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Mitochondria from frozen thawed bull sperm are capable of
Mitochondria from frozen-thawed bull sperm are capable of producing energy using pyruvate and lactate as substrates through oxidative pathways (Beorlegui et al., 1997). Based on the present study, heparin capacitated sperm had a lesser lactate concentration in the incubation medium, supporting the hypothesis that the carbon skeletons of these substrates are derived for translocation across the inner mitochondrial membrane to complete oxidation through the respiratory pathway. In this context, heparin capacitation allows pyruvate to be metabolized through the Krebs cycle in cryopreserved bull sperm, in a manner similar to that observed in epididymal mouse sperm (Van Dop et al., 1977). In contrast, hyaluronic e3 ligases leads to a metabolic pattern where the carbon skeletons are used by LDH, generating more lactate in the incubation medium and not deriving pyruvate to produce energy through the Krebs cycle and the respiratory chain, confirmed by the low oxygen consumption generated by this inducer, determined in previous research in our laboratory. Sperm motility is supported by ATP supplied to the dynein/ATPase present in the flagellum (Garrett et al., 2008). There are marked differences between the sperm of various species in the energy metabolism necessary for motility; ATP can be produced by glycolysis and oxidative respiration, both of which occur in bull sperm (Krzyzosiak et al., 1999). The phosphocreatine/creatine-shuttle is a physiological adaptation to overcome the diffusion limitations of adenosine nucleotides, especially ADP, to facilitate long-distance energy transport, connecting sites of ATP production with subcellular sites of ATP utilization (Wallimann et al., 2011). In the present research, it was found CK enzyme activity increases with hyaluronic acid while it decreases when heparin is used as a capacitation inducer. The less CK activity in heparin-capacitated samples would indicate a predominance of mitochondrial oxidation, evidenced by the greater oxygen consumption determined previously. In contrast, the increase in CK activity in hyaluronic acid capacitated sperm, is linked with a lesser oxygen consumption and, therefore, with a lesser respiratory chain activity (Fernández and Cordoba, 2014a). In cryopreserved sperm of bulls, hyaluronic acid induces the use of creatine phosphate to provide energy, for example for sperm motility and induction of intracellular signals related to kinases. Sperm capacitation is dependent on the modulation of key enzymes, such as protein kinase A, protein kinase C, tyrosine kinase and adenylate cyclase (AC). Transmembrane and soluble forms of AC facilitate the synthesis of cAMP (Lackey and Gray, 2015). In mammals, AC activation increases cAMP concentration that induces protein kinase A activity and protein phosphorylation such as kinases and phosphatases (Signorelli et al., 2012). The role and presence of mAC in sperm has been a subject of controversy, but one defining property of mAC is that it is regulated by members of the heterotrimeric G protein family (Wertheimer et al., 2013). In bull sperm capacitated with heparin, an increase in intracellular calcium activates soluble AC increasing cAMP and then inducing protein kinase A, leading to intracellular signals involved in capacitation induction (Parrish et al., 1994). In the present study, the inhibition of mAC by 2′,5′-dideoxyadenosine did not completely block capacitation produced by hyaluronic acid, suggesting that mAC is not involved in the hyaluronic acid intracellular signaling mechanism under these conditions. Meanwhile, capacitation was blocked in heparin treatment with mAC inhibitor, suggesting that heparin requires the activity of this regulatory enzyme to modulate capacitation, as was observed in our previous research (Fernández and Córdoba, 2014a). Both heparin and hyaluronic acid have membrane receptors (McCauley et al., 1996, Bergqvist et al., 2005). Different extracellular ligands, such as heparin, are known to activate G protein and mAC (Fraser et al., 2005, Taussig and Gilman, 1995). In the present study conditions, the mild inhibition by 2′,5′-dideoxyadenosine observed in hyaluronic acid capacitation indicates that there is a less sensitivity to the blocking hyaluronic acid effect than heparin effect. Previous studies have shown the involvement of tyrosine kinase and protein kinase C enzymes in the intracellular signaling system of hyaluronic acid (Bourguignon et al., 2001, Hall et al., 2001). Results of the present study indicate that the binding of hyaluronic acid with the sperm membrane induces signals that would not be related to mAC activation to the same extent as heparin induction.