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7. Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation

Author

Davila, M Plaza, Muñoz, P Martin, Bolaños, J M Gallardo, Stout, T A E, Gadella, B M, Tapia, J A, da Silva, C Balao, Ferrusola, C Ortega, Peña, F J, dES/dFAH FR, LS Klinische Reproductie, LS Voortplanting Inwendige Ziekten, Sub Biologie van de mannelijke gameet, and Sub Reproductie mannelijk

Abstract

To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also significantly reduced mitochondrial membrane potential. Sperm motility almost completely ceased after the inhibition of mitochondrial respiration and both percentage of motile sperm and sperm velocity were reduced in the presence of mitochondrial uncouplers. Inhibition of ATP synthesis resulted in the loss of sperm membrane integrity and increased the production of reactive oxygen species by degenerating sperm. Inhibition of glycolysis by deoxyglucose led to reduced sperm velocities and reduced ATP content, but not to loss of membrane integrity. These results suggest that, in contrast to many other mammalian species, stallion spermatozoa rely primarily on oxidative phosphorylation to generate the energy required for instance to maintain a functional Na(+)/K(+) gradient, which is dependent on an Na(+)-K(+) antiporter ATPase, which relates directly to the noted membrane integrity loss. Under aerobic conditions, however, glycolysis also provides the energy required for sperm motility.

Published
2016

14. Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes.

Author

Ferrusola, C. Ortega, Fernández, L. González, Morrell, J. M., Sandoval, C. Salazar, García, B .Macías, Rodríguez-Martinez, H., Tapia, J. A., and Peña, F. J.

Subjects
PEROXIDATION, CRYOPRESERVATION of organs, tissues, etc., SPERMATOZOA, STALLIONS, ANIMAL reproduction
Abstract

Lipid peroxidation (LPO) of stallion spermatozoa was assessed in fresh semen and in samples of the same ejaculates after freezing and thawing. Particular attention was paid to individual differences in the susceptibility to LPO and its possible relationship with freezability. Innate levels of LPO were very low in fresh spermatozoa but increased after thawing, a change that was largely stallion-dependent. The level of LPO in fresh spermatozoa was not correlated with that of the thawed spermatozoa. Negative correlations existed between LPO and intact membranes post-thaw (r=-0.789, P<0.001), and also between LPO and spermatozoa with high mitochondrial membrane potential (δψm) post-thaw (r=-0.689, P<0.001). LPO was also highly and significantly correlated with caspase activity. The correlation between caspase activity in ethidium positive cells and LPO was r=0.772, P<0.001. This LPO is unlikely to represent, per se, a sign of cryopreservation-induced injury, but it is apparently capable of triggering 'apoptotic-like changes' that could result in the sub-lethal cryodamage often seen among surviving spermatozoa [ABSTRACT FROM AUTHOR]

Published
2009

15. Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols?

Author

Peña FJ, García BM, Samper JC, Aparicio IM, Tapia JA, and Ferrusola CO

Subjects
Animals, Apoptosis, Cryopreservation methods, Male, Osmotic Pressure, Oxidative Stress, Reactive Oxygen Species metabolism, Cryopreservation veterinary, Horses, Spermatozoa
Abstract

We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Published
2011
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16. Freezing stallion semen with the new Cáceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability.

Author

Rodríguez AM, Ferrusola CO, García BM, Morrell JM, Martínez HR, Tapia JA, and Peña FJ

Subjects
Acrosome physiology, Animals, Cell Survival physiology, Cryopreservation methods, Flow Cytometry veterinary, Male, Membrane Potential, Mitochondrial physiology, Microscopy, Phase-Contrast veterinary, Semen Preservation methods, Sperm Motility physiology, Statistics, Nonparametric, Cryopreservation veterinary, Cryoprotective Agents, Horses physiology, Semen Preservation veterinary, Spermatozoa physiology
Abstract

Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p<0.05) and velocity (p<0.05), membrane integrity (increases ranging from 11 to 14%, p<0.05) and mitochondrial membrane potential (p<0.05). It is concluded that this new extender should be included in a freezeability test to determine the best extender for each individual., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Published
2011
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17. Membrane lipids of the stallion spermatozoon in relation to sperm quality and susceptibility to lipid peroxidation.

Author

García BM, Fernández LG, Ferrusola CO, Salazar-Sandoval C, Rodríguez AM, Martinez HR, Tapia JA, Morcuende D, and Peña FJ

Subjects
Animals, Caspases metabolism, Cell Membrane chemistry, Cell Membrane ultrastructure, Fatty Acids analysis, Male, Membrane Fluidity, Membrane Potential, Mitochondrial, Spermatozoa ultrastructure, Horses, Lipid Peroxidation, Membrane Lipids analysis, Spermatozoa chemistry, Spermatozoa physiology
Abstract

Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p<0.05) of this fatty acid in his sperm plasma membranes., (© 2010 Blackwell Verlag GmbH.)

Published
2011
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