Relevance of Cysteine-RIch Secretory Proteins for male fertility

CURCI, LUDMILA; ROJO DANIELA; CUASNICU P S; BRUCKMAN NG; RUBINSTEIN M; WEIGEL MUÑOZ M; DA ROS, V.

Congreso; Gordon Research Conference; 2019

Cysteine-Rich Secretory Protein (CRISP) 1, 2, 3 and 4 are mainly expressed in the reproductive tract and have key roles in mammalian fertilization. In spite of this, mutant mice lacking either CRISP1, 2 or 4 are fertile. To investigate the functional relevance of CRISP3, we developed Crisp3 knockout mice using the novel CRISPR/Cas9 technique. With the idea of also generating double Crisp1-/-/Crisp3-/- mice, sgRNAs targeting exon 2 of Crisp1 and Crisp3 were selected by bioinformatic analysis. These sgRNAs were microinjected together with Cas9 mRNA into C57BL/6 mouse zygotes and then transferred to pseudopregnant females. Mice carrying the targeted mutations were identified by PCR of genomic DNA and SDS-PAGE. Germline transmission of the mutations was confirmed by DNA sequencing and mice carrying heterozygous null-allele mutations in Crisp3 or in Crisp1 and Crisp3 were selected as breeders to obtain homozygous mutants. Expression analysis of CRISP proteins in mutant mice performed by Western blot revealed that both Crisp3-/-and Crisp1-/-/Crisp3-/- animals lack CRISP1 whereas CRISP2 and CRISP4 were not affected in either colony. Male fertility rates evaluated by natural mating were significantly reduced in both colonies, suggesting that the normal fertility observed in single knockouts involves compensatory mechanisms between homologous CRISP proteins. Since both colonies were deficient in CRISP1 and CRISP3, we decided to continue the studies using only Crisp1-/-/Crisp3-/- (double KO) mice i.e. those mice that had mutations in both genes. In order to elucidate the mechanisms underlying the lower fertility rates observed in these males, we analyzed the percentage of fertilized eggs recovered from the ampulla of superovulated females mated by double KO or control males. As no differences in the in vivo fertilization rates between these groups were observed, the recovered fertilized oocytes from each group were incubated in vitro for 5 days to analyze their subsequent development. Results showed that the percentage of oocytes from mutant males that reached the blastocysts stage in vitro was significantly lower than controls, suggesting that CRISP1 and CRISP3 may be important for early embryo development. We believe these studies support the relevance of the CRISP family for male fertility and will contribute to a better understanding of how paternal factors could impact on embryo development.