# ATR Deficiency Impairs DNA Damage Repair and Accelerates Cellular Senescence in Bovine Mammary Epithelial Cells, Leading to Lactation Dysfunction

## TL;DR

This study shows that reduced ATR protein in dairy cows impairs DNA repair and causes mammary gland aging, leading to lower milk production.

## Contribution

The novel finding is that ATR deficiency directly causes lactation dysfunction through DNA repair impairment and cellular senescence in bovine mammary cells.

## Key findings

- ATR silencing increases senescence markers and disrupts lactation signaling pathways in bovine mammary epithelial cells.
- Low ATR levels correlate with reduced milk yield and elevated DNA repair protein expression in low-yield dairy cows.
- ATR deficiency leads to cell cycle arrest, apoptosis, and increased SASP factors in mammary epithelial cells.

## Abstract

As parity increases, the health of dairy cows’ mammary glands declines, leading to reduced milk yield and a shorter productive lifespan. Variation in mammary gland health is the primary cause of differences in milk production. Therefore, studying the differences in mammary gland health among dairy cows is crucial for promoting healthy livestock farming. Our study demonstrates that with increasing parity, differences in mammary gland health manifest in signaling pathways related to immune and inflammatory responses, as well as DNA damage repair. Alterations in ATR and PIG3, which affect DNA damage repair in mammary gland cells, contribute to the polarized milk yield. A reduction in ATR results in cell cycle arrest and apoptosis in bovine mammary epithelial cells (BMECs), an increase in senescence-associated secretory phenotype (SASP) content, decreased mTOR expression, elevated STAT3 protein levels, and significantly reduced mRNA levels of CSN2 and CSN3.

Mammary glands in cows are highly dynamic, making genomic stability particularly crucial. Continuous lactation and self-renewal of these glands are primary contributors to genomic instability. Results: We employed transcriptomic and proteomic methods to analyze the expressional characteristics in the mammary glands of cows with varying levels of milk production. Our findings indicated differences in relevant pathways, including DNA damage repair and apoptosis, which are influenced by increasing parity. Notably, ATR protein levels in the mammary glands of low-yield dairy cows were reduced. Following in vitro silencing of ATR, β-galactosidase content increased in aging mammary epithelial cells, with cell cycle arrest in the G2 and S phases. Secretory phenotypes associated with aging, including IL-6, IL-10, IL-1β, INF-γ, and IL-2, were elevated, along with increased TNF-α content. The expressions of DNA repair-related proteins, including PIG3, PARP1, and Cleaved caspase3, were upregulated, and SP1 expression was decreased. Furthermore, the expressions of cytochrome C and BAK increased, and ATR silencing inhibited mTOR and STAT5 lactation signaling pathways, resulting in elevated STAT3 protein levels associated with mammary gland degeneration. Conclusions: This study emphasizes the significance of the ATR protein in the mammary glands of dairy cows, contributing valuable insights into maintaining their health and presenting a novel perspective on strategies to enhance their lifespan.