RESUMEN
Metformin is a leading antidiabetic drug that is used worldwide in the treatment of diabetes mellitus. This biguanide exerts metabolic and pleiotropic effects in somatic cells, although its invitro actions on human spermatozoa remain unknown. The present study investigated the effects of metformin on human sperm function. Human spermatozoa were incubated in the presence or absence of 10mM metformin for 8 or 20h, and motility was measured by computer-aided sperm analysis (CASA); other parameters were evaluated by flow cytometry. Metformin significantly reduced the percentage of motile, progressive and rapid spermatozoa and significantly decreased sperm velocity. Metformin did not affect viability, mitochondrial membrane potential (MMP) or mitochondrial superoxide anion generation of human spermatozoa at any time studied. However, metformin clearly inhibited the protein kinase (PK) A pathway and protein tyrosine phosphorylation at 8 and 20h, key regulatory pathways for correct sperm function. In summary, metformin treatment of human spermatozoa had a detrimental effect on motility and inhibited essential sperm signalling pathways, namely PKA and protein tyrosine phosphorylation, without affecting physiological parameters (viability, MMP, mitochondrial superoxide anion generation). Given the growing clinical use of metformin in different pathologies in addition to diabetes, this study highlights an adverse effect of metformin on spermatozoa and its relevance in terms of human fertility in patients who potentially could be treated with metformin in the future.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipoglucemiantes/farmacología , Metformina/farmacología , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Humanos , Masculino , Mitocondrias/metabolismo , Fosforilación/efectos de los fármacos , Análisis de Semen , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Superóxidos/metabolismo , Tirosina/metabolismoRESUMEN
Metformin is clinically used to treat diabetes. Given its role-impacting metabolism, metformin has been also added to semen cryopreservation media showing specie-dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin-including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ-population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.
Asunto(s)
Metformina/farmacología , Preservación de Semen/veterinaria , Motilidad Espermática/efectos de los fármacos , Sus scrofa/fisiología , Acrosoma/efectos de los fármacos , Animales , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Refrigeración/veterinaria , Análisis de Semen/veterinaria , Preservación de Semen/métodos , Espermatozoides/efectos de los fármacosRESUMEN
The aim of this study was to determine the prevalence and risk factors for human papillomavirus (HPV) infection in the Southern region of the State of Bahia, evaluating the performance of alternative complementary methods for cervical lesion detection. Cervical samples from women who attended healthcare units were collected and diagnosed by visual inspection, cervical cytology and nested polymerase chain reaction (PCR). Moreover, hemi-nested PCR was performed to detect different HPV genotypes. The prevalence of HPV infection was 47·7%, with genotype 16 detected in most cases. Infection was associated with dyspareunia and bleeding (P < 0·001, odds ratio (OR) 5·6, 95% confidence interval (CI) 2·815-11·14) and hormonal contraceptive use (P = 0·007, OR 2·33, 95% CI 1·25-4·34). There was a positive correlation between positive PCR and positive visual inspection, cervical cytology and symptoms reported. Furthermore, visual inspection was twice as specific, and had a greater positive predictive value than cytology. We showed a high prevalence of HPV infection in Southern Bahia, with HPV 16 being the most common type, and visual inspection being most effective at detecting HPV lesions, corroborating the suggestion that it can be applied in routine gynecologic examinations for low-income populations.
Asunto(s)
Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Cuello del Útero/citología , Estudios Transversales , Detección Precoz del Cáncer , Femenino , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pobreza , Prevalencia , Factores de Riesgo , Adulto JovenRESUMEN
AMP-activated kinase (AMPK) plays a key function in maintaining cellular energy homeostasis. We recently identified and localized AMPK protein in human spermatozoa and showed that inhibition of AMPK activity significantly modified human sperm motility. Recently, AMPK has gained great relevance as prime target for pharmacological approaches in several energy-related pathologies and therefore pharmacological research is aimed to develop direct AMPK-activating compounds such as A769662. Our aim was to investigate the effect of A769662 in essential functional processes of human spermatozoa. Human spermatozoa were incubated in the presence or absence of the AMPK activator A769662 for different incubation times (0-20 h) and motility was evaluated by CASA system whereas other functional parameters were evaluated by flow cytometry. A769662 treatment significantly reduces the percentages of motile, progressive, and rapid spermatozoa starting at 2 h. Moreover, AMPK activator in human spermatozoa causes a significant reduction in any velocity measured, which is concomitant to a significant decrease in the percentage of rapid spermatozoa, both at short- (2-3 h) and long-time treatment (20 h). Treatment of human spermatozoa with A769662 does not significantly alter any of the following functional parameters: sperm viability, mitochondrial membrane potential, phosphatidylserine translocation to the outer leaf of plasma membrane, acrosome membrane integrity, or mitochondrial superoxide anion production. In summary, our results suggest that AMPK in human spermatozoa contributes to the regulation of sperm motility, without affecting basic physiological parameters of human spermatozoa (viability, mitochondrial membrane potential or reactive oxygen species production, acrosome membrane integrity, phosphatidylserine exposure at plasma membrane). As sperm motility is required in the female reproductive tract to achieve fertilization, we conclude that AMPK is an essential regulatory kinase of the human spermatozoa function. This conclusion needs to be taken into account when AMPK is elected as prime target in pharmacological approaches for several energy-related pathologies.
Asunto(s)
Pironas/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Tiofenos/farmacología , Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Compuestos de Bifenilo , Regulación hacia Abajo , Humanos , Masculino , Espermatozoides/enzimologíaRESUMEN
Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode's basal medium or TBM) for 3, 8 and 24h or in capacitating conditions (Tyrode's complete medium or TCM) for 4h at 38.5°C or 40°C (Heat stress) in the presence or absence of 5 or 20µM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7±4.1 vs. 1.9±1.1; % mean±SEM), PM (3.1±0.9 vs. 0) and high mitochondrial membrane potential (19.5±2.2 vs. 11.8±0.8) decreased significantly in sperm incubated at 40°C for 24h in the presence of 20µM 17-AAG (control vs. 20µM 17-AAG, respectively; p<0.05). In sperm incubated at 38.5°C only a mild decrease in TM was observed (48.7±3.1 vs. 32.1±4.8; control vs. 20µM 17-AAG, respectively; p<0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Potencial de la Membrana Mitocondrial , Motilidad Espermática , Espermatozoides/fisiología , Animales , Calor , Masculino , Espermatozoides/citología , Estrés Fisiológico , PorcinosRESUMEN
Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.
Asunto(s)
Proteína Quinasa C/metabolismo , Espermatozoides/enzimología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Masculino , Fosforilación/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Porcinos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Some studies of polymorphisms in prostate cancer (PCa) analyze individuals in a uniform manner, regardless of genetic ancestry. However, PCa aggressiveness differs between subjects of African descent and those of European extraction. Thus, genetic ancestry analysis may be used to detect population stratification in case-control association studies. We genotyped 11 ancestry informative markers to estimate the contributions of African, European, and Amerindian ancestries in a case-control sample of 213 individuals from Bahia State, Northeast Brazil, including 104 PCa patients. We compared this data with self-reported ancestry and the stratification of cases by PCa aggressiveness according to Gleason score. A larger African genetic contribution (44%) was detected among cases, and a greater European contribution (61%) among controls. Self-declaration data revealed that 74% of PCa patients considered themselves non-white (black and brown), and 41.3% of controls viewed themselves as white. Our data showed a higher degree of European ancestry among fast-growing cancer cases than those of intermediate and slow development. This differs from many previous studies, in which the prevalence of African ancestry has been reported for all grades. Differences were observed between degrees of PCa aggressiveness in terms of genetic ancestry. In particular, the greater European contribution among patients with high-grade PCa indicates that a population's genetic structure can influence case-control studies. This investigation contributes to our understanding of the genetic basis of tumor aggressiveness among groups of different genetic ancestries, especially admixed populations, and has significant implications for the assessment of inter-population heterogeneity in drug treatment effects.
Asunto(s)
Población Negra/genética , Etnicidad/genética , Genoma Humano , Neoplasias de la Próstata/genética , Anciano , Brasil , Estudios de Casos y Controles , Frecuencia de los Genes/genética , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Pigmentación/genética , Población Blanca/genéticaRESUMEN
Detailed molecular mechanisms mediating signal transduction cascades that regulate boar sperm function involving Ser/Thr and tyrosine phosphorylation of proteins have been reviewed previously. Therefore, this review will focus in those kinase pathways identified recently (<10 years) in boar spermatozoa that regulate different functional spermatozoa processes. AMP-activated protein kinase (AMPK) is a cell energy sensor kinase that was first identified in mammalian spermatozoa in 2012, and since then it has emerged as an essential regulator of boar sperm function. Signaling pathways leading to AMPK activation in boar sperm are highlighted in this review (PKA, CaMKKα/ß, and PKC as well as Ca(2+) and cAMP messengers as upstream regulators). Interestingly, stimuli considered as cell stress (hyperosmotic stress, inhibition of mitochondrial activity, absence of intracellular Ca(2+)) markedly activate AMPK in boar spermatozoa. Moreover, AMPK plays a remarkable and necessary regulatory role in mammalian sperm function, controlling essential boar sperm functional processes such as motility, viability, mitochondrial membrane potential, organization and fluidity of plasma membrane, and outer acrosome membrane integrity. These mentioned processes are all required under fluctuating environment of spermatozoa when transiting through the female reproductive tract to achieve fertilization. An applied role of AMPK in artificial insemination techniques is also suggested as during boar seminal doses preservation at 17 °C, physiological levels of AMPK activity markedly increase (maximum on Day 7) and result essential to maintain the aforementioned fundamental sperm processes. Moreover, regulation of sperm function exerted by the glycogen synthase kinase 3 and Src family kinase pathways is summarized.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Espermatozoides/fisiología , Porcinos/fisiología , Animales , Masculino , Motilidad Espermática/fisiologíaRESUMEN
In this study a Bayesian network (BN) has been built for the study of the objective motility of Tinca tinca spermatozoa (spz). Semen from eight 2-year-old sexually mature male tenchs was obtained and motility analyses were performed at 6-17, 23-34 and 40-51 s after activation, using computer-assisted sperm analysis (CASA) software. Motility parameters rendered by CASA were treated with a two-step cluster analysis. Three well-defined sperm subpopulations were identified, varying the proportion of spermatozoa contained in each cluster with time and male. Cluster, cinematic and time variables were used to build the BN to study the probabilistic relationships among variables and how each variable influenced the final sperm classification into one of three predefined clusters. Both network structure and conditional probabilities were calculated based on the collected data set. Results shown that almost all the variables were directly or indirectly related to each other. By doing probabilistic inference we observed that the cluster distribution corresponded to the definition provided by the cluster analysis. Also, velocity and time variables determined the cluster to which each spermatozoon belonged with a high degree of accuracy. Thus, BNs can be applied in the study of sperm motility. The construction of a BN that include fertility data opens a new way to try to clarify the roles of motility and other sperm quality indicators in fertilization.
Asunto(s)
Análisis de Semen , Semen/citología , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Animales , Teorema de Bayes , Análisis por Conglomerados , Cyprinidae , Masculino , Recuento de Espermatozoides , Espermatozoides/clasificaciónRESUMEN
We recently demonstrated that AMPK inhibition in spermatozoa regulates motility, plasma membrane organization, acrosome integrity and mitochondrial membrane potential. As AMPK activity varies in different energy conditions induced by sperm environment, this work investigates the functional effects of AMPK activation in boar spermatozoa. Spermatozoa were incubated under non-stimulating (TBM) or Ca(2+) and [Formula: see text]-stimulating (TCM) media in the presence/absence of AMPK activator, A769662, for different times. AMPK activity, evaluated as Thr(172) phosphorylation by western blot, is effectively increased by A769662 in spermatozoa. AMPK activation significantly reduces the percentage of motile spermatozoa under Ca(2+) and/or [Formula: see text]-stimulating conditions. Moreover, AMPK activation in spermatozoa incubated in TBM or TCM significantly reduces curvilinear VCL, straight-line VSL and average VAP velocities, which subsequently lead to a significant decrease in the percentage of rapid spermatozoa (VAP > 80 µm/s). The effect of AMPK activation on motility is intensified by the absence of BSA in the incubation medium. AMPK activation for a short time prevents the decline in cell viability and in the sperm population displaying high mitochondrial membrane potential which is induced by Ca(2+) and [Formula: see text]. Sustained (24 h) AMPK activation under TBM or TCM significantly increases both lipid disorganization and phosphatidylserine externalization in the sperm plasma membrane, and diminishes the acrosome membrane integrity. In summary, AMPK activation modifies essential sperm processes such as motility, viability, mitochondrial membrane potential, acrosome membrane integrity, and organization and fluidity of plasma membrane. As these spermatozoa processes are required under different environmental conditions when transiting through the female reproductive tract to achieve fertilization, we conclude that balanced levels of AMPK activity are essential for regulating sperm function.
Asunto(s)
Adenilato Quinasa/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Animales , Compuestos de Bifenilo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fosforilación , Pironas/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Sus scrofa , Tiofenos/farmacologíaRESUMEN
During boar semen liquid preservation, extender is one of the factors that influence storage tolerance of spermatozoa. However, there are few studies about intra-breed variation in the preservation of semen quality during storage in different extenders. Similarly, boar breed is generally not considered a possible factor influencing variation in the semen storage tolerance in a particular extender. The aim of this study was to compare boar semen storage potential, in terms of the ability to maintain sperm viability and motility, of two currently used long-term extenders, MR-A and XCell. Extended semen from two breeds, Iberian and Duroc that had been stored at 17°C for up to 7 days was used. Intra- and inter-breed effect was studied. On Days 1, 4 and 7 (Day 0=day of semen collection), motility parameters and the percentage of total motile sperm and progressively motile sperm using a CASA system was evaluated. Viability (SYBR-14/PI) was evaluated by flow cytometry. Within each breed and for each storage day, there were differences between extenders, although semen tolerance to preservation was more influenced by the extender in the Iberian than in the Duroc breed. Neither breed nor extender influenced the percentage of viable spermatozoa during the storage time. Moreover, differences in motility parameters were observed between breeds, although the differences were greater when the XCell extender was used. In conclusion, both extender and breed influence motility characteristics of liquid-stored boar semen, so both aspects have to be considered in the design of comparative studies about stored boar semen quality from different breeds or with different extenders. Further studies are needed to corroborate these findings.
Asunto(s)
Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Espermatozoides/fisiología , Porcinos/fisiología , Animales , Citometría de Flujo/veterinaria , Masculino , Preservación de Semen/métodos , Estadísticas no ParamétricasRESUMEN
Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 µm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.
Asunto(s)
Reacción Acrosómica/fisiología , Calcio/metabolismo , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Espermatozoides/metabolismo , Porcinos/metabolismo , Animales , Regulación de la Expresión Génica/fisiología , Masculino , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/genética , Receptores Acoplados a Proteínas G/genética , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Porcinos/genéticaRESUMEN
Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 µM MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P > 0.05) throughout the trial. Although the findings of the present study revealed that 1 µM MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen.
Asunto(s)
Antioxidantes/farmacología , Melatonina/farmacología , Preservación de Semen/métodos , Semen/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Porcinos/fisiología , Animales , Relación Dosis-Respuesta a Droga , Etanol/farmacología , Masculino , Factores de TiempoRESUMEN
Both the study and the relationship between sperm design and sperm function have been a target of several researchers. In our study we have evaluated the relationship between the morphometry of sperm head and midpiece as well as the relationship between morphometry of these two spermatic components and sperm motion characteristics in the boar. Analysis of regression (lineal and multiple) and principal components analysis were used for the study of these relationships. Semen samples from five Iberian boars were taken for analysis. Analysis of morphometry was assessed by CASMA system and motility by CASA system. Sperm midpiece showed a significant relationship (positive or negative, depending on the morphometric parameter evaluated) with sperm head. VSL, LIN, STR, BCF and VAP showed a significant relationship with several head and midpiece morphometric parameters. Finally, through the analysis of multiple lineal regression we obtained several statistical models that predict STR, LIN, VCL, ALH, BCF, PC1 and PC2 (the last two variables have been obtained from a principal components analysis) as a function of one, two or three morphometric parameters. Our results suggest a co-evolution of sperm head and midpiece and in addition that sperm motion characteristics of porcine spermatozoa are influenced by morphometry of head and midpiece.
Asunto(s)
Semen/citología , Motilidad Espermática/fisiología , Espermatozoides/citología , Porcinos , Animales , Masculino , Análisis de Componente PrincipalRESUMEN
Computer-assisted sperm morphometry analysis was used to determine the effects of cryopreservation on boar sperm head and midpiece morphometry. Sperm-rich fractions were collected from five mature boars. Three microscope slides were prepared from single extended sperm samples prior freezing and post-thawing. All slides were stained with Hemacolor, and 250 sperm images were obtained from each slide. The sperm head dimensions for length, width, area, perimeter and four shape factors and sperm-midpiece dimensions for area, width, angle and distance were determined in each spermatozoa. The effects of sperm freezing on sperm dimensions within and among boars were determined. A previous discriminant analysis of the results was able to correctly classify a 78.3 and 82% of fresh and frozen-thawed spermatozoa respectively. Sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for length, width, area and perimeter. Sperm midpieces were also significantly smaller in cryopreserved spermatozoa for width and area. The highest changes in morphometric dimensions after the freeze-thawing process were found in the midpiece of spermatozoa. The variability of morphometric measurements only was significantly different between fresh and thawed samples for head rugosity and midpiece area. The effects of cryopreservation on morphometric parameters were similar in the boars, which allow us to conclude that cryopreservation process does not have a different effect in each individual boar. In summary, morphometric changes associated with the cryopreservation process on boar spermatozoa do not apparently depends on an effect at individual level.
Asunto(s)
Criopreservación , Espermatozoides/citología , Animales , Masculino , Motilidad Espermática , PorcinosRESUMEN
Sperm functions are critically controlled through the phosphorylation state of specific proteins. Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase with two different isoforms (alpha and beta), the enzyme activity of which is inhibited by serine phosphorylation. Recent studies suggest that GSK3 is involved in the control of bovine sperm motility. Our aim was to investigate whether GSK3 is present in porcine spermatozoa and its role in the function of these cells. This work shows that both isoforms of GSK3 are present in whole cell lysates of porcine sperm and are phosphorylated on serine in spermatozoa stimulated with the cAMP analog, 8Br-cAMP. A parallel increase in serine phosphorylation of the isoform GSK3alpha, but not in the isoform GSK3beta, is observed after treatments that also induce a significant increase in porcine sperm velocity parameters. Therefore, a significant positive correlation among straight-line velocity, circular velocity, average velocity, rapid-speed spermatozoa, and GSK3alpha serine phosphorylation levels exists. Inhibition of GSK3 activity by alsterpaullone leads to a significant increase in the percentage of rapid- and medium-speed spermatozoa as well as in all sperm velocity parameters and coefficients. Moreover, pretreatment of porcine spermatozoa with alsterpaullone significantly increased the percentage of capacitated porcine spermatozoa and presents no effect in the number of acrosome-reacted porcine spermatozoa. Our work suggests that the isoform GSK3alpha plays a negative role in the regulation of porcine sperm motility and points out the possibility that sperm motile quality might be modulated according the activity state of GSK3alpha.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Serina/metabolismo , Motilidad Espermática/fisiología , Porcinos/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Benzazepinas/farmacología , Western Blotting/métodos , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Glucógeno Sintasa Quinasa 3/análisis , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Calor , Indoles/farmacología , Masculino , Microscopía de Contraste de Fase , Fosforilación , Motilidad Espermática/efectos de los fármacos , Estimulación QuímicaRESUMEN
Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.
Asunto(s)
Supervivencia Celular/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Capacitación Espermática , Espermatozoides/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Androstadienos/metabolismo , Animales , Cromonas/metabolismo , AMP Cíclico/metabolismo , Humanos , Masculino , Morfolinas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/metabolismo , Sus scrofa , WortmaninaRESUMEN
The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.
Asunto(s)
Cabeza del Espermatozoide , Espermatozoides/citología , Coloración y Etiquetado/normas , Porcinos , Animales , Procesamiento de Imagen Asistido por Computador , Masculino , Reproducibilidad de los Resultados , Manejo de Especímenes/normas , Recuento de EspermatozoidesRESUMEN
Protein kinase C-delta (PKC-delta) becomes activated in pancreatic acini in response to cholecystokinin (CCK) and plays a pivotal role in the exocrine pancreatic secretion. Rottlerin, a polyphenolic compound, has been widely used as a potent and specific PKC-delta inhibitor. However, some recent studies showed that rottlerin was not effective in inhibiting PKCdelta activity in vitro and that may display unspecific effects. The aims of this work were to investigate the specificity of rottlerin as an inhibitor of PKC-delta activity in intact cells and to elucidate the biochemical causes of its unspecificity. Preincubation of pancreatic acini with rottlerin (6 microM) inhibited CCK-stimulated translocation, tyrosine phosphorylation (TyrP) and activation of PKC-delta in pancreatic acini in a time-dependent manner. Rottlerin inhibited amylase secretion stimulated by both PKC-dependent pathways (CCK, bombesin, carbachol, TPA) and also by PKC-independent pathways (secretin, VIP, cAMP analogue). CCK-stimulation of MAPK activation and p125(FAK) TyrP which are mediated by PKC-dependent and -independent pathways were also inhibited by rottlerin. Moreover, rottlerin rapidly depleted ATP content in pancreatic acini in a similar way as the mitochondrial uncouplers CCCP and FCCP. All studied inhibitory effects of rottlerin in pancreatic acini were mimicked by FCCP (agonists-stimulated amylase secretion, p125(FAK) TyrP, MAPK activation and PKC-delta TyrP and translocation). Finally, rottlerin as well as FCCP display a potent inhibitory effect on the activation of other PKC isoforms present in pancreatic acini. Our results suggest that rottlerin effects in pancreatic acini are not due to a specific PKC-delta blockade, but likely due to its negative effect on acini energy resulting in ATP depletion. Therefore, to study the role of PKC-delta in cellular processes using rottlerin it is essential to keep in mind that may deplete ATP levels and inhibit different PKC isoforms. Our results give reasons for a more careful choice of rottlerin for PKC-delta investigation.