GATA4 modulates autophagy, apoptosis and tight junction marker remodeling in Bactrian camel Sertoli cells

Bactrian camels exhibit distinct seasonal estrus, with periodic testicular functional fluctuations regulating their reproductive activity. As a key transcription factor in reproductive modulation, the specific role of GATA4 in the testicular microenvironment of Bactrian camels remains unclear. This study investigates the expression patterns of GATA4 in the testicular tissue during estrus and anestrus phases, alongside its potential regulatory effects on Sertoli cells (SCs) function. Utilizing mRNA sequencing, we analyzed testicular samples in estrus (n = 3) and anestrus (n = 3), identifying 291 differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that GATA4 was involved in reproduction-related processes, including tight junction formation, autophagy, and cell cycle regulation. Notably, validation showed that GATA4 expression was significantly upregulated in testicular tissue during the anestrus period, with its protein localized primarily in SCs. Gain- and loss-of-function assays revealed that altered GATA4 expression is associated with changes in autophagy-related marker profiles, evidenced by a lower LC3II/LC3I ratio, decreased Beclin-1, and increased P62 expression level. Silencing GATA4 induced opposite alterations in these autophagy-associated molecular markers, and these molecular changes are associated with modified mTOR/NF-κB pathway expression. Additionally, GATA4 influences SCs apoptosis and G0/G1 cell cycle arrest and these cellular phenotypic changes are accompanied by alterations in the PI3K/AKT pathway, which correlates with changed levels of BAX, Caspase-3, BCL2, and CDK1. And moreover, GATA4 modulates the expression of the tight junction markers ZO-1 and Occludin, and these remodeling events are associated with modified WNT1/β-catenin signaling. In conclusion, GATA4 exhibits dynamic expression patterns throughout the reproductive cycle, which correlates with the modulation of multiple biological processes in SCs, including autophagy-related marker remodeling, apoptosis regulation, and tight junction marker remodeling. This study's findings provide a key molecular target for elucidating the seasonal reproductive mechanism and reproductive regulation of Bactrian camels and enrich the current understanding of reproductive regulatory mechanisms in this species.