Structural Basis of Egg Coat-Sperm Recognition at Fertilization

Abstract

SummaryRecognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1–3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Mammalian egg coat modifications and the block to polyspermy

“…Although high‐resolution structural information is available on both mouse ZP2 ZP‐N1 (Raj et al, 2017) and ZP‐C (Bokhove et al, 2016; Figure 2), it is unknown how cleavage of the protein's ZP‐N2 domain mechanistically induces ZP hardening and blocks sperm‐binding. Together with ZP‐N1 and ZP‐N3 (Figure 1), ZP2 ZP‐N2 is thought to constitute protrusions that project from the ZP filament core (Monné et al, 2008; Wassarman & Mortillo, 1991).…”

“…On the basis of the appearance of multiple bands of ~20–35 kDa that reacted with antibodies raised against its ZP‐N1 domain (Burkart et al, 2012; Greenhouse, Castle, & Dean, 1999), it was suggested that post‐fertilization processing of mouse ZP2 is not limited to the LADE sequence, but also involves additional cleavages at positions 54 DE 55 and 127 DD 128 (Burkart et al, 2012). Considering the disulfide bond pattern of ZP2 ZP‐N1 (Raj et al, 2017), proteolysis at these secondary sites should release a peptide of ~8.7 kDa that would also carry an N ‐glycan attached to N83 (Boja et al, 2003); as a result, ZP2f would be expected to show a ~10‐kDa shift in SDS‐PAGE migration relative to ZP2, even when analyzed in nonreducing conditions. Consistent with the fact that such a shift was not observed experimentally (Bleil et al, 1981), it was recently shown that the heterogeneity of the N‐terminus of ZP2f is caused by variable N ‐glycosylation (Tokuhiro & Dean, 2018).…”

“…Second, the putative sperm‐binding site of ZP2 contains a glycosylation site (N83 in the mouse; Boja et al, 2003), that is conserved in both species. Although the corresponding N ‐glycan is not required for secretion of mouse ZP2 ZP‐N1 (Raj et al, 2017) or important for sperm–oocyte recognition (Tokuhiro & Dean, 2018), species‐specific glycosylation of the sperm‐binding site of ZP‐N1 in the humanized oocytes may indirectly interfere with processing of the adjacent human ZP‐N2 domain by mouse ovastacin. Third, the C‐terminal part of ovastacin contains an additional domain of ~150 amino acids that differ from sequences found in the corresponding region of other astacins (Quesada et al, 2004).…”

“…As mentioned above, native mouse ZP3 is N ‐glycosylated at five positions (Boja et al, 2003; Figure 1) but these glycans are neither required for intracellular trafficking and incorporation into the ZP (Hoodbhoy et al, 2006; Roller & Wassarman, 1983) nor important for gamete interaction (Florman & Wassarman, 1985). On the contrary, as also discussed above, N ‐glycosylation is necessary for secretion of site ZP2 (Roller & Wassarman, 1983), which also carries an N ‐glycan attached to N83 within its putative sperm‐binding site (Boja et al, 2003; Raj et al, 2017); however, this carbohydrate is not essential for gamete recognition (Tokuhiro & Dean, 2018), although its possible involvement in ZP hardening remains to be investigated. In contrast with these findings on mouse fertilization, residues such as mannose, fucose, GlcNAc, and the sialyl‐Lewis X sequence [NeuAcα2–3Galβ1–4(Fucα1–3)GlcNAc] of N ‐ and O ‐glycans have been suggested to play a role in human egg–sperm binding (Miranda et al, 1997; Oehninger, Patankar, Seppala, & Clark, 2009; Pang et al, 2011).…”

From preformation to reformulation—the then and now of sperm selection

“…Slithering through the forest of egg coat materialsknown affectionately to the human ART community as the zona pellucida-is not simply a case of sink or swim! Rather, reports on the molecular structure of egg coat materials, including the mammalian zona pellucida component ZP2, have now revealed the presence of a sophisticated and apparently evolutionarily conserved mechanism for sperm penetration [4]. The proposed gating mechanism involves interaction of sperm lysins with ZP2 effecting creation of pores through which motile sperm can pass.…”