STUDY OF THE MECHANISMS INVOLVED IN Crisp2-/- SPERM FERTILIZING DEFECTS

CARVAJAL G; BRUKMAN NG; CUASNICÚ PS; DA ROS VG

Jornada; XVIII Jornadas Anuales Multidisciplinarias; 2016

Sociedad Argentina de Biología (SAB)

Testicular Cysteine-RIch Secretory Protein 2 (CRISP2)is present in sperm and participates in gamete fusion and penetration of both zonapellucida (ZP) and cumulus oophorus during fertilization. Inaddition, Crisp2-/- sperm exhibit lower percentages ofhyperactivation, a vigorous motility required for the penetration of the eggcoats, and higher intracellular Ca2+(iCa2+) levels aftercapacitation, compared to controls. In the present work, we furtherinvestigated the mechanisms underlying these Crisp2-/- sperm defects.ZP-intact eggs treated with reduced glutathione to destabilize the ZP prior totheir insemination, produced a significant increase in the fertilization levelscorresponding to Crisp2-/- sperm, supporting that the lowerfertilizing ability of these cells is linked to their lower levels ofhyperactivation. We then evaluated iCa2+levels by flow cytometry inmutant sperm incubated in capacitating media containing either an inhibitor ofCatSper, the main sperm membrane Ca2+ channel, or lowerconcentration of CaCl2. Exposure of sperm to any of these conditionsproduced a reversion of the deregulated iCa2+ of Crisp2-/-cells, indicating that the higher iCa2+ levels of the mutant spermare dependent on extracellular Ca2+. However, when these treatedsperm exhibiting normal iCa2+were subjected to in vitrofertilization, no improvement in the fertilization rates was observed.Together, these observations support the relevance of CRISP2 for the regulationof the complex and fine-tuned Ca2+ regulating system operating inthe fertilizing sperm.