scRNA-seq deciphers molecular mechanisms of endocrine disruptor 4-nonylphenol impairing spermatogenesis in mice.

4-Nonylphenol (NP) is an environmental endocrine disruptor widely used in consumer products. Previous studies have shown that NP can interfere with hormone synthesis and metabolism in humans and animals, leading to male reproductive dysfunction. This study utilized the scRNA-seq method to evaluate cell populations and their heterogeneity, aiming to elucidate the toxic mechanisms of NP exposure on testicular cells. We demonstrate, for the first time, the transcriptomic characteristics of testicular single cells in adolescent mice exposed to NP. Adolescent mice, initially exposed at 4 weeks of age, were subsequently analyzed at sexual maturity after a continuous exposure period of 3 months. The blank control and NP-exposed groups underwent scRNA-seq analysis, identifying ten cell populations. The results showed that after NP exposure, the number of germline cells was remarkably reduced compared to the control group. NP exposure significantly decreased the protein expression of the four common differentially expressed genes (DEGs) (Cmtm2b, Rpl28, Adam32, and Pgam2). The DEGs enriched in the GO functions of the four germline cell types were spermatogenesis and spermatid development. KEGG analysis showed that the DEGs were enriched in the oxidative phosphorylation, and ROS signaling pathways. Further analysis of intercellular interactions revealed that NP exposure altered intercellular communication between germ cells, with the NECTIN3-NECTIN2 receptor-ligand interactions activating between spermatogonia, Sertoli, and Leydig cells. Germ cells bind to Sertoli and Leydig cells via NECTIN3-NECTIN2 receptor ligands. Somatic cells bind to RS and ES through GRN-SORT1 receptor ligands. CADM1-CADM1 receptor-ligand interactions enhances between germ and Sertoli cells. Our study provides new insights into the potential impacts of NP on spermatogenesis and sperm function, emphasizing the importance of environmental hormones in male fertility issues.