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

Q: Why study INSL3 in Dabie Mountain cattle follicular development?

Reproductive efficiency in Dabie Mountain cattle is relatively low, and ovarian follicle developmental stage is a key factor. The study investigates how INSL3 regulates granulosa-cell growth and development to improve understanding of follicular mechanisms.

Q: How were candidate genes identified in the study?

Granulosa cells were collected from small ( 8 mm) follicles, followed by RNA extraction and transcriptomic sequencing. Differentially expressed genes were then screened across comparison groups.

Sep 30, 2026

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Dabie Mountain cattle exhibit strong feed tolerance, disease resistance, and meat quality, yet lower reproductive efficiency limits their development. This study characterized follicle developmental patterns across small (<5 mm), medium (5–8 mm), and big (>8 mm) stages and screened candidate genes using granulosa-cell transcriptomic sequencing. Differential expression identified key up- and down-regulated genes, highlighting INSL3 and its interaction with STAR. Functional assays showed INSL3 knockdown in granulosa cells enhanced proliferation, increased S-phase entry, and elevated proliferation gene expression, supporting a regulatory role in ovarian follicular development.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages15

Reading Time38 min

AccessFree to download

1. Introduction

Follicular development background in Dabie Mountain cattle

Ovarian follicle structure and follicular waves

Article New Insights into the Mechanism of Insulin-like Peptide 3 (INSL3) Regulating the Growth and Development of Bovine Follicular Granulosa Cells Hongxian Li , Fenglou He, Xinye Li, Junjie Nie, Hasnain Ali Khan, Chao Chen * and Jinling Hua * Academic Editor: Jan Tesarik Received: 1 December 2025 Revised: 26 December 2025 Accepted: 29 December 2025 Published: 30 December 2025 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license. College of Animal Science, Anhui Science and Technology University, Fengyang 233100, China; [lihongxian20221@outlook.com](lihongxian20221@outlook.com) (H.L.) * Correspondence: [chenchao@ahstu.edu.cn](chenchao@ahstu.edu.cn) (C.C.); [huajl@ahstu.edu.cn](huajl@ahstu.edu.cn) (J.H.); Tel.: +86-13297979859 (C.C.);+86-15155057079 (J.H.) 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. Keywords: Dabie Mountain cattle; INSL3; follicular development; granulosa cells; proliferation 1. Introduction Dabie Mountain cattle is one of the unique local yellow cattle breeds in China, belonging to the dual-purpose and meat use type of yellow cattle breeds. Under suitable feeding conditions, high-quality snowflake beef can be produced, which has the characteristics of tolerance to extensive feeding, strong disease resistance, and delicious meat [1] . But its reproductive rate is relatively low, with cows starting to heat at an average age of 1.5 and agestation period of about 274 days

FAQs

Why study INSL3 in Dabie Mountain cattle follicular development?

Reproductive efficiency in Dabie Mountain cattle is relatively low, and ovarian follicle developmental stage is a key factor. The study investigates how INSL3 regulates granulosa-cell growth and development to improve understanding of follicular mechanisms.

How were candidate genes identified in the study?

Granulosa cells were collected from small (<5 mm), medium (5–8 mm), and big (>8 mm) follicles, followed by RNA extraction and transcriptomic sequencing. Differentially expressed genes were then screened across comparison groups.

What effect does INSL3 knockdown have on granulosa cells?

INSL3 expression was highest in small follicles and nearly absent in big follicles. After si-INSL3 knockdown, CCK-8 assays showed increased cell viability and number, flow cytometry indicated reduced G1-phase proportion and increased S-phase cells, and proliferation-related genes (e.g., Myc, PCNA, Cyclin D1) were upregulated.

Sep 30, 2026

Uploaded by Aran