Gulimire·ABUDUREYIMU1, SHI Xiao-Xiao1, CHEN Ying1, TANG Shu-Hong3, WU Yang-Sheng1, HUANG Jun-Cheng2, WANG Li-Qin1,*, LIN Jia-Peng1,*
1 Xinjiang Key Laboratory of Reproductive Regulation and Breeding for Ruminant Livestock/Key Laboratory of Animal Biotechnology, Urumqi 830011, China; 2 Xinjiang Uygur Autonomous Region Sheep and Goat Engineering Research Center, Urumqi 830011, China; 3 Aksu Regional Zootechnical Extension Centre, Aksu 843000, China
Abstract：Follicular atresia is a key physiological process affecting ovarian reserve and fertility. This study aimed to delineate the molecular mechanisms underlying atretic granulosa cells (AGC) in sheep (Ovis aries) and to evaluate the role of protein lactylation regulation in this process. Based on Gene Expression Omnibus (GEO), differentially expressed genes (DEGs) between AGC and healthy granulosa cells (HGC) were identified and subjected to KEGG pathway enrichment. Weighted gene co-expression network analysis (WGCNA) was performed to construct gene modules associated with the atresia phenotype. Candidate regulators were further mined by integrating protein-protein interaction (PPI) networks with a lactylation-related gene set. Ovine HGC and AGC were collected to measure lactate dehydrogenase (LDH) levels, and key genes were validated by RT-qPCR and Western blot. In vitro, granulosa cells (GCs) were treated with Na-lactate and transduced to overexpress LGALS1, yielding 4 groups: GC, GC+Na-lactate, GC+Na-lactate+OE-NC, and GC+Na-lactate+OE-LGALS1. Cell proliferation (CCK-8), apoptosis (TUNEL), migration/invasion (Transwell), and LDH release (ELISA) were assessed. Adhesion/EMT (epithelial-mesenchymal transition) markers (αVβ3, E-cadherin, N-cadherin, ICAM-1, VCAM-1) were examined by RT-qPCR/Western blot, and pathway proteins (FAK, p-FAK, Src, p-Src, MMP-2, MMP-9) were examined by Western blot. Results showed that a total of 726 DEGs were identified and were enriched mainly in cell adhesion molecule pathways. WGCNA revealed a module closely associated with atretic GCs, from which 22 hub genes were obtained via PPI analysis. Intersecting these with 12 lactylation-related DEGs pinpointed LGALS1 as a key candidate gene, which was highly expressed in HGC and functionally linked to cell adhesion, immune regulation, and apoptosis. In primary samples, the LDH level in the AGC group was extremely significantly higher than that in the HGC group (P<0.001), whereas the expression level of LGALS1 was extremely significantly decreased (P<0.001). In vitro experiments showed that Na-lactate treatment decreased proliferation, increased apoptosis, and weakened migration/invasion, accompanied by downregulation of E-cadherin, upregulation of αVβ3/N-cadherin/ICAM-1/VCAM-1, and activation of the FAK/Src-MMP axis. Under the same conditions, LGALS1 overexpression enhanced proliferation, reduced apoptosis, impaired migration and invasion capacity, upregulated E-cadherin, downregulated αVβ3 and related markers, and suppressed p-FAK, p-Src, MMP-2, and MMP-9. Experimental results indicated that LGALS1 might serve as a lactylation-associated key regulator in the granulosa-cell atresia process. This study provides a potential molecular target and theoretical basis for elucidating adhesion-mediated mechanisms of follicular atresia and for developing fertility-preserving interventions.
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