SPERM MATURATION DEFECTS IN CRISP1/CRISP4 DOUBLE KNOCK OUT MICE

CARVAJAL G; VISACOVSKY NICOLAS; WEIGE MUÑOZ, M; CUASNICU P S

Jornada; Jornadas de la sociedad argentina de biologia; 2018

To become fertilization competent, sperm must undergo both epididymal maturation in the male tract and capacitation in the female tract. During epididymal maturation, sperm undergo a series of biochemical and physiological changes that involve the incorporation of new molecules synthetized by the epididymal epithelium as well as post-translational modifications. CRISP1 and CRISP4 are two members of the Cysteine Rich Secretory Protein (CRISP) family that associate with the sperm surface during epididymal transit and are key mediators of fertilization. Double KO males (DKO) for these proteins are subfertile and exhibit sperm fertilizing defects and both an immature epididymal epithelium and abnormal luminal pH. To gain insights into the mechanisms underlying this phenotype, in the present work, we analyzed different sperm parameters associated with epididymal maturation. Results showed that DKO animals presented altered cauda sperm motility and a higher percentage of sperm with cytoplasmic droplets. Using Fluo4-AM and flow cytometry, we observed that while cauda epididymal sperm presented no changes in basal intracellular calcium concentrations compared to controls, they exhibited different calcium levels after calcium ionophore exposure, supporting changes in calcium homeostasis in the DKO cells. Moreover, flow cytometry analysis using merocyanine dye and DiSBAC2(3) probe to evaluate membrane fluidity and membrane potential, respectively, showed lower values for both parameters in DKO sperm compared to controls. Altogether, these results reveal defects in the maturation state of DKO caudal sperm supporting the relevance of CRISP1 and CRISP4 for sperm epididymal maturation.