ALKBH5 facilitates Srgn stability via m6A demethylation to disrupt blood-testis barrier integrity in the high-fat diet mice.

Obesity has become an important factor affecting male fertility and is often accompanied by abnormal levels of fatty acids, especially saturated fatty acids (SFAs). Palmitic acid (PA; C16:0) is the most frequent SFA in high-fat diet (HFD) and impairs blood-testis barrier (BTB) integrity and decreases sperm quality. RNA N6-methyladenosine (m6A) is involved in lipid metabolism and affects BTB integrity and spermatogenesis. However, it is unclear whether RNA m6A modification plays a role in the BTB damage caused by PA. Herein, we unraveled that PA induced the upregulation of ALKBH5 in TM4 and primary Sertoli cells and this upregulation of ALKBH5 was involved in PA-induced BTB disruption. Conversely, suppressing Alkbh5 expression could alleviate the BTB disruption induced by PA. Moreover, we found Srgn might be a potential downstream target of Alkbh5-mediated BTB integrity suppression. Mechanistically, the Alkbh5 upregulation caused by PA disrupted BTB integrity via enhancing Srgn stability in a RNA m6A dependent manner, and Srgn impaired the integrity of the BTB by activating the NF-κB and MAPK pathways and thereby elevating the expression of Ccl2 and TNF-α in TM4 and primary Sertoli cells. Our study revealed an important role of the regulatory network of ALKBH5-m6A-Srgn in the PA-induced BTB disruption and provided potential therapeutic strategies for improving fertility potential in obese patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-026-06189-y.