Membrane potential hyperpolarization induces cytoplasmic alkalinization of mouse sperm

To gain fertilization ability sperm must undergo a series of physiological modification inthe female tract called capacitation, which leads to the acquisition of hyperactivatedmotility (HA) and the ability to undergo acrosome reaction (AR). Membrane potential(Em) hyperpolarization and alkalinization of the intracellular pH are necessary for HAand AR to take place. It has been reported either pharmacologically or genetically(potassium channel Slo3 knockout mice) that hyperpolarization is necessary andsufficient for sperm acrosomal responsiveness. In addition, intracellular alkalinizationallows the opening of CatSper channels which lead to an increase in intracellular calciumthat is fundamental for the HA of sperm. The aim of this work was to elucidate theinterplay between Em and pH in mouse sperm. We used BCECF-AM and DISC3(5), probesthat measures changes in cytoplasmic pH and Em, respectively. First, we studied thedependence of pH and Em, by co-incubating sperm with both probes followed by FlowCytometry analysis. We observed that all the cells were grouped into two populations:1) low pH and depolarized Em or 2) high pH and hyperpolarized Em. We also determinedthat sperm incubated under non-capacitating conditions with the addition of 1 μMvalinomycin displayed a robust increase in pH suggesting that changes in Em arenecessary for the rise in pH observed during capacitation. This increase was abrogatedin the presence of high K+ in the extracellular space. In addition, we studied the kineticsof this changes by assessing Em and pH at different time points of capacitation (0 to 60min). Our results showed that there is a rapid pH alkalinization during the first 15 min ofincubation in capacitating medium that is followed by a transient acidification at 30 min.Alkalinization is then restored at 45 min and remain unchanged. Thus, our resultssuggested that changes in the Em induces modifications in intracellular pH during mousesperm capacitation.Fil: 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: Jabloñski, Martina. 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: Gilio, Nicolas. 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: 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: Krapf, Dari. 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: Mariano Gabriel Buffone. 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; ArgentinaLXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; IX Reunión Anual de la Asociación Argentina de Nanomedicinas y VI Reunión Científica Regional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de Ciencia y Tecnología de Animales de LaboratorioAsociación Argentina de Nanomedicina

Restoration of antitumor immunity through anti-MICA antibodies elicited with a chimeric protein

Background Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity.Methods We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect.Results Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment.Conclusions Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors

Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation

Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca2+), and bicarbonate (HCO3 −). In this work, sperm were double stained with propidium iodide and the Ca2+ dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca2+ concentration ([Ca2+]i) in individual live sperm. An increase in [Ca2+]i was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca2+]i within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca2+]i after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca2+ and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca2+]i. In addition, it was determined that the capacitation-associated [Ca2+]i increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca2+]i. Surprisingly, a minimum increase in [Ca2+]i was also observed in CatSper KO sperm suggesting the existence of other Ca2+ transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+]i very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca2+.Fil: 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: D'alotto Moreno, Tomas. 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: Martín Hidalgo, David. University of Massachussets; Estados UnidosFil: Ritagliati, Carla. 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: 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: 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: 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: Schiavi Ehrehaus, Liza. 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: Gilio, Nicolas. 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: Visconti, Pablo E.. University of Massachussets; Estados UnidosFil: 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