| 1. |
- Munoz, P. Martin, et al.
(författare)
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Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH center dot) only induces spermptosis
- 2018
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Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 53:1, s. 54-67
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2-methyl-1,4-naphthoquinone (menadione), or hydroxyl radical formation after FeSO4 exposure. Either exposure induced significant increases (p amp;lt; 0.05) in two markers of lipid peroxidation: 8-iso-PGF(2) and 4-hydroxynonenal (4-HNE). While both treatments induced changes indicative of spermptosis (caspase-3 activation and decreased mitochondrial membrane potential) (p amp;lt; 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.
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| 2. |
- Ortega Ferrusola, C., et al.
(författare)
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Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na
- 2017
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Ingår i: Andrology. - : WILEY. - 2047-2919 .- 2047-2927. ; 5:6, s. 1174-1182
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Tidskriftsartikel (refereegranskat)abstract
- In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+-K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (pamp;lt;0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (pamp;lt;0.05) in intracellular Na+. These changes occurred in relation to activation of caspase 3 (pamp;lt;0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+-K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+-K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.
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| 3. |
- Balao da Silva, C. M., et al.
(författare)
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Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA
- 2016
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Ingår i: Reproduction in domestic animals. - : WILEY-BLACKWELL. - 0936-6768 .- 1439-0531. ; 51:1, s. 18-25
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Tidskriftsartikel (refereegranskat)abstract
- To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre-and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.
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| 4. |
- Ortega-Ferrusola, C., et al.
(författare)
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Flow cytometry in Spermatology: A bright future ahead
- 2017
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Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 52:6, s. 921-931
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Forskningsöversikt (refereegranskat)abstract
- Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high-throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information. The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single-cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry-based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.
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| 5. |
- Ortiz-Rodriguez, Jose Manuel, et al.
(författare)
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The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoa
- 2019
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Ingår i: Biology of Reproduction. - : OXFORD UNIV PRESS INC. - 0006-3363 .- 1529-7268. ; 101:1, s. 208-222
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is considered amajor mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P amp;lt; 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation-reduction potential of spermatozoa, with sulfasalazine only preventing this effect on fresh spermatozoa that were incubated for 3 h at 37 degrees C, but not in frozen-thawed spermatozoa. While sulfasalazine reduced the motility of frozen-thawed spermatozoa, it increased motility in fresh samples. The present findings provide new and relevant data on the mechanism regulating the redox status of spermatozoa and suggest that a different redox regulatory mechanism exists in cryopreserved spermatozoa, thus providing new clues to improve current cryopreservation technologies and treat male factor infertility. Summary Sentence The SLC7A11 antiporter that exchanges cystine by intracellular glutamate is present and functional in stallion spermatozoa, but cryopreserved spermatozoa may present altered functionality.
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| 6. |
- Ortiz-Rodriguez, Jose M., et al.
(författare)
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Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse
- 2019
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 14:6
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Tidskriftsartikel (refereegranskat)abstract
- Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P amp;lt; 0.01), and (ii) genes for which the fold change was amp;gt;= 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.
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