[Ca2+]_i oscillations in human sperm are triggered in the flagellum by membrane potential- sensitive activity of CatSper

Study question: How are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated?Summary answer: P4-induced [Ca2+]i oscillations are generated in the flagellum by membrane-potential (Vm)-dependent Ca2+-influx through CatSper channels, which then induce secondary Ca2+ mobilisation at the sperm head/neck region.What is known already: A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Though pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required.Study design, size, duration: This was a laboratory study, that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1,000 individual cells over a period of 2 years.Participants/materials, setting, methods: Semen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp.Main results and the role of chance: P4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1-2 s). K+-ionophore valinomycin (1 μM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly ‘reset’ upon manipulation of [K+]o. Pretreatment of sperm with valinomycin ([K+]o=5.4 mM) had no effect on the P4-induced [Ca2+] transient (P=0.95; 8 experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n=257; P=5*10-55 compared to control) and significantly reduced both amplitude and frequency of persisting oscillations (p=0.0001). Upon valinomycin washout oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+] the inhibitory effect of valinomycin was reduced and was dependent on EK (P=10-25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P<0.01). The CatSper inhibitor RU1968 (4.8 and 11 μM) caused immediate and reversible arrest of activity in36% and 96% of oscillating cells respectively (P<10-10). 300 μM quinidine which blocks the sperm K+ current (Ksper) completely inhibited [Ca2+]i oscillations.Large scale data: n/aLimitations, reasons for caution: This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm.Wider implications of the findings: [Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the generation (or failure) and regulation of these oscillations.Study funding/competing interest(s): ET was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). The authors have no conflicts of interest

Effects of semen processing on sperm function: Differences between swim-up and density gradient centrifugation

Purpose: Andrology research has evolved notoriously in the latest years, particularly since male factor contribution to couple infertility has been undoubtedly demonstrated. However, sperm function investigations results are sometimes contradictory, probably as a result of the use of different sperm processing techniques. In this work, we underwent a systematic functional comparison of human sperm samples simultaneously processed by swim-up and density gradient centrifugation, which are the preferred sperm processing methods used in basic and clinical laboratories. Materials and Methods: To compare functional characteristics of sperm isolated by swim-up and density gradient centrifugation followed by incubation at different times under capacitating conditions. Results: Semen samples processed in parallel by these two procedures resulted in sperm preparations with significant differences in redox state, spontaneous intracellular calcium oscillations, hyperactivation, protein tyrosine phosphorylation, and acrosome reaction responsivity to calcium ionophore. Such differences showed time-dependent specific patterns for spontaneous intracellular calcium oscillations, hyperactivation and protein tyrosine phosphorylation. Sperm retrieved by density gradient centrifugation showed more hyperactivation and tyrosine phosphorylation than swim-up sperm, suggesting a higher degree of capacitation. Conclusions: Our results account for functional differences observed in spermatozoa processed with these two methods and therefore may contribute to a better interpretation of outcomes obtained in different laboratories as well as to improve experimental designs aimed to study sperm physiology and fertility potential.Fil: Hernández Silva, Gabriela. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; MéxicoFil: López Torres, Aideé S.. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; MéxicoFil: Maldonado Rosas, Israel. Centro de Innovación Tecnológica y Medicina Reproductiva; MéxicoFil: Mata Martínez, Esperanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Larrea, Fernando. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; MéxicoFil: Torres Flores, Víctor. Universidad Nacional Autónoma de México; MéxicoFil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; MéxicoFil: Chirinos, Mayel. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; Méxic

TRPM8, a Versatile Channel in Human Sperm

BACKGROUND:The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca(2+) dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+) ([Ca(2+)]i). Ca(2+) triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. PRINCIPAL FINDINGS:Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM) and 80% by BCTC (1.6 microM). Activation of TRPM8 either by temperature or menthol induced [Ca(2+)]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM) and BCTC (1.6 microM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. CONCLUSIONS:This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis

Expression and differential cell distribution of low-threshold Ca2+ channels in mammalian male germ cells and sperm

AbstractNumerous sperm functions including the acrosome reaction (AR) are associated with Ca2+ influx through voltage-gated Ca2+ (CaV) channels. Although the electrophysiological characterization of Ca2+ currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (CaV3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of CaV3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three CaV3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only CaV3.1 and CaV3.2 were detected in the head, suggesting its participation in the AR. CaV3.1 and CaV3.3 were found in the principal and the midpiece of the flagella. All CaV3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of CaV3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit CaV3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) CaV channels caused small but significant motility alterations. Antibodies to HVA channels detected CaV1.3 and CaV2.3 in human sperm flagella

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3-dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3 transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na/HCO3 cotransporter (NBC) and epithelial Na channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3 influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3 also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.Fil: Puga Molina, Lis del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pinto, Nicolás Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Torres, Nicolás I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: González Cota, Ana, L.. University of Washington; Estados UnidosFil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Balestrini, Paula Ania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Romarowski, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Santi, Celia M.. University of Washington; Estados UnidosFil: Treviño, Claudia L.. Universidad Nacional Autónoma de México; MéxicoFil: Darszon, Alberto. Universidad Nacional Autónoma de México; MéxicoFil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Prevalence and trends of markers of hepatitis B virus, hepatitis C virus and human Immunodeficiency virus in Argentine blood donors

BACKGROUND: Transfusion-transmitted infections are a major problem associated with blood transfusion. The aim of this study was to determine prevalence and trends of HBV, HCV and HIV in blood donors in Argentina. METHODS: A retrospective study was carried out in blood donors of 27 transfusion centers covering the whole country over a period of eight years (2004-2011). Serologic screening assays for HBsAg, anti-HBc, anti-HCV, and anti-HIV were performed in all centers and nucleic acid amplification testing (NAT) was performed in 2 out of the 27 centers. RESULTS: The 2,595,852 samples tested nationwide from 2004 to 2011 showed that the prevalence of HBsAg decreased from 0.336% to 0.198% (p < 0.0001), that of anti-HBc from 2.391% to 2.007% (p < 0.0001), that of anti-HCV from 0.721% to 0.460%, (p < 0.0001) and that of anti-HIV from 0.208% to 0.200 (p = 0.075). The prevalence of HBV, HCV and HIV was unevenly distributed among the different regions of the country. Two out of 74,838 screening- negative samples were positive in NAT assays (1 HIV-RNA and 1 HCV-RNA); moreover, HBV-DNA, HCV-RNA and HIV-RNA were detected in 60.29, 24.54 and 66.67% of screening-positive samples of the corresponding assays. As regards donors age, positive HBV-DNA and HCV-RNA donors were significantly older than healthy donors (46.6, 50.5 and 39.5 y respectively, p < 0.001). CONCLUSIONS: Argentina has a low prevalence of HBsAg, anti-HCV and anti-HIV in blood donors, with a decreasing trend for HBsAg, anti-HBc and anti-HCV but not for anti-HIV over the last 8 years. The uneven distribution of transfusion-transmitted infections prevalence among the different regions of the country highlights the need to implement regional awareness campaigns and prevention. The discrepancy between samples testing positive for screening assays and negative for NAT assays highlights the problem of blood donors who test repeatedly reactive in screening assays but are not confirmed as positive upon further testing. The uneven distribution of age between healthy donors and NAT-positive donors could be related to changes in risks of these pathogens in the general population and might be attributed to a longer exposure to transmission risk factors in elderly people.Fil: Flichman, Diego Martin. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blejer, Jorgelina L.. Fundación Hemocentro; ArgentinaFil: Livellara, Beatriz I.. Hospital Italiano; ArgentinaFil: Ré, Viviana Elizabeth. Universidad Nacional de Cordoba. Facultad de Medicina. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bartoli, Sonia. Centro regional de Hemoterapia Jujuy; ArgentinaFil: Bustos, Juan A.. Banco de sangre San Jorge; ArgentinaFil: Ansola, Claudia P.. Provincia de Mendoza. Servicio de Hemoterapia; ArgentinaFil: Hidalgo, Susana. Hospital Dr. Enrique Vera Barros; ArgentinaFil: Cerda, Martín E.. Hospital Dr. Lucio Molas; ArgentinaFil: Levin, Alicia E.. Provincia de Mendoza. Servicio de Hemoterapia; ArgentinaFil: Huenul, Adriana. Hospital Artémides Zatti; ArgentinaFil: Riboldi, Victoria. Hospital Regional Río Gallegos; ArgentinaFil: Treviño, Elena M. C.. Universidad Nacional de Córdoba; ArgentinaFil: Salamone, Horacio J.. Fundación Favaloro; ArgentinaFil: Nuñez, Felix A.. Hospital Italiano; ArgentinaFil: Fernández, Robert J.. Fundación Hemocentro; ArgentinaFil: Reybaud, Juan F.. Fundación Favaloro; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Anopheles darlingi polytene chromosomes: revised maps including newly described inversions and evidence for population structure in Manaus

Salivary gland polytene chromosomes of 4th instar Anopheles darlingi Root were examined from multiple locations in the Brazilian Amazon. Minor modifications were made to existing polytene photomaps. These included changes to the breakpoint positions of several previously described paracentric inversions and descriptions of four new paracentric inversions, two on the right arm of chromosome 3 and two on the left arm of chromosome 3 that were found in multiple locations. A total of 18 inversions on the X (n = 1) chromosome, chromosome 2 (n = 7) and 3 (n = 11) were scored for 83 individuals from Manaus, Macapá and Porto Velho municipalities. The frequency of 2Ra inversion karyotypes in Manaus shows significant deficiency of heterozygotes (p < 0.0009). No significant linkage disequilibrium was found between inversions on chromosome 2 and 3. We hypothesize that at least two sympatric subpopulations exist within the An. darlingi population at Manaus based on inversion frequencies

¿Es igual el comportamiento de los espermatozoides de humano que de ratón?

Publicación trimestral, Enero, Febrero, Marzo. Año 2016, N.4. Páginas 5 y 6.El metabolismo del espermatozoide es muy particular y distinto al de las otras células del cuerpo, lo que lo convierte en una célula única y al mismo tiempo fascinante ya que contiene los elementos celulares mínimos, pero suficientes que le permiten moverse, desplazarse y sobrevivir durante varias horas dentro del tracto genital femenino, antes de llegar al óvulo.En este proceso participan muchas proteínas y nosotros nos hemos interesado en un tipo particular llamado anhidrasas carbónicas. Las anhidrasas carbónicas (ACs) son enzimas (proteínas que llevan a cabo reacciones químicas a gran velocidad) que están presentes en todos los seres vivos y catalizan una reacción muy importante para la supervivencia de las células. A pesar de la gran importancia de las anhidrasas carbónicas en la fisiología de todas las células, hasta ahora son escasos los estudios en los que se ha analizado su presencia y/o su función en los espermatozoides de mamífero. Por lo tanto, en este trabajo nos dimos a la tarea de investigar estas interrogantes en los espermatozoides de humano y de ratón. Comparamos la fisiología de los espermatozoides de ratón y de humano, ya que, aunque el ratón ha sido hasta ahora un modelo experimental de mamífero que ha permitido obtener información muy valiosa. Los resultados de este trabajo demostraron que las enzimas anhidrasas carbónicas juegan un papel muy importante en el funcionamiento de los espermatozoides. Adicionalmente, este trabajo establece los antecedentes para enfocar nuestras investigaciones futuras al análisis detallado de las anhidrasas carbónicas, como por ejemplo, su interacción con otras proteínas que son importantes en la vida del espermatozoide