The CK catalyzes the reversible conversion of creatine into

The CK catalyzes the reversible conversion of creatine into creatine phosphate using ATP and thereby releasing ADP. The CK isoenzymes, specifically localized in places where there is a demand and production of energy, are linked to a creatine/creatine phosphate shuttle (Wallimann et al., 1998). This CK shuttle is a source of extra-mitochondrial ATP and is responsible for the energy transfer between mitochondria and cytosol. The CK activity is an indicator of normal spermatogenesis and maturation and a predictor of fertilizing potential in human sperm (Huszar et al., 1997). Sperm capacitation is a complex process modulated by intracellular signaling pathways, such as adenylate cyclase, protein kinase A and protein tyrosine kinases (Signorelli et al., 2012). Adenylate cyclase increases intracellular cAMP, capacitation and tyrosine protein phosphorylation in mouse sperm (Adeoya-Osiguwa and Fraser, 2000). A membrane-associated isoform of adenylate cyclase (mAC) produces cAMP regulating sperm capacitation in mammals (Fraser et al., 2005). The cAMP production was inhibited in mouse sperm by 2′,5′-dideoxyadenosine, a specific mAC inhibitor (Baxendale and Fraser, 2003). It was reported that bull sperm incubated with heparin displays a reproducible pattern of protein tyrosine phosphorylation regulated by a cAMP-dependent pathway (Galantino-Homer et al., 1997). In contrast, in previous research, it was suggested that hyaluronic Bafetinib sale capacitation would not be modulated by a mAC (Fernández and Córdoba, 2014a).
Materials and methods
Results In heparin and hyaluronic acid-treated samples capacitation percentages were determined and then confirmed by inducing the acrosome reaction with lysophosphatidylcholine as evaluated using CTC. Capacitation was greater with heparin than with hyaluronic acid treatment (0.05) (Table 1), and the percentage of true acrosome reacted-sperm was 30.57±3.60% and 18.44±6.70% for heparin and hyaluronic acid, respectively, in the presence of lysophosphatidylcholine (0.05). A mAC specific inhibitor added to sperm samples only blocked capacitation induction produced by heparin (0.05). In contrast, capacitation was not completely inhibited in sperm samples with hyaluronic acid and 2′,5′-dideoxyadenosine (<0.05) (Table 1). Membrane and acrosome integrity evaluation, however, were not different among samples (>0.05; Table 1). To assess differences in sperm metabolism using heparin or hyaluronic acid as in vitro capacitation inducers, LDH and CK activities and lactate concentration in the incubation medium were determined by spectrophotometry. The LDH activity was different in heparin-capacitated sperm compared with hyaluronic acid treatment (0.38±0.09U/108sp and 1.02±0.40U/108sp, respectively; 0.05). Moreover, LDH activity was less in samples capacitated with heparin and there was a significant increase in samples treated with hyaluronic acid compared with the control (0.05; Figs. 1 A and 2 A). The CK activity in heparin-capacitated sperm was less than with the hyaluronic acid treatment, 0.20±0.04U/108sp and 0.37±0.07U/108sp, respectively (0.05). Furthermore, CK activity was less in heparin-capacitated sperm and greater in hyaluronic acid-capacitated samples compared with the control (0.05; Figs. 1 A and 2 A). The addition of a mAC inhibitor (2′,5′-dideoxyadenosine) in samples treated with hyaluronic acid resulted in a decrease in LDH activity in contrast to heparin-capacitated sperm (<0.05; Figs. 1 A and 2 A). The 2′,5′-dideoxyadenosine did not, however, cause any change in CK activity in the presence of heparin (>0.05), but in hyaluronic acid treated samples there was less CK activity (0.05; Figs. 1 A and 2 A). Lactate concentration in the incubation medium was greater in hyaluronic acid-capacitated samples than with heparin treatment (1.03±0.48mM/106sp and 0.41±0.04mM/106sp, respectively; P<0.05). Also, with heparin treatment there was less hyaluronic acid and greater lactate concentrations in the incubation medium compared with the control (0.05; Figs. 1 B and 2 B).