Novel insights into human CRISP2: localization in reproductive tissues and sperm, and molecular characterization†.

CRISP2 is enriched in the male reproductive system of mammals and plays roles in spermatogenesis, sperm motility, and fertilization. Although extensively investigated in rodents and boars, human CRISP2 (hCRISP2) remains poorly studied, particularly concerning its localization in testicular and epididymal tissues and its molecular features. In this study, we used immunofluorescence to determine the localization of hCRISP2 in testis, epididymis, and ejaculated sperm. While no expression was observed in the epididymal epithelium, hCRISP2 was detected at different stages during spermatogenesis. Specifically, hCRISP2 was found in the nucleus of primary spermatocytes and of both round and early elongated spermatids. In elongated spermatids, it was additionally observed in the cytoplasm, the flagellum, and the equatorial segment of the acrosome (EqS). The presence of aggregated material with hCRISP2 immunoreactivity in the apical pole of Sertoli cells suggests that most of the hCRISP2 involved in spermatogenesis is phagocytized by these cells during spermiation. In ejaculated sperm, hCRISP2 was found in the cytoplasmic droplet, flagellum, and EqS, consistent with its described roles in sperm motility and gamete fusion. Native and denaturing electrophoresis combined with western blot analyses depicted the ability of hCRISP2 to form stable high molecular weight complexes, and mass spectrometry revealed that these complexes likely consist exclusively of hCRISP2. Furthermore, we showed that hCRISP2 undergoes only limited post-translational modifications. These findings shed light into the dynamic localization of hCRISP2 throughout spermatogenesis and in ejaculated sperm, as well as its molecular features, enhancing our understanding of its functional roles and relevance for male fertility.