Relevance of cystein-rich secretory proteins for fertilization, fertility and early embryo development

Abstract

CRISP1 and CRISP3 are two members of the Cysteine Rich Secretory Protein family expressed in the male and female mammalian reproductive tracts. In spite of the important roles of CRISP1 in fertilization, CRISP1 knockout mice are fertile, suggesting compensatory mechanisms among CRISP proteins. As the functional role of CRISP3 in reproduction still remains unclear, in this work we investigated the relevance of CRISP3 for both fertilization and fertility by generating single Crisp3 and double Crisp1/Crisp3 knockout mice. Methods: Crisp3-/- and Crisp1-/-/Crisp3-/- (DKO) mice were generated by using the novel CRISPR/Cas9 technique. DNA sequencing was used to confirm the null-allele mutations and Western blot for evaluating protein expression. Fertility was evaluated by mating males with one females and in vivo fertilization by analyzing the percentage of fertilized eggs recovered from the ampulla of mated females. Finally, early development was evaluated as the percentage of fertilized eggs reaching the blastocysts stage 5 days after in vitro incubationResults: Male and female fertility were significantly reduced in both Crisp3-/- and DKO animals. Protein expression analysis revealed the lack of CRISP1 protein not only in DKO but also in Crisp3-/- mice. Subsequent experiments were carried out using only those mice exhibiting mutations in both genes. Examination of in vivo fertilization to elucidate the mechanisms underlying fertility defects showed significantly lower fertilization rates in DKO females than in controls but normal fertilization rates when control females were mated by DKO males. Interestingly, the eggs normally fertilized by DKO sperm exhibited a significantly lower ability to reach the blastocyst stage compared to controls, supporting an additional role for CRISP1 and CRISP3 in early embryo development.Conclusion: Our results confirm the relevance of CRISP1 and CRISP3 for both fertilization and fertility and may contribute to a better understanding of how paternal factors could impact on embryo development.