An update on the characterization of human sperm CRISP2: analysis of its localization, post-translational modifications, and oligomerization

The Cystein-RIch Secretory Protein (CRISP) family gathers proteins recognized as key players in male fertility. Although most of the knowledge on CRISPs comes from rodent models, some information is available regarding the three human CRISPs. While CRISP1 and 3 are secreted by the epididymal epithelium and associated with the membrane of the spermatozoa, CRISP2 is expressed within the spermatozoa, appearing at the round spermatid stage during spermatogenesis. CRISP2 has been localized within the acrosome, the neck and, in some studies, the flagellum. Finally, unlike 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 CRISP2 in human spermatozoa. First, using immunofluorescence confocal imaging, we compared the localization of CRISP2 to the one of acrosomal and perinuclear theca markers. Indeed, recently, CRISP2 was shown to be restricted to the flagellum and the perinuclear theca in boars. Then, we analyzed CRISP2 quaternary organization by native-PAGE and showed that it forms highly stabilized aggregates of high molecular weight under native conditions. Finally, we investigated the presence of phosphorylation (so far undescribed) by Western blot and re-assessed the presence of glycosylation through lectin blot targeting O- and N-glycosylation.
Together, these results help to better describe CRISP2 from a biochemical point of view, and therefore, improve our understanding of its function in the fertilization process.