Towards a Molecular Understanding of the Sperm Protein CRISP2 in Humans - A Study of Its Localization, Interaction Partners, and Oocyte Binding

Human fertility relies on complex interactions between gametes, yet the molecular mechanisms behind these events remain largely unclear. Among the proteins involved, the Cysteine-Rich Secretory Protein family (CRISPs) has emerged as crucial in reproductive biology due to their predominant expression in reproductive tissues. Within this family, CRISP2 is unique. Indeed, while other CRISPs associate with sperm during epididymal transit, CRISP2 originates within the testes and remains throughout sperm maturation.
This thesis aims to characterize human CRISP2 (hCRISP2) using complementary approaches. Initially, the precise localization of hCRISP2 during spermatogenesis and in mature sperm was investigated. Immunofluorescence and biochemical analyses demonstrated that hCRISP2 forms stable complexes and clarified its localization across different developmental stages and sperm compartments.
One of the major objectives of this thesis was to determine if hCRISP2 interacts specifically with the oocyte surface. Binding sites for CRISP1 have been identified on rodent and human oocytes, whereas CRISP2 binding has only been confirmed in rodents. In this study, peptides based on a conserved twelve-amino acid motif derived from hCRISP1 and hCRISP2 were tested and showed oocyte binding. However, control experiments suggested potential non-specific interactions. To address this, recombinant full-length hCRISP2 proteins were produced in bacterial and mammalian systems. Unfortunately, these proteins could not be adequately purified for binding assays. A pentameric hCRISP2-based probe was also developed and tested. Although successfully validated using the sperm protein IZUMO1, this probe did not demonstrate specific binding for hCRISP2.
Finally, the thesis aimed to identify potential interaction partners of hCRISP2. A comprehensive list of candidate proteins, termed the human oocyte surfaceome, was established. A plasmid library encoding these candidates was generated and employed in cell-based binding assays and computational modeling with AlphaFold Multimer. Additionally, yeast two-hybrid screening identified intracellular partners. These approaches revealed numerous candidate proteins, highlighting the complexity of hCRISP2’s role in sperm biology and fertilization.
Collectively, this work advances our molecular understanding of hCRISP2 and introduces robust experimental methods valuable for studying other sperm proteins and exploring crucial aspects of fertilization, such as the fertilization synapse. A deeper comprehension of the molecular mechanisms governing gamete physiology and fertilization will be essential for developing more accurate diagnostic tools, innovative therapeutic approaches, and novel contraceptive strategies.