that compared with the siNC group, the expression levels of <i>Myc</i>, <i>PCNA</i>, <i>Cytochrome C</i>, and <i>Cyclin D1</i> were significantly increased in the si-INSL3 group. In conclusion, knockdown of <i>INSL3</i> affects follicular development in <i>Dabie Mountain cattle</i> by regulating granulosa cell proliferation in the ovaries, providing new insights into the regulatory mechanisms of follicular development in cattle.