[en] The Cystein-RIch Secretory Protein (CRISP) family gathers proteins recognized as key players in male fertility. Although most of the knowledge on the CRISPs comes from rodent models, three CRISPs have been identified in humans. While CRISP1 and 3 are secreted by the epididymal epithelium and associate on the surface of the spermatozoa, CRISP2 is expressed inside the spermatozoa, appearing at the round spermatid stage during spermatogenesis. However, its exact localization inside the mature spermatozoon is debated. Contrary to CRISP1 and 3, CRISP2 is thought to be non-glycosylated, but the presence of other post-translational modifications has not yet been investigated.
In the present study, we contributed to the characterization of native CRISP2 in human spermatozoa. First, immunofluorescence imaging preceded by epitope retrieval allowed us to evaluate the localization of CRISP2. Then, CRISP2 was extracted from spermatozoa with various extraction buffer and analyzed by SDS-page and native-page followed by Western blot analyses to assess the presence of disulfide bonds and of CRISP2 oligomers. Finally, native CRISP2 was immunoprecipitated from sperm after capacitation (a maturation of the spermatozoa required for oocyte fertilization) to study the presence of potential post-translational modifications (PTM), in our case, phosphorylation and glycosylation.
