Fertilization defects in sperm from Cysteine-rich protein 2 (Crisp2) knockout mice.

COHEN DJ; DA ROS VG; BRUKMAN N; MIYATA H; CUASNICU PS; CARVAJAL G; IKAWA M

Congreso; The Betsheva de Rothschild Seminar on Challenges and Frontiers in Mammalian Reproduction.; 2016

Fertilization defects in sperm from Cysteine-rich secretory protein 2 (Crisp2) knockout miceN.G. Brukman, G. Carvajal, D.J. Cohen, H. Miyata, M. Ikawa, V.G. Da Ros, and P.S. CuasnicúTesticular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility. To further investigate the role of CRISP2 in sperm physiology and fertilization, we analyzed the reproductive phenotype of males from a mice line carrying a deletion in Crisp2 gene. Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability. Crisp2-/- sperm also exhibit lower percentages of hyperactivation, a vigorous motility required for the penetration of the egg coats, and higher intracellular Ca2+ (iCa2+) levels after capacitation, compared to controls. Interestingly, zona pellucida-intact eggs treated with reduced glutathione to destabilize the zona pellucida prior to their insemination, produced a significant increase in the fertilization levels corresponding to Crisp2-/- sperm, supporting that the lower fertilizing ability of these cells is linked to their lower levels of hyperactivation. In addition, we evaluated iCa2+ levels by flow cytometry in mutant sperm incubated in capacitating media containing either an inhibitor of CatSper, the main sperm membrane Ca2+ channel, or lower concentration of CaCl2. Exposure of sperm to any of these conditions produced a reversion of the deregulated iCa2+ of Crisp2-/- cells, indicating that the higher iCa2+ levels of the mutant sperm are dependent on the extracellular Ca2+. Together, these observations support the relevance of CRISP2 for the regulation of the complex and fine-tuned Ca2+ regulating system operating in the fertilizing sperm.