Prenatal cadmium exposure and male infertility in mice: A multi-generational mechanistic study

Male infertility affects approximately one in seven couples worldwide. Prenatal cadmium (Cd) exposure has been shown to affect offspring phenotypes and increase susceptibility to diseases later in life. However, the effects of prenatal Cd exposure on multi-generational offspring fertility and the mechanisms remain unknown. A novel murine multi-generational (F1-F3 offspring) male subfertility model induced by prenatal Cd exposure was developed. The levels of testosterone and steroidogenic enzymes were also lower in these offspring's testes. The ubiquitin-dependent degradation of NR4A1, the upstream transcription factor regulating steroidogenic enzymes, was enhanced across generations upon prenatal Cd exposure. After treatment with MG132, an inhibitor of the ubiquitin-proteasome system, the levels of NR4A1 and steroidogenic enzymes were higher in offspring testes with prenatal Cd exposure. Based on the analysis of the UbiBrowser database and testicular global transcriptome, RAPSN was identified as a novel ubiquitin E3 ligase containing the RING-H2_Rapsyn domain that mediates multi-generational testicular NR4A1 ubiquitination. m⁶A epitranscriptome analysis revealed that prenatal Cd exposure upregulated RAPSN expression in multi-generational offspring testes, and was attributed to a higher level of m⁶A modification of Rapsn mRNA. Furthermore, there was a lower level of YTHDC2, a m⁶A reader, in the multi-generational offspring testes with prenatal Cd exposure. Prenatal and postnatal testicular YTHDC2 overexpression reduced the stability of m⁶A-methylated Rapsn mRNA to downregulate RAPSN expression in F1-F3 testes. Overall, YTHDC2 reduction-mediated increment in m⁶A-methylated Rapsn mRNA contributed to prenatal Cd-enhanced multi-generational susceptibility to male subfertility.