Inhibition of forward and reverse transport of Ca2+ via Na+/Ca2+ exchangers (NCX) prevents sperm capacitation.

BACKGROUND: While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated. RESULTS: Partial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation. CONCLUSIONS: Inhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

The achievement of boar sperm in vitro capacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels.

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed in vitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4 h significantly (p < 0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4 h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4 h significantly (p < 0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

Efeito da inibição da óxido nítrico sintase induzível na capacitação in vitro de espermatozoides bovinos / Effect of inhibition of inducible nitric oxide synthase on in vitro capacitation of bovine spermatozoa

Avaliaram-se o papel do óxido nítrico (NO) por meio da inibição da enzima óxido nítrico sintase induzível (iNOS), após a adição da aminoguanidina (AG), na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M) de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO) à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (P<0,05). A adição de SNP ao meio de cultivo com 0,1M de AG somente reverteu a integridade da membrana após 300min. A inibição da síntese de NO pela adição de AG não alterou a atividade mitocondrial. A percentagem de oócitos penetrados com espermatozoides tratados com 0,01 e 0,1M de AG diminuiu 20,3 e 100 por cento, respectivamente, em relação aos não tratados (controle) (P<0,05), contudo houve aumento de 15 por cento na percentagem de oócitos desnudados penetrados com espermatozoides capacitados em presença de 0,1M de AG. Conclui-se que a inibição da síntese de NO pela AG diminuiu a qualidade espermática durante a capacitação de espermatozoides bovinos in vitro, exceto a atividade mitocondrial. Somente a integridade da membrana foi revertida após adição de NO, sugerindo diferentes vias de ação do NO na qualidade espermática ao longo da capacitação in vitro de espermatozoides bovinos.(AU)

The role of nitric oxide (NO) was evaluated by inhibition of inducible nitric oxide synthase (iNOS), with aminoguanidine (AG) on motility, vigor, and plasmatic membrane integrity of bovine spermatozoa culture after 15, 60, 120, 180, 240, and 300min and on mitochondrial activity and capacitation after 300min, respectively. Different concentrations, 0.001, 0.01, and 0.1M of AG were added during the heparin induced capacitation and sodium nitroprusside (SNP, NO donor-500μM) to the deleterious concentration. The addition of 0.1M of AG diminished progressive motility, spermatic vigor, and membrane integrity (P<0.05). SNP addition to the 0.1M of AG did revert only plasmatic membrane integrity after 300min. Mitochondrial activity was not influenced by addition of AG. Percentage of penetrated oocytes after addition of 0.01 and 0.1M of AG diminished, 20.3 and 100 percent, respectively, in relation to the control oocytes (P<0.05). However, an increase of 15 percent was observed when denuded oocytes were used with 0.1M AG treated sperm (P<0.05). It was concluded that the inhibition of NO synthesis with aminoguanidine diminished sperm quality during in vitro capacitation of bovine spermatozoa, except the mitochondrial activity. Only membrane integrity was reverted with the addition of NO to culture medium, suggesting different pathways of NO action on bovine sperm quality during in vitro capacitation.(AU)

Efeito da inibição da óxido nítrico sintase induzível na capacitação in vitro de espermatozoides bovinos / Effect of inhibition of inducible nitric oxide synthase on in vitro capacitation of bovine spermatozoa

Avaliaram-se o papel do óxido nítrico (NO) por meio da inibição da enzima óxido nítrico sintase induzível (iNOS), após a adição da aminoguanidina (AG), na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M) de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO) à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (P<0,05). A adição de SNP ao meio de cultivo com 0,1M de AG somente reverteu a integridade da membrana após 300min. A inibição da síntese de NO pela adição de AG não alterou a atividade mitocondrial. A percentagem de oócitos penetrados com espermatozoides tratados com 0,01 e 0,1M de AG diminuiu 20,3 e 100 por cento, respectivamente, em relação aos não tratados (controle) (P<0,05), contudo houve aumento de 15 por cento na percentagem de oócitos desnudados penetrados com espermatozoides capacitados em presença de 0,1M de AG. Conclui-se que a inibição da síntese de NO pela AG diminuiu a qualidade espermática durante a capacitação de espermatozoides bovinos in vitro, exceto a atividade mitocondrial. Somente a integridade da membrana foi revertida após adição de NO, sugerindo diferentes vias de ação do NO na qualidade espermática ao longo da capacitação in vitro de espermatozoides bovinos.

The role of nitric oxide (NO) was evaluated by inhibition of inducible nitric oxide synthase (iNOS), with aminoguanidine (AG) on motility, vigor, and plasmatic membrane integrity of bovine spermatozoa culture after 15, 60, 120, 180, 240, and 300min and on mitochondrial activity and capacitation after 300min, respectively. Different concentrations, 0.001, 0.01, and 0.1M of AG were added during the heparin induced capacitation and sodium nitroprusside (SNP, NO donor-500μM) to the deleterious concentration. The addition of 0.1M of AG diminished progressive motility, spermatic vigor, and membrane integrity (P<0.05). SNP addition to the 0.1M of AG did revert only plasmatic membrane integrity after 300min. Mitochondrial activity was not influenced by addition of AG. Percentage of penetrated oocytes after addition of 0.01 and 0.1M of AG diminished, 20.3 and 100 percent, respectively, in relation to the control oocytes (P<0.05). However, an increase of 15 percent was observed when denuded oocytes were used with 0.1M AG treated sperm (P<0.05). It was concluded that the inhibition of NO synthesis with aminoguanidine diminished sperm quality during in vitro capacitation of bovine spermatozoa, except the mitochondrial activity. Only membrane integrity was reverted with the addition of NO to culture medium, suggesting different pathways of NO action on bovine sperm quality during in vitro capacitation.