Disrupted inhibin B synthesis due to Sox9 hyper-palmitoylation in Sertoli cells impairs spermatogenesis via paracrine effects.

BACKGROUND: Male subfertility is a global health concern, with spermatogenic dysfunction being a critical cause. Abnormally high level of palmitic acid (PA), a main component of dietary saturated fatty acid, has been reported to be implicated in the spermatogenic dysfunction, accompanied with a decrease of inhibin B (INHB). However, the mechanism underlying PA-induced downregulation of INHB, and the specific function of INHB in the spermatogenesis microenvironment, remain unclear. Since PA is the main substrate of palmitoylation, a common post-translational lipid modification, we investigated the role of palmitoylation in INHB synthetic defects and subsequent dyszoospermia induced by PA in this study. METHODS: Mice were treated with PA for 30 days to establish a high PA model, and a palmitoylation inhibitor 2-bromopalmitate (2BP) was used for spermatogenesis rescuing. Concentrations and motilities of sperms in the cauda epididymides were analyzed, and pathological examinations were performed to assess spermatogenic function. Hormone levels were detected using ELISA. Primary mouse Sertoli cells and TM4 Sertoli cell line were used for in vitro exploration of mechanisms. Acyl biotin exchange assay was used to explore protein palmitoylation. Co-culture of TM4 and GC1 cells was used to explore the effects of Sertoli cell-secreted INHB on spermatogonia in a paracrine manner. RESULTS: In this study, we found that excessive PA downregulates testicular INHB levels by suppressing expression of its βB subunit (InhβB) in Sertoli cells, with hyper-palmitoylation of the transcription factor SRY-box containing gene 9 (Sox9) serving as a key regulatory node in this process. We further identified the palmitoyl transferase ZDHHC16 as the primary enzyme responsible for PA-induced Sox9 hyper-palmitoylation. Furthermore, INHB was shown to promote spermatogonial proliferation and differentiation in a paracrine manner within the spermatogenic microenvironment, thereby mediating the modulation of spermatogenesis by palmitoylation in Sertoli cells. CONCLUSION: Overall, this study demonstrated that INHB synthesis can be suppressed by PA-induced hyper-palmitoylation of Sox9, and decreased secretion of INHB by Sertoli cells directly leads to spermatogenic dysfunction in the testis microenvironment. These findings highlight Sox9 palmitoylation as a candidate target for treatment of dyszoospermia accompanied with dyslipidemia, and underscore the critical role of INHB in regulating spermatogenesis within the testicular microenvironment.