# Study on the Function of ID2 Gene in Granulosa Cells of Ovaries of Hetian Sheep and Its Correlation Analysis with Lambing Traits

## TL;DR

This study explores how the ID2 gene affects fertility in Hetian sheep and finds that it influences reproductive traits like litter size.

## Contribution

The study identifies ID2 gene polymorphisms and their functional impact on granulosa cells in Hetian sheep, offering a new candidate for improving sheep fertility.

## Key findings

- Four SNPs in the ID2 gene were significantly associated with increased litter size in Hetian sheep.
- ID2 overexpression promotes granulosa cell proliferation and alters reproductive hormone levels.
- ID2 affects key genes in the TGF-β/BMP-SMAD signaling pathway, suggesting a role in ovarian function.

## Abstract

Sheep are an important source of meat, wool, and income for farmers, but some breeds, such as the Hetian sheep in Xinjiang, produce only a small number of lambs each year. Improving their ability to have more lambs would benefit farmers and local communities. In this study, we focused on a gene called ID2, which helps control how cells grow and develop. We found that this gene is especially active in the ovaries of Hetian sheep, where it likely influences fertility. By studying the DNA of 157 sheep, we discovered several genetic differences in ID2 that were linked to larger litter sizes. We also tested how the gene works in ovarian cells and found that it makes these cells grow faster and changes the levels of important reproductive hormones. These findings show that ID2 plays an important role in sheep reproduction. This information can help scientists and breeders develop better strategies to select sheep with stronger fertility traits, leading to improved flock productivity and better support for the sheep industry.

Reproductive efficiency is a key determinant of sheep productivity, yet Hetian sheep remain limited by relatively low fecundity despite their adaptability to harsh environments. The inhibitor of DNA binding 2 (ID2) gene is known to regulate cell proliferation and differentiation, but its specific role in sheep reproduction is not well understood. This study aimed to characterize the ID2 gene in Hetian sheep and to assess its functional association with ovarian granulosa cells and litter size. The coding sequence of ovine ID2 was cloned and analyzed using bioinformatics tools. Tissue-specific expression patterns were measured by quantitative PCR at different pubertal stages. A total of 157 ewes were genotyped to identify single-nucleotide polymorphisms (SNPs) and their association with litter size. Functional studies were performed by lentiviral overexpression of ID2 in granulosa cells, with effects evaluated using CCK-8 proliferation assays, ELISA for hormone secretion, and RT-qPCR for related gene expression. ID2 was highly expressed in the ovary, particularly during puberty. Four SNPs (g.18202368 A>T, g.18202372 G>A, g.18202431 G>C, g.18202472 G>C) were significantly associated with increased litter size. Overexpression of ID2 promoted granulosa cell proliferation, increased progesterone, decreased estradiol, and altered expression of key genes in the TGF-β/BMP-SMAD signaling pathway. The ID2 gene plays a crucial role in ovarian function and reproductive regulation in Hetian sheep. Its polymorphisms and functional impact on granulosa cells suggest that ID2 is a promising candidate gene for marker-assisted selection to improve reproductive efficiency in sheep.