EFFECT OF PROTEIN KINASE C AND TYROSINE KINASE INHIBITION ON OXIDATIVE METABOLISM IN CRYOPRESERVED BOVINE SPERM IN VITRO CAPACITATION

FERNÁNDEZ SILVINA; CÓRDOBA M.

Jornada; IV Jornadas de INITRA; 2014

Ins. de Investigacion en Reproduccion animal INITRA

EFFECT OF PROTEIN KINASE C AND TYROSINE KINASE INHIBITION ON OXIDATIVE METABOLISM IN CRYOPRESERVED BOVINE SPERM IN VITRO CAPACITATION   Fernández S AND Córdoba M                 Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Unidad ejecutora UBA-Conicet de Investigaciones en Producción Animal (INPA), Cátedra de Química Biológica, Facultad de Ciencias Veterinarias ? Universidad de Buenos Aires   Glycosaminoglycan heparin is present in genital tract of female cattle and can also inducein vitro sperm capacitation. During capacitation, it has been identified signaling pathways mediated by proteinkinase C (PKC), protein kinase andtyrosine kinase (TK). Malate dehydrogenase NAD+ dependent(MDH-NAD+) is a Krebs cycle enzyme, an indicator of mitochondrial oxidative activity. The objective was to determine MDH-NAD+ activity and capacitation of cryopreservedbovine sperm incubated with heparin and PKC andTK inhibitors. GF-109203X and genistein were used as inhibitors of PKC and TK, respectively. MDH-NAD+ activity was determined at 340nm spectrophotometrically, capacitation was evaluated by chlortetracycline epifluorescence technique (CTC) and acrosomal integrityand viability bytrypan blue stain/DIC. Data were analyzed by ANOVAand Tukey test (P<0.05). Activity of MDH-NAD+ registered for heparin capacitated sperm (5.42 ± 1.32 U/108 sp x 10-2)was significantly higher compared tocontrol. In samples incubated with heparin and inhibitors of PKC and TK, enzyme activity was lower than that obtained in heparin capacitated samples (P<0.05). Sperm capacitation induced by heparin (32.75 ± 6.49 %) determined by CTC, was significantly higher than samples treated with heparin and GF (12.50 ± 3.16 %), heparinand genistein (14.25 ± 4.06 %) and for the control (8.50± 2.56 %) (P<0.05). Percentage of live spermatozoaand with acrosome integrity showed no significant differencesbetween treatments and control. Protein kinase Cand tyrosine kinase are involved in the intracellular signal mechanism of heparin capacitation and modulate malate dehydrogenase NAD+ dependent activity in cryopreserved bovine sperm in vitro capacitation.