AKAP12 maintains blood-testis barrier integrity and resists BPAF-induced male fertility impairment by inhibiting Src-mediated ZO-1 degradation

Most existing studies have focused on the direct reproductive toxicity of Bisphenol AF (BPAF), whereas the endogenous protective mechanisms within testicular tissues that defend against BPAF remain unclear. Blood-testis barrier (BTB), established by Sertoli cells, isolates the seminiferous tubule to provide an immune-privileged microenvironment that restricts toxins and supports spermatogenesis. As a scaffold protein orchestrating multiple signaling kinases, A-kinase anchor protein 12 (AKAP12) plays a critical role in various biological barriers. However, its functional relevance to the BTB has yet to be documented. Sertoli cell-specific Akap12 -deficient male mice were generated and exposed to BPAF via oral gavage for 28 days. Male reproductive function and sperm quality were evaluated by assessing fertility, sperm motility. BTB integrity was assessed using a biotin tracer assay and transmission electron microscopy (TEM). The effects of Src inhibition on ZO-1 phosphorylation and degradation were further evaluated in Akap12 -deficient Sertoli cells. We show that Sertoli cell-specific Akap12- deficient male mice exhibit markedly reduced fertility following BPAF exposure. Mechanistically, AKAP12 maintains BTB integrity by sequestering Src kinase. Lack of AKAP12 enhances Src (Tyr416) phosphorylation, which results in increased tyrosine phosphorylation of zonula occludens-1 (ZO-1), and ubiquitin-dependent degradation and subsequent destabilization of tight junction proteins, including Occludin and Claudin-11. Conversely, Src inhibitor administration effectively reverses AKAP12 deficiency-triggered ZO-1 phosphorylation and degradation in Sertoli cells. AKAP12 deficiency also disrupts extracellular matrix-integrin homeostasis. This study demonstrates that AKAP12 protects against BPAF-induced male fertility impairment by sequestering Src kinase, preserving tight junction integrity, and maintaining the BTB. Our findings extend AKAP12 research into reproductive toxicology and offer new insights into gene-environment interactions.