ALKBH5 in mouse testicular Sertoli cells regulates Cdh2 mRNA translation to maintain blood-testis barrier integrity

Background: RNA N⁶-methyladenosine (m⁶A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m⁶A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood-testis barrier (BTB) through cell junctions between adjacent Sertoli cells.

Methods: ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m⁶A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m⁶A immunoprecipitation-quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation-qPCR, western blot, coimmunoprecipitation, and polysome fractionation-qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure.

Results: ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m⁶A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice.

Conclusions: Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.