Sperm surface protein disulfide isomerase ERp57 is crucial for mammalian fertilization but functions independently of IZUMO1

BACKGROUND: Human fertilization requires fusion of spermatozoon and oocyte membranes to form a diploid zygote, beginning with adhesion mediated by spermatozoon IZUMO1 and oocyte JUNO. Current models propose that IZUMO1 dimerizes after interacting with JUNO, possibly triggered by a protein disulfide isomerase. It has been proposed that protein disulfide isomerase ERp57 is the trigger for IZUMO1 dimerization, a mechanism supported by parallels in viral entry, but direct evidence is lacking. METHODS: In vitro fertilization studies were performed for both mice and humans using ERp57 inhibitors to confirm the importance of ERp57 in mammalian fertilization. Additionally, for this study, we generated a sperm-specific ERp57 conditional knockout mouse model and performed in vivo and in vitro fertilization experiments. Biophysical assays, including dynamic light scattering and a fluorescence-based dissociation assay, were developed and utilized to investigate interactions between ERp57 and IZUMO1. Structural modeling was used to supplement the ERp57 and IZUMO1 interaction findings. RESULTS: Here, we reveal that ERp57 is crucial for mammalian fertilization but does not show evidence of any direct interaction with IZUMO1. ERp57 inhibition significantly reduces fertilization in human and mouse in vitro assays, and ERp57 spermatozoa conditional knockout (scKO) males exhibit severe hypofertility in vivo and in vitro. ERp57 localizes to the equatorial segment of human spermatozoa following the acrosome reaction, consistent with a role in gamete interaction. However, ERp57-deficient spermatozoa fail to accumulate in the perivitelline space, pointing to a role upstream of membrane fusion. Additionally, ERp57 neither promotes IZUMO1 dimerization nor facilitates dissociation of the IZUMO1-JUNO complex. Structural modeling predicted no significant interaction between ERp57 and IZUMO1, supporting experimental findings. CONCLUSIONS: These findings establish ERp57 as critical for mammalian fertilization but challenge existing assumptions about its mechanistic involvement in gamete membrane fusion. Our research contributions provide key new mechanistic insights that reexamine and reshape the current paradigms surrounding the fundamental process of sperm-egg fusion. By addressing a long-standing bottleneck in the field, our work opens new avenues of investigation that could finally lead to the identification of the elusive human sperm-egg fusogen.