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Contents The aim of this work was to determine the enzymatic activity of phosphofructokinase ( PFK), malate dehydrogenase ( MDH) and isocitrate dehydrogenase ( IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline ( CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction ( AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa. [ABSTRACT FROM AUTHOR]

The aim of this work was to quantify NO, O and ONOO production during heparin-induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin-dependent capacitation, O uptake, and NO production. Conversely, O production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O consumption (5.9 ± 0.6 nmol/min × 10 cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10 cells), and a five-fold increase in O production (1.3 ± 0.07 nmol/min × 10 cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O generation and the second-order rate constant of the reaction between these species. To conclude, heparin-induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O production by a membrane-bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation. [ABSTRACT FROM AUTHOR]