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

## TL;DR

This study explores how the INSL3 gene affects the growth of granulosa cells in cattle follicles, revealing its role in regulating cell proliferation and follicular development.

## Contribution

The study identifies INSL3 as a key regulator of granulosa cell proliferation and follicular development in Dabie Mountain cattle through transcriptomic and functional analyses.

## Key findings

- INSL3 expression is highest in small follicles and decreases in larger follicles.
- Knockdown of INSL3 increases granulosa cell viability and proliferation.
- INSL3 knockdown alters cell cycle distribution and upregulates proliferation-related genes like Myc and PCNA.

## Abstract

Dabie Mountain cattle 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 Dabie Mountain cattle 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 DEFB, FAM124A, and RASSF10. Additionally, 227 commonly down-regulated DEGs were identified, including genes such as INSL3, GAS7, and PAQR7. Protein interaction network analysis revealed an interaction between INSL3 and STAR. Bovine ovarian granulosa cells (GCs) were collected to investigate the effect of the INSL3 on GCs proliferation. The results revealed that INSL3 expression was highest in small follicles and was almost absent in big follicles. Subsequently, the INLS3 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 INSL3 expression (p < 0.01), si-INSL3 (239) for follow-up research. CCK-8 was used to assess cell proliferation, revealing that INSL3 knockdown significantly enhanced GCs viability and number at 24, 48, and 72 h (p < 0.05). Flow cytometry was used to detect cell cycle distribution. The results showed that knockdown of INSL3 expression significantly decreased the proportion of G1 phase cells and significantly increased the number of S phase cells (p < 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 Myc, PCNA, Cytochrome C, and Cyclin D1 were significantly increased in the si-INSL3 group. In conclusion, knockdown of INSL3 affects follicular development in Dabie Mountain cattle by regulating granulosa cell proliferation in the ovaries, providing new insights into the regulatory mechanisms of follicular development in cattle.