A new role for CRISP1 in the fertilization process

ERNESTO JI; CALB D; WEIGEL MUÑOZ M; COHEN D.J; CUASNICU PS

Montreal

Congreso; 36th Annual Meeting of American Society of Andrology; 2011

American Society of Andrology

Epididymal protein CRISP1 is the first described member of the CRISP family with members present mainly in the male reproductive tract. Evidence from our group obtained using in vitro fertilization assays and knock out (KO) mice generated in our laboratory, supports the participation of CRISP1 in sperm capacitation as well as in sperm interaction with both the zona pellucida and the oolema. In the present work, CRISP1 KO mice were used to investigate the presence of the protein in the female reproductive tract and its potential role in the fertilization process. RT-PCR analysis revealed the presence of CRISP1 mRNA in the uterus, ovary, oviduct, and cumulus cells of wild type (WT) but not KO mice. Western blot (Wb) and indirect immunofluorescence studies using a specific polyclonal anti-CRISP1 antibody confirmed the presence of the protein only in the tissues and cumulus cells from WT females. In view of these observations, we evaluated the possible participation of female CRISP1 in fertilization by in vitro fertilization assays performed using cumuls-oocyte complexes (COCs) and sperm from both WT and KO mice. Results showed that when WT sperm were used for insemination, significantly (p< 0,0001) lower levels of fertilization were observed for KO compared to WT COCs (33% vs 67%). This decrease was even more evident when KO COCs were exposed to KO instead than to WT (10 % vs 33%) sperm. Considering that KO sperm fertilize WT COCs normally, the use of a more restrictive system where both COCs and sperm lack CRISP1, allows the detection of a deficiency of KO sperm to penetrate the cumulus. Together, these results confirm that sperm are exposed to CRISP proteins in both the male and female reproductive tracts and support the existence of a new role for CRISP1 in the fertilization process.