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SUMMARYThis work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca 2+ signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O 2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca 2+ labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca 2+ signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca 2+. The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca 2+ -induced peak, O 2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 +/- 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 +/- 0.1 mIU/mg protein to 0.60 +/- 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly dependent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15-0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose transporter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins involved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at concrete points in the boar-sperm surface.

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Several studies have shown that dopamine and other catecholamines are present in oviduct luminal fluid. We recently reported that dopamine type 2 receptors (DRD2) are present in a wide range of mammalian sperm, suggesting a role for dopaminergic signaling in events such as fertilization, capacitation, and sperm motility. In the present study, we used Western blot analysis to show that boar sperm express DRD2 and that their activation with dopamine (100 nM) has a positive effect on cell viability that can be correlated with AKT/PKB phosphorylation. Bromocriptine (100 nM) and dopamine (100 nM and 10 muM) increased tyrosine phosphorylation during the capacitation period. Immunofluorescence analysis indicated that DRD2 localization is dynamic and depends on the capacitation stage, colocalizing with tyrosine phosphorylated proteins in the acrosome and midpiece region of capacitated boar sperm. This association was confirmed by coimmunoprecipitation analysis. We also showed that bromocriptine (100 nM) and low-concentration dopamine (100 nM and 10 muM) increased total and progressive motility of sperm. However, high concentrations of dopamine (1 mM) decreased tyrosine phosphorylation and motility in in vitro sperm capacitation assays. This can be explained by the presence of the dopamine transporters (DAT, official symbol SLC6A3) in sperm, as demonstrated by Western blot analysis and immunocytochemistry. Taken together, our results support the idea that dopamine may have a fundamental role during sperm capacitation and motility in situ in the female upper reproductive tract.

Incubation of boar spermatozoa in a capacitation medium with oligomycin A, a specific inhibitor of the F0 component of the mitochondrial ATP synthase, induced an immediate and almost complete immobilisation of cells. Oligomycin A also inhibited the ability of spermatozoa to achieve feasible in vitro capacitation (IVC), as measured through IVC-compatible changes in motility patterns, tyrosine phosphorylation levels of the acrosomal p32 protein, membrane fluidity and the ability of spermatozoa to achieve subsequent, progesterone-induced in vitro acrosome exocytosis (IVAE). Both inhibitory effects were caused without changes in the rhythm of O2 consumption, intracellular ATP levels or mitochondrial membrane potential (MMP). IVAE was accompanied by a fast and intense peak in O2 consumption and ATP levels in control spermatozoa. Oligomycin A also inhibited progesterone-induced IVAE as well as the concomitant peaks of O2 consumption and ATP levels. The effect of oligomycin on IVAE was also accompanied by concomitant alterations in the IVAE-induced changes on intracellular Ca2+ levels and MMP. Our results suggest that the oligomycin A-sensitive mitochondrial ATP-synthase activity is instrumental in the achievement of an adequate boar sperm motion pattern, IVC and IVAE. However, this effect seems not to be linked to changes in the overall maintenance of adequate energy levels in stages other than IVAE.

The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.

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