The epididymis contributes to sperm DNA integrity and early embryo development through Cysteine-Rich Secretory...

Numerous reports showed that the epididymis plays key roles in the acquisition of sperm fertilizing ability but its contribution to embryo development remains less understood. Female mice mated with males with simultaneous mutations in Crisp1 and Crisp3 genes exhibited normal in vivo fertilization but impaired embryo development. In this work, we found that this phenotype was not due to delayed fertilization, and it was observed in eggs fertilized by epididymal sperm either in vivo or in vitro. Of note, eggs fertilized in vitro by mutant sperm displayed impaired meiotic resumption unrelated to Ca²⁺ oscillations defects during egg activation, supporting potential sperm DNA defects. Interestingly, cauda but not caput epididymal mutant sperm exhibited increased DNA fragmentation, revealing that DNA integrity defects appear during epididymal transit. Moreover, exposing control sperm to mutant epididymal fluid or to Ca²⁺-supplemented control fluid significantly increased DNA fragmentation. This, together with the higher intracellular Ca²⁺ levels detected in mutant sperm, supports a dysregulation in Ca²⁺ homeostasis within the epididymis and sperm as the main factor responsible for embryo development failure. These findings highlight the contribution of the epididymis beyond fertilization and identify CRISP1 and CRISP3 as novel factors essential for sperm DNA integrity and early embryo development.