RESUMEN
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.
Asunto(s)
Calcio , Intercambiador de Sodio-Calcio , Capacitación Espermática , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Intercambiador de Sodio-Calcio/metabolismo , Intercambiador de Sodio-Calcio/efectos de los fármacos , Calcio/metabolismo , Porcinos , Espermatozoides/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Motilidad Espermática/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.
Asunto(s)
Reacción Acrosómica , Acrosoma , Calcio , Plomo , Motilidad Espermática , Espermatozoides , Masculino , Espermatozoides/efectos de los fármacos , Calcio/metabolismo , Motilidad Espermática/efectos de los fármacos , Animales , Acrosoma/efectos de los fármacos , Plomo/toxicidad , Reacción Acrosómica/efectos de los fármacos , AMP Cíclico/metabolismo , Bovinos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Análisis de Semen , Daño del ADN/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Compuestos Organometálicos/farmacologíaRESUMEN
To acquire the ability to fertilize the egg, mammalian spermatozoa must undergo a series of changes occurring within the highly synchronized and specialized environment of the female reproductive tract, collectively known as capacitation. In an attempt to replicate this process in vitro, various culture media for mouse sperm were formulated over the past decades, sharing a similar overall composition but differing mainly in ion concentrations and metabolic substrates. The widespread use of the different media to study the mechanisms of capacitation might hinder a comprehensive understanding of this process, as the medium could become a confounding variable in the analysis. In this context, the present side-by-side study compares the influence of four commonly used culture media (FD, HTF and two TYH versions) on mouse sperm capacitation. We evaluated the induction of protein kinase A phosphorylation pathway, motility, hyperactivation and acrosome reaction. Additionally, in vitro fertilization and embryo development were also assessed. By analyzing these outcomes in two mouse colonies with different reproductive performance, our study provides critical insights to improve the global understanding of sperm function. The results obtained highlight the importance of considering variations in medium composition, and their potential implications for the future interpretation of results.
Asunto(s)
Reacción Acrosómica , Medios de Cultivo , Fertilización In Vitro , Capacitación Espermática , Espermatozoides , Animales , Capacitación Espermática/efectos de los fármacos , Masculino , Ratones , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Espermatozoides/metabolismo , Fertilización In Vitro/métodos , Femenino , Reacción Acrosómica/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Fosforilación , Fertilización , Desarrollo Embrionario/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismoRESUMEN
OBJECTIVE: Oxidative stress is a mechanism of cadmium-induced reproductive dysfunction. Carpolobia lutea is a free radical scavenger. Our study investigated the potential protective effects of Carpolobia lutea root methanol extract against cadmium-induced reproductive toxicity. METHODS: We obtained the Carpolobia lutea root in Akure, and it was authenticated at the Forestry Research Institute of Nigeria (FRIN) herbarium, Ibadan, Nigeria, with FHI number 109784. We used Soxhlet extraction to obtain its methanol extract. We used thirty male Wistar rats (150-170g) in this study, (n=5 per group), and treated them as follows: Control (1 ml/kg normal saline), Cd (2 mg/kg), Cd+MCL (2 mg/kg+100 mg/kg), Cd+MCL (2 mg/kg+200 mg/kg), MCL (100 mg/kg), MCL (200 mg/kg). We administered Carpolobia lutea orally for 8 weeks. We administered a single dose of 2 mg/kg of cadmium intraperitoneally. We assessed the sperm profile using a computer-aided sperm analyzer. Under microscopy, we determined the sperm acrosome reaction and the DNA damage. We measured the seminal fructose level using spectrophotometry, and the data were analyzed using ANOVA at p<0.05. RESULTS: Cd+MCL (2mg/kg+200 mg/kg) significantly increased sperm count (339.0±25.0 vs. 29.0±4.5 million/mL), motility (80.0±0.2 vs. 55.0±4.9%), viability (68.7±2.7 vs. 31.3±2.9%) and decreased abnormal sperm (28.3±1.7 vs. 43.3±2.5%), relative to the cadmium group. Cd+MCL (2mg/kg+200 mg/kg) significantly increased acrosome reaction (68.0±7.5 vs. 15.2±2.4%) and seminal fructose level (0.49±0.06 vs. 0.28±0.06 mmol/L) relative to the cadmium group. Cd+MCL (2mg/kg+200 mg/kg) significantly decreased sperm DNA damage (14.1±1.6 vs. 35.9±5.3%) in relation to the cadmium group. CONCLUSIONS: Carpolobia lutea root extract improves the sperm variables of rats exposed to cadmium.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Antioxidantes/farmacología , Cadmio/toxicidad , Extractos Vegetales/farmacología , Espermatozoides/efectos de los fármacos , Animales , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Análisis de Semen , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Testículo/efectos de los fármacosRESUMEN
Exocytosis of spermatozoon's secretory vesicle, named acrosome reaction (AR), is a regulated event that plays a central role in fertilization. It is coupled to a complex calcium signaling. Ceramide is a multitasking lipid involved in exocytosis. Nevertheless, its effect on secretion is controversial and the underlying cellular and molecular mechanisms remain unknown. Human spermatozoa are useful to dissect the role of ceramide in secretion given that the gamete is not capable to undergo any trafficking mechanisms other than exocytosis. We report for the first time, the presence of sphingolipid metabolism enzymes such as neutral-sphingomyelinase and ceramide synthase in sperm. Ceramidases are also present and active. Both the addition of cell-permeable ceramide and the rise of the endogenous one, increase intracellular calcium acting as potent inducers of exocytosis. Ceramide triggers AR in capacitated spermatozoa and enhances the gamete response to progesterone. The lipid induces physiological ultrastructural changes in the acrosome and triggers an exocytosis-signaling cascade involving protein tyrosine phosphatase 1B and VAMP2. Real-time imaging showed an increment of calcium in the cytosol upon ceramide treatment either in the absence or in the presence of extracellular calcium. Pharmacological experiments demonstrate that at early stages the process involves ryanodine receptors, CatSper (calcium channel of sperm), and store-operated calcium channels. We set out the signaling sequence of events that connect ceramide to internal calcium mobilization and external calcium signals during secretion. These results allow the coordination of lipids and proteins in a pathway that accomplishes secretion. Our findings contribute to the understanding of ceramide's role in regulated exocytosis and fertilization.
Asunto(s)
Reacción Acrosómica/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Espermatozoides/efectos de los fármacos , Proteína 2 de Membrana Asociada a Vesículas/genética , Acrosoma/efectos de los fármacos , Acrosoma/metabolismo , Reacción Acrosómica/efectos de los fármacos , Adulto , Calcio/química , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Ceramidas/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/genética , Exocitosis/genética , Fertilización/genética , Humanos , Masculino , Canal Liberador de Calcio Receptor de Rianodina/genética , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/genética , Espermatozoides/patologíaRESUMEN
Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.
Asunto(s)
Calgranulina B/metabolismo , Epitelio/metabolismo , Oviductos/metabolismo , Capacitación Espermática , Interacciones Espermatozoide-Óvulo , Reacción Acrosómica/efectos de los fármacos , Adulto , Animales , Sitios de Unión , Epitelio/efectos de los fármacos , Femenino , Humanos , Masculino , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oviductos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/farmacología , Semen/efectos de los fármacos , Semen/metabolismo , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/metabolismoRESUMEN
Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.
Asunto(s)
Medios de Cultivo/farmacología , Fármacos para la Fertilidad Masculina/farmacología , Caballos , Análisis de Semen/veterinaria , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Aminopiridinas/farmacología , Animales , Cafeína/farmacología , Fertilización In Vitro/veterinaria , Fluoresceínas/farmacología , Masculino , Potencial de la Membrana Mitocondrial , Aglutinina de Mani/farmacología , Fosforilación , Procaína/farmacología , Semen/efectos de los fármacos , Análisis de Semen/métodos , Espermatozoides/efectos de los fármacosRESUMEN
O objetivo do trabalho foi avaliar o desempenho e a variabilidade de resultados das provas funcionais de integridade acrossômica (IA) e a reação acrossômica induzida (RAI) em touros jovens da raça Nelore, bem como verificar suas associações com outros parâmetros andrológicos. Foram avaliadas 16 amostras de sêmen congelado de touros jovens da raça Nelore utilizando-se esfregaços em lâminas pelo método de Azul Trypan / Giemsa. A média geral da integridade acrossômica pós-congelação foi de 85 ± 6%, com redução para 76 ± 8% após quatro horas de incubação em banho maria a 37º C com meio de TALP HEPES com albumina. Os testes de RAI tiveram diferenças acentuadas entre os touros do experimento, com média 18 ± 9 %, com números máximos e mínimos de 44% a 3%. Os resultados sugerem que ambos os indutores Heparina e Heparina + Lisofosfatidilcolina (LPC) foram eficientes e devem ser utilizados nas avaliações de RAI. Na comparação entre touros de alta a baixa RAI quanto a diversos parâmetros andrológicos, houve diferença (p<0,05) apenas para motilidade pré-congelação entre os grupos, com resultados superiores para os touros de baixa RAI (≤ 17%). Quanto as correlações dos parâmetros andrológicos e as provas funcionais da qualidade espermática (IA e RAI), somente a motilidade espermática esteve negativamente correlacionada a RAI (0,51, p<0,05). Concluiu-se que os testes de integridade acrossômica e reação acrossômica induzida são importantes testes complementares para avaliação da qualidade funcional dos espermatozoides, uma vez que apresentam alta variabilidade entre touros e baixa associação com outros parâmetros andrológicos. Entretanto, os mesmos devem ser interpretados dentro dos limites de avaliação dos processos associados a qualidade espermática, e não como um indicador amplo e único da fertilidade ou capacidade fecundante de touros.
The aim of this study was to evaluate the performance and variability of the results of functional tests for assessing acrosome integrity and heparin-induced acrosome reaction (IAR) in young Nellore bulls, as well as to verify their associations with other andrological parameters. Smears were prepared from 16 frozen semen samples of young Nellore bulls and stained with Trypan blue/Giemsa. The overall average post-freezing acrosome integrity was 85 ± 6%, with a reduction to 76 ± 8% after 4 hours of incubation and no large variation between bulls. Marked differences between bulls of the experiment were observed for the heparin IAR tests, with an average of 18 ± 9% (range 44% to 3%). The results suggest that both heparin and heparin + lysophosphatidylcholine were efficient inducers that should be used together in IAR evaluations. Comparison of the different andrological parameters between bulls with high and low IAR showed a difference (p<0.05) only for pre-freezing motility, with superior results for low IAR bulls (≤ 17%). There were no correlations between the andrological parameters and functional tests of sperm quality (acrosome integrity and IAR). Only pre-freezing sperm motility was negatively correlated with IAR (0.51, p<0.05). In conclusion, the acrosome integrity and IAR tests are important complementary tests to evaluate the functional quality of sperm since they exhibit high variability between bulls and low association with other andrological parameters. However, the tests should be interpreted within the limits of evaluation of the processes associated with sperm quality and not as a broad indicator of bull fertility or reproductive capacity.
Asunto(s)
Masculino , Animales , Bovinos , Acrosoma , Análisis de Semen/métodos , Análisis de Semen/veterinaria , Heparina/administración & dosificación , Reacción Acrosómica/efectos de los fármacosRESUMEN
O objetivo do trabalho foi avaliar o desempenho e a variabilidade de resultados das provas funcionais de integridade acrossômica (IA) e a reação acrossômica induzida (RAI) em touros jovens da raça Nelore, bem como verificar suas associações com outros parâmetros andrológicos. Foram avaliadas 16 amostras de sêmen congelado de touros jovens da raça Nelore utilizando-se esfregaços em lâminas pelo método de Azul Trypan / Giemsa. A média geral da integridade acrossômica pós-congelação foi de 85 ± 6%, com redução para 76 ± 8% após quatro horas de incubação em banho maria a 37º C com meio de TALP HEPES com albumina. Os testes de RAI tiveram diferenças acentuadas entre os touros do experimento, com média 18 ± 9 %, com números máximos e mínimos de 44% a 3%. Os resultados sugerem que ambos os indutores Heparina e Heparina + Lisofosfatidilcolina (LPC) foram eficientes e devem ser utilizados nas avaliações de RAI. Na comparação entre touros de alta a baixa RAI quanto a diversos parâmetros andrológicos, houve diferença (p<0,05) apenas para motilidade pré-congelação entre os grupos, com resultados superiores para os touros de baixa RAI (≤ 17%). Quanto as correlações dos parâmetros andrológicos e as provas funcionais da qualidade espermática (IA e RAI), somente a motilidade espermática esteve negativamente correlacionada a RAI (0,51, p<0,05). Concluiu-se que os testes de integridade acrossômica e reação acrossômica induzida são importantes testes complementares para avaliação da qualidade funcional dos espermatozoides, uma vez que apresentam alta variabilidade entre touros e baixa associação com outros parâmetros andrológicos. Entretanto, os mesmos devem ser interpretados dentro dos limites de avaliação dos processos associados a qualidade espermática, e não como um indicador amplo e único da fertilidade ou capacidade fecundante de touros.(AU)
The aim of this study was to evaluate the performance and variability of the results of functional tests for assessing acrosome integrity and heparin-induced acrosome reaction (IAR) in young Nellore bulls, as well as to verify their associations with other andrological parameters. Smears were prepared from 16 frozen semen samples of young Nellore bulls and stained with Trypan blue/Giemsa. The overall average post-freezing acrosome integrity was 85 ± 6%, with a reduction to 76 ± 8% after 4 hours of incubation and no large variation between bulls. Marked differences between bulls of the experiment were observed for the heparin IAR tests, with an average of 18 ± 9% (range 44% to 3%). The results suggest that both heparin and heparin + lysophosphatidylcholine were efficient inducers that should be used together in IAR evaluations. Comparison of the different andrological parameters between bulls with high and low IAR showed a difference (p<0.05) only for pre-freezing motility, with superior results for low IAR bulls (≤ 17%). There were no correlations between the andrological parameters and functional tests of sperm quality (acrosome integrity and IAR). Only pre-freezing sperm motility was negatively correlated with IAR (0.51, p<0.05). In conclusion, the acrosome integrity and IAR tests are important complementary tests to evaluate the functional quality of sperm since they exhibit high variability between bulls and low association with other andrological parameters. However, the tests should be interpreted within the limits of evaluation of the processes associated with sperm quality and not as a broad indicator of bull fertility or reproductive capacity.(AU)
Asunto(s)
Animales , Masculino , Bovinos , Análisis de Semen/métodos , Análisis de Semen/veterinaria , Acrosoma , Heparina/administración & dosificación , Reacción Acrosómica/efectos de los fármacosRESUMEN
When levonorgestrel (LNG) is given for emergency contraception during the follicular phase, it not only inhibits or delays ovulation, but also induces changes in endometrial secretions that modulate sperm functionality. In order to characterize the female reproductive tract secreted molecules that may affect human spermatozoa, we analyzed changes in the protein content of uterine flushings obtained from women during the periovulatory phase of a control and a LNG-treated menstrual cycle. Lectin affinity analysis and 2D gel electrophoresis of uterine samples showed changes in protein glycosylation patterns and the presence of 31 differentially expressed proteins (8 upregulated and 23 downregulated). Mass spectrometry and Western blot analyses of the differential expressed proteins showed lactotransferrin (LTF) as one of the upregulated molecules by LNG. In this study, LTF exhibited significant dose-related effects on sperm functionality, particularly a decrease of calcium ionophore-induced acrosome reaction and protein tyrosine phosphorylation. Overall, the results indicated that LNG promoted changes in the proteome of uterine secretions that might compromise human sperm capacitation. These data further support the participation of other mechanisms of action of LNG as emergency contraceptive, in addition to those on ovulation.
Asunto(s)
Agentes Anticonceptivos Hormonales/uso terapéutico , Fase Folicular/efectos de los fármacos , Lactoferrina/metabolismo , Lactoferrina/farmacología , Levonorgestrel/uso terapéutico , Espermatozoides/efectos de los fármacos , Útero/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Adulto , Ionóforos de Calcio/farmacología , Femenino , Fase Folicular/metabolismo , Glicosilación , Humanos , Masculino , Ovulación/efectos de los fármacos , Fosforilación , Espermatozoides/metabolismo , Tirosina/metabolismo , Útero/metabolismo , Adulto JovenRESUMEN
Male chronic alcohol abuse causes testicular failure and infertility. We analyzed the effects of moderate sub-chronic alcohol intake on sperm morphology, capacitation, fertilization and sperm head decondensation. CF-1 male mice were administered 15% ethanol in drinking water for 15 days; control mice received ethanol-free water. Similar patterns of tyrosine phosphorylation were observed in capacitated spermatozoa of control and treated males. Percentage of hyperactivation (H) and spontaneous (SAR) and progesterone-induced (IAR) acrosome reaction significantly decreased at 120 and 150 min of capacitation in treated males compared to controls (H: 14.1 ± 2.5 vs 23.7 ± 2.6, P < 0.05; SAR-T120 min: 17.9 ± 2.5 vs 32.9 ± 4.1, P < 0.01; IAR-150 min: 43.3 ± 3.5 vs 73.1 ± 1.1, P < 0.001, n = 6). During in vitro fertilization (2.5, 3.5 and 4.5 h post-insemination), there was an increased percentage of fertilized oocytes (with a decondensed sperm head and one or two pronuclei) in treated males (P < 0.001, n = 7). After 60 min of in vitro decondensation with glutathione plus heparin, the percentage of decondensed sperm heads was significantly higher in treated males than in controls (mean ± s.d.: 57.1 ± 5.6 vs 48.3 ± 4.5, P < 0.05, n = 5). The percentage of morphologically normal sperm heads was significantly decreased in treated males with respect to controls (P < 0.001, n = 9). These results show that short-term moderate alcohol consumption in outbred mice affect sperm morphology, hyperactivation, acrosomal exocytosis, and the dynamics of in vitro fertilization and in vitro sperm nuclear decondensation.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Etanol/administración & dosificación , Oocitos/efectos de los fármacos , Capacitación Espermática/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Animales , Antiinfecciosos Locales/administración & dosificación , Fertilización In Vitro , Masculino , Ratones , Oocitos/patologíaRESUMEN
The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm-zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Camélidos del Nuevo Mundo/fisiología , Motilidad Espermática/efectos de los fármacos , Zona Pelúcida/efectos de los fármacos , Animales , Bovinos , Medios de Cultivo , Femenino , Sangre Fetal , Masculino , Suero , Capacitación Espermática/efectos de los fármacos , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , EspermatozoidesRESUMEN
Ca2+ -activated Cl- channels (CaCCs) are anionic channels that regulate many important physiological functions associated with chloride and calcium flux in some somatic cells. The molecular identity of CaCCs was revealed to be TMEM16A and TMEM16B (also known as Anoctamin or ANO1 and ANO2, respectively) in all eukaryotes. A recent study suggests the presence of TMEM16A in human sperm and a relationship with the rhZP-induced acrosome reaction. However, to the best of our knowledge, little is known about the role of TMEM16A in other spermatic processes such as capacitation or motility. In this study, we evaluated the effects of two TMEM16A antagonists on capacitation, acrosome reaction, and motility in guinea pig sperm; these antagonists were T16Ainh-A01, belonging to a second generation of potent antagonists of TMEM16A, and niflumic acid (NFA), a well-known antagonist of TMEM16A (CaCCs). First of all, we confirmed that the absence of Cl- in the capacitation medium changes motility parameters, capacitation, and the progesterone-induced acrosome reaction. Using a specific antibody, TMEM16A was found as a protein band of â¼120 kDa, which localization was in the apical crest of the acrosome and the middle piece of the flagellum. Inhibition of TMEM16A by T16Ainh-A01 affected sperm physiology by reducing capacitation, blocking the progesterone-induced acrosome reaction under optimal capacitation conditions, inhibiting progressive motility, and the acquisition of hyperactivated motility, diminishing [Ca2+ ]i, and increasing [Cl- ]i. These changes in sperm kinematic parameters provide new evidence of the important role played by TMEM16A in the production of sperm capable of fertilizing oocytes.
Asunto(s)
Anoctamina-1/antagonistas & inhibidores , Pirimidinas/farmacología , Capacitación Espermática/efectos de los fármacos , Motilidad Espermática/efectos de los fármacos , Tiazoles/farmacología , Reacción Acrosómica/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/farmacología , Calcio/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Cloruros/metabolismo , Cobayas , Masculino , Ácido Niflúmico/farmacologíaRESUMEN
During transit through the female reproductive tract, sperm encounter metabolites and environmental conditions that modulate various processes leading to fertilization. Intracellular Ca2+ dynamics regulate the acrosome reaction (AR), which involves exocytosis of the acrosomal granule, a prerequisite for successful fertilization. We explored the ability of progesterone, prostanglandin-E1, and GABA to induce Ca2+ mobilization and AR in single human spermatozoa capacitated under external pH (pHe) conditions found in different regions of the female reproductive tract (pHe 6.5, 7.4 and 8.0). The highest percentage of AR induction, regardless of the inducer, occurred when sperm were capacitated at pHe 7.4. Interestingly, at pHe 6.5 a high percentage of cells exhibit Ca2+ oscillations, which prevent AR. These oscillations involve extracellular and intracellular Ca2+ channels. Pharmacological inhibition of Ca2+ oscillations restores the ability of spermatozoa to undergo the AR when exposed to progesterone, even if capacitated at pHe 6.5.
Asunto(s)
Reacción Acrosómica/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Concentración de Iones de Hidrógeno , Progesterona/administración & dosificación , Espermatozoides/química , Espermatozoides/fisiología , Reacción Acrosómica/efectos de los fármacos , Células Cultivadas , Humanos , Masculino , Espermatozoides/efectos de los fármacosRESUMEN
Epididymal sperm protein CRISP1 has the ability to both regulate murine CatSper, a key sperm calcium channel, and interact with egg-binding sites during fertilization. In spite of its relevance for sperm function, Crisp1-/-mice are fertile. Considering that phenotypes can be influenced by the genetic background, in the present work mice from the original mixed Crisp1-/- colony (129/SvEv*C57BL/6) were backcrossed onto the C57BL/6 strain for subsequent analysis of their reproductive phenotype. Whereas fertility and fertilization rates of C57BL/6 Crisp1-/- males did not differ from those reported for mice from the mixed background, several sperm functional parameters were clearly affected by the genetic background. Crisp1-/- sperm from the homogeneous background exhibited defects in both the progesterone-induced acrosome reaction and motility not observed in the mixed background, and normal rather than reduced protein tyrosine phosphorylation. Additional studies revealed a significant decrease in sperm hyperactivation as well as in cAMP and protein kinase A (PKA) substrate phosphorylation levels in sperm from both colonies. The finding that exposure of mutant sperm to a cAMP analog and phosphodiesterase inhibitor overcame the sperm functional defects observed in each colony indicated that a common cAMP-PKA signaling defect led to different phenotypes depending on the genetic background. Altogether, our observations indicate that the phenotype of CRISP1 null males is modulated by the genetic context and reveal new roles for the protein in both the functional events and signaling pathways associated to capacitation.
Asunto(s)
Fertilidad/genética , Fertilización/genética , Glicoproteínas de Membrana/genética , Reproducción/genética , Espermatozoides/metabolismo , Reacción Acrosómica/efectos de los fármacos , Reacción Acrosómica/genética , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Antecedentes Genéticos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Progesterona/farmacología , Motilidad Espermática/genética , Espermatozoides/efectos de los fármacosRESUMEN
STUDY QUESTION: Are human spermatozoa able of chemorepulsive behaviour? SUMMARY ANSWER: Capacitated human spermatozoa are able to be chemorepelled by synthetic Progesterone Receptor Ligands (sPRL, known as contraceptives) and zinc (a cation released by the oocyte upon fertilization). WHAT IS KNOWN ALREADY: Moving cells can be oriented towards or against a molecular gradient, processes called chemoattraction and chemorepulsion, respectively, which have been described in unicellular organisms such as amoebas and bacteria, to organismic cells such macrophages and developmental cells. In the case of spermatozoa, chemoattraction may help the finding of an oocyte and has been widely studied in various invertebrate and mammalian species; however, chemorepulsion has not yet been verified in spermatozoa. STUDY DESIGN, SIZE, DURATION: This is an in vitro study involving human, rabbit and mouse spermatozoa which were used to perform 3-30 experiments per treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were obtained by masturbation from healthy donors who gave written consent. Only those samples exhibiting normal semen parameters according to current WHO criteria were included in the study. Rabbit spermatozoa were obtained by artificial vagina whereas mice spermatozoa were obtained from epididymis. The sperm selection assay (SSA), originally designed to evaluate sperm chemoattraction towards progesterone (P), and a video-microscopy and computer motion analysis system were used to test sperm chemorepulsion. Additional kinetic parameters were also determined by video-microscopy and computer motion analysis. In some experiments, the level of induced acrosome-reacted spermatozoa was determined. Rabbit mating manipulation was achieved to perform the sperm-oocyte co-incubation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Sperm accumulation in the well containing 100 pg/ml of sPRL was lower than the culture medium negative control (P < 0.05). The percentage of sperm persistence against the well containing 100 pg/ml ulipristal acetate (UPA) (P = 0.001), and the percentage of sperm showing a repulsive pattern of movement (a linear trajectory followed by a transitional one after turning against the UPA), were higher than the culture medium negative control (P = 0.049). Sperm accumulation was diminished when spermatozoa where exposed to a homogeneous distribution of 100 pg/ml sPRL combined with a chemotactic gradient of progesterone (P), with respect to the culture medium negative control (P < 0.05). These results were reverted when non-capacitated spermatozoa were used to perform the same experimental settings. The accumulation of spermatozoa against 100 pg/ml sPRL was lower than the culture medium negative control also in rabbits and mice (P < 0.05). The relative number of rabbit spermatozoa arriving to the vicinity of the oocyte was diminished under the presence of 100 pg/ml UPA (P = 0.004). Sperm accumulation in the well containing zinc was decreased compared to the culture medium negative control (P < 0.05). A homogeneous distribution of zinc combined with a gradient of 10 pM P, was lower than the culture medium negative control (P = 0.016). The results were quite reproducible with two different methodologies (accumulation assay and video-microscopy combined with computer motion analysis), in three mammalian species. LIMITATIONS REASONS FOR CAUTION: The experiments were performed in vitro. Even though a quite complete characterization of sperm chemorepulsion was provided, the molecular mechanism that governs sperm repulsion is currently under investigation. WIDER IMPLICATIONS OF THE FINDINGS: Since the chemorepelled spermatozoa are those physiologically ready to fertilize the oocyte, these findings may have both biological and clinical implications, preventing either polyspermy under natural conditions or fertilization under pharmacological treatment with sPRL. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by the Universidad Nacional de Cordoba (Argentina). The authors declare that they do not have competing financial interests. TRIAL REGISTRATION NUMBER: N/A.
Asunto(s)
Fertilización/efectos de los fármacos , Progesterona/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Zinc/farmacología , Reacción Acrosómica/efectos de los fármacos , Reacción Acrosómica/fisiología , Animales , Fertilización/fisiología , Humanos , Masculino , Ratones , Conejos , Capacitación Espermática/efectos de los fármacos , Capacitación Espermática/fisiología , Motilidad Espermática/fisiología , Espermatozoides/fisiologíaRESUMEN
Levonorgestrel (LNG), a synthetic 19 nor-testosterone derivative, is widely used for emergency contraception. It is well known that LNG prevents ovulation only when given prior to the surge of serum luteinizing hormone (LH) during the periovulatory phase of the menstrual cycle. This observation suggests that LNG, given its contraceptive efficacy, has additional effects other than those affecting ovulation. In this study, we have evaluated the effects on human sperm functionality of uterine flushings (UF) obtained from women at day LH + 1 of a control cycle (CTR-LH + 1) and after receiving LNG (LNG-LH + 1) two days before the surge of LH. Human sperm from normozoospermic donors were incubated with UF and protein tyrosine phosphorylation, sperm motility, acrosome reaction as well as zona pellucida (ZP) binding capacity were assessed. A significant decrease in total motility and tyrosine phosphorylation accompanied by an increase on spontaneous acrosome reaction was observed when sperm were incubated in the presence of LNG-LH + 1. None of these effects were mimicked by purified glycodelin A (GdA). Moreover, the addition of UF obtained during the periovulatory phase from LNG-treated women or the presence of purified GdA significantly decreased sperm-ZP binding. The data were compatible with changes affecting sperm capacitation, motility and interaction with the ZP. These results may offer evidence on additional mechanisms of action of LNG as an emergency contraceptive.
Asunto(s)
Líquidos Corporales , Anticonceptivos Femeninos/uso terapéutico , Levonorgestrel/uso terapéutico , Espermatozoides/efectos de los fármacos , Irrigación Terapéutica , Útero/patología , Reacción Acrosómica/efectos de los fármacos , Adulto , Líquidos Corporales/efectos de los fármacos , Líquidos Corporales/fisiología , Anticonceptivos Femeninos/farmacología , Femenino , Humanos , Técnicas In Vitro , Levonorgestrel/farmacología , Masculino , Capacitación Espermática/efectos de los fármacos , Espermatozoides/fisiologíaRESUMEN
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.
Asunto(s)
Reacción Acrosómica/efectos de los fármacos , Isocitrato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/metabolismo , Fosfofructoquinasas/metabolismo , Capacitación Espermática/efectos de los fármacos , Espermatozoides/enzimología , Animales , Bicarbonatos/farmacología , Femenino , Líquido Folicular/fisiología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Malato Deshidrogenasa/antagonistas & inhibidores , Masculino , Fosfofructoquinasas/antagonistas & inhibidores , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Sus scrofa , Tartronatos/farmacologíaRESUMEN
Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm, and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work, we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3- -entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility, and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH, and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. J. Cell. Physiol. 232: 1404-1414, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Álcalis/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Potenciales de la Membrana , Capacitación Espermática , Reacción Acrosómica/efectos de los fármacos , Benzoatos/metabolismo , Movimiento Celular/efectos de los fármacos , Cloruros/metabolismo , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Humanos , Concentración de Iones de Hidrógeno , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Isoquinolinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Capacitación Espermática/efectos de los fármacos , Sulfonamidas/farmacología , Tiazolidinas/metabolismoRESUMEN
ããBACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. OBJECTIVE: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. MATERIALS AND METHODS: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. RESULTS: The statistical analysis did not reveal any significant modification of seminal parameters. CONCLUSION: Within the concentrations tested, quercetin supplementation in equine freezing extender did not affect progressive motility, mitochondrial functionality, acrosome reaction, membrane integrity or DNA fragmentation index in post-thaw equine semen.