Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa

[EN] We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.SIThe authors received financial support for this study from the Ministerio de Economía y Competitividad-FEDER, Madrid, Spain, grants AGL2013-43211-R, AGL2017-83149-R Junta de Extremadura-FEDER (European Regional Development Fund) (GR 18008 and IB16030). PMM is supported by a pre-doctoral grant from the Ministerio de Educación, Cultura y Deporte, Madrid Spain FPU13/03991. COF is supported by a post-doctoral grant from the Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671, JMO-R holds a PhD grant from Valhondo Calaaf Foundation, Cáceres Spain

A Rare Case of a Primary Unilateral Low-Grade Paratesticular Leiomyosarcoma in a 2 Years Old Dog

A 2 years old dog was brought to the clinic with complains of testicular enlargement. The tissue was diffusely affected as confirmed by ultrasonographic examination, being the right testicle atrophied and the right epididymis enlarged, with loss of echotexture and presence of several anechogenic areas. The situation required the excision of the referred testicle and epididymis. Final diagnose made by histopathological analysis was primary unilateral low-grade paratesticular leiomyosarcoma. Scarce bibliography is found on this matter, with several cases reported on human, and none in dog. This case report is therefore an important milestone on the area of small animal oncology directly related to the reproductive tissue

Gold nanoparticles inhibit steroid-insensitive asthma in mice preserving histone deacetylase 2 and NRF2 pathways

Background: Gold nanoparticles (AuNPs) can inhibit pivotal pathological changes in experimental asthma, but their effect on steroid-insensitive asthma is unclear. The current study assessed the effectiveness of nebulized AuNPs in a murine model of glucocorticoid (GC)-resistant asthma. Methods: A/J mice were sensitized and subjected to intranasal instillations of ovalbumin (OVA) once a week for nine weeks. Two weeks after starting allergen stimulations, mice were subjected to Budesonide or AuNP nebulization 1 h before stimuli. Analyses were carried out 24 h after the last provocation. Results: We found that mice challenged with OVA had airway hyperreactivity, eosinophil, and neutrophil infiltrates in the lung, concomitantly with peribronchiolar fibrosis, mucus production, and pro-inflammatory cytokine generation compared to sham-challenged mice. These changes were inhibited in mice treated with AuNPs, but not Budesonide. In the GCresistant asthmatic mice, oxidative stress was established, marked by a reduction in nuclear factor erythroid 2-related factor 2 (NRF2) levels and catalase activity, accompanied by elevated values of thiobarbituric acid reactive substances (TBARS), phosphoinositide 3-kinases δ (PI3Kδ) expression, as well as a reduction in the nuclear expression of histone deacetylase 2 (HDAC2) in the lung tissue, all of which sensitive to AuNPs but not Budesonide treatment. Conclusion: These findings suggest that AuNPs can improve GC-insensitive asthma by preserving HDAC2 and NRF2

Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described

Assessing the failure in transfer of passive immunity in beef calves: two case reports

Introduction: Failure in Transfer of Passive Immunity (FTPI) is a condition that occurs frequently in the bovine species when inadequate ingestion or absorption of enough good-quality colostrum takes place. In bovine kept in extensive conditions, FTPI diagnosis is usually made by complete physical examination, with not enough time for laboratory analysis, and treatment is based on the administration of colostrum replacement products or transfusion. Objectives: To evaluate the transfer of passive immunity in meat calves kept in extensive conditions. Methods: We hereby describe two case reports of calves presenting clinical signs compatible with FTPI and their biochemical analysis, including IgG determination by an ELISA test. Results: The results indicated that both calves presented a very low serum IgG concentration (calf 1: 0.7mg/ml and calf 2: 1mg/ml), confirming FTPI. The animals were treated with a plasma transfusion from a crossbreed cow. At 24 hours after the plasma transfusion, serum IgG concentration raised slightly on both animals, but after 48 hours serum IgG reduced to a concentration similar to the pre-transfusion values. Conclusions: We conclude that plasma transfusion can be an effective treatment for FTPI in clinical cases in extensive conditions but it has to be monitored assuring that serum IgG concentration reaches normal values. Immunological status of donors should be assessed before plasma collection and transfer.info:eu-repo/semantics/publishedVersio

Avaliação da falha de transferência de imunidade passiva em vitelos de carne: dois casos clínicos

Introduction: Failure in Transfer of Passive Immunity (FTPI) is a condition that occurs frequently in the bovine species when inadequate ingestion or absorption of enough good-quality colostrum takes place. In bovine kept in extensive conditions, FTPI diagnosis is usually made by complete physical examination, with not enough time for laboratory analysis, and treatment is based on the administration of colostrum replacement products or transfusion. Objectives: To evaluate the transfer of passive immunity in meat calves kept in extensive conditions. Methods: We hereby describe two case reports of calves presenting clinical signs compatible with FTPI and their biochemical analysis, including IgG determination by an ELISA test. Results: The results indicated that both calves presented a very low serum IgG concentration (calf 1: 0.7mg/ml and calf 2: 1mg/ml), confirming FTPI. The animals were treated with a plasma transfusion from a crossbreed cow. At 24 hours after the plasma transfusion, serum IgG concentration raised slightly on both animals, but after 48 hours serum IgG reduced to a concentration similar to the pre-transfusion values. Conclusions: We conclude that plasma transfusion can be an effective treatment for FTPI in clinical cases in extensive conditions but it has to be monitored assuring that serum IgG concentration reaches normal values. Immunological status of donors should be assessed before plasma collection and transfer.Introdução: A Falha de Transferência de Imunidade Passiva (FTIP) é uma condição que ocorre frequentemente na espécie bovina após ingestão ou absorção inadequada de quantidades suficientes de colostro de qualidade adequada. Em bovinos mantidos em extensivo, o diagnóstico é habitualmente realizado através de um exame clínico completo, não existindo tempo suficiente para uma análise laboratorial, e o tratamento é baseado na administração de substitutos de colostro ou transfusão. Objetivos: Avaliar a transferência de imunidade passiva em vitelos de carne em cultivo extensivo. Métodos: No presente trabalho descrevem-se dois casos clínicos de vitelos com sinais clínicos compatíveis com FTIP, assim como a sua análise bioquímica, incluindo determinação de igg através de um teste ELISA. Resultados: Ambos os vitelos apresentavam uma concentração sérica muito baixa de igg (vitelo 1: 0.7mg/ml e vitelo 2: 1mg/ml), confirmando a FTIP. Os animais foram tratados com uma transfusão de plasma de uma vaca cruzada. Após 24 horas da transfusão de plasma, a concentração de igg aumentou ligeiramente em ambos os animais, mas após 48 horas da transfusão a concentração reduziu para valores semelhantes aos verificados antes do tratamento. Conclusões: A transfusão de plasma pode ser um tratamento eficaz para a FTIP de casos clínicos em condições de extensivo, mas esta terá de ser monitorizada garantindo que a concentração sérica de igg atinge valores adequados. O estado imunitário das dadoras deve ser avaliado antes da colheita e transfusão de plasma.Introducción: El Fallo de Transferencia de Inmunidad Pasiva (FTIP) es una condición que ocurre frecuentemente en la especie bovina, después de la inadecuada ingestión o absorción de calostro de calidad adecuada. En bovinos mantenidos en extensivo, el diagnóstico de FTIP es frecuentemente realizado con un examen físico completo, sin tiempo suficiente para realizar un análisis de laboratorio, y el tratamiento se basa en la administración de sustitutos de calostro o transfusión. Objetivos: Evaluar la transferencia de inmunidad pasiva en terneros de carne. Métodos: En este trabajo se describen dos casos clínicos de becerros con signos clínicos compatibles con FTIP y sus análisis bioquímicos, incluyendo determinación de IgG con un test ELISA. Resultados: Los resultados indicaron que los dos becerros presentaban una concentración sérica muy baja de IgG (vitelo 1: 0.7mg/mL y vitelo 2: 1mg/mL), confirmando FTIP. Los animales fueron tratados con una transfusión de plasma de una vaca cruzada. Tras 24 horas de la transfusión de plasma, la concentración de IgG incrementó ligeramente en ambos animales, pero tras 48 horas de la transfusión la concentración se redujo para valores semejantes a los verificados antes del tratamiento. Conclusiones: La transfusión de plasma puede ser un tratamiento eficaz para la FTIP de casos clínicos en condiciones de extensivo, pero tendrá de ser monitorizada garantizando que la concentración sérica de IgG alcanza valores adecuados. El estadio inmunitario de las donantes debe ser evaluado antes de la recogida y transfusión de plasma

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility.

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence

Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3.

BackgroundThe population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature.ObjectivesThus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation.Materials and methodsThawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 μM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor).ResultsRosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (pConclusionWe provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology

Significant correlations between different indicators of the production of ROS and membrane intactness and mitochondrial functionality.

<p>** p<0.01, n.s non-significant</p><p>Mitosox+ spermatozoa show mitochondrial production of superoxide radical, H₂DCFDA—percentage of spermatozoa showing hydrogen peroxide production, CellRox (Live Cells)—percentage of live spermatozoa showing ROS production, HE—percentage of spermatozoa showing superoxide production, Intact sperm—percentage of spermatozoa with completely intact membranes, YoPro+—percentage of spermatozoa with intact membranes but with increased permeability, JC- high percentage of spermatozoa with high mitochondrial membrane potential.</p><p>Significant correlations between different indicators of the production of ROS and membrane intactness and mitochondrial functionality.</p

Effect of the incubation of stallion spermatozoa in BWW complete media (BWW GLUC PYR) devoid of glucose (BWW PYR) or devoid of glucose and pyruvate (BWW) on on the ATP content of stallion spermatozoa.

<p>ATP was determined as described in the materials and methods. The results are given as the means ± SD.</p