PRSS38 Is a Novel Sperm Serine Protease Involved in Human and Mouse Fertilization

Abstract

Sperm proteases are involved in several gamete interaction events leading to fertilization.This report presents a detailed analysis on the expression and localization of serine proteasePRSS38 in human and in mouse spermatozoa and its involvement in fertilization-related events, using bioinformatics, cellular, biochemical, molecular and functionalapproaches. Bioinformatics analyses included genomics and data analysis, prediction ofprotein subcellular localization and post-translational modifications, Self-OrganizingMaps (SOMs) unsupervised training with other serine proteases, protein modeling (AlphaFold) and genetic variant analysis. For cellular, biochemical and functional studies,human semen samples were obtained from healthy normozoospermic volunteers, andcauda epididymal sperm were collected from adult Balb-c/C57 mice. PRSS38 presencewas detected in human and mouse sperm protein extracts by Western immunobloting.Sperm PRSS38 subcellular localization was determined by fluorescence immunocytochemistry.Human sperm-oocyte interaction events were assessed by means of themouse Cumulus Penetration Assay (CPA) using mouse COCs, the human HemizonaAssay (HZA), and the ZP-free hamster egg Sperm Penetration Assay (SPA). Mousesperm-oocyte interactions were evaluated by means of in vitro fertilization (IVF) withCOCs and denuded oocytes. PRSS38 is proposed to be a GPI-anchored serine protease(active site: His-100, Asp-150 and Ser-245) based on bioinformatics analyses. Usingcommercial antibodies, protein forms of the expected Mr (human: 31 kDa; mouse: 32 and24 kDa) were specifically immunodetected in protein sperm extracts. Immunocytochemicalanalysis revealed a specific PRSS38 signal in the human sperm acrosomal region,equatorial segment, and flagellum. Mouse sperm PRSS38 was immunolocalized inthe equatorial segment and hook. Human sperm preincubation with specific antibodiesresulted in inhibition (P<0.05) of CPA, HZA, and SPA. Mouse sperm preincubation withPRSS38 antibodies impaired (P<0.05) homologous IVF using COCs and denuded oocytes.Genetic variants affecting residues involved in the GPI anchor and the catalytic triadwere found in individuals from the general population whose PRSS38 protease functioncould be altered. This study provides, for the first time, an integrated analysis of PRSS38in human and mouse sperm, contributing our understanding of mammalian fertilization and male infertility.