Alexa Fluor 488-phalloidin, M199 medium, gentamicin and fungizone were purchased from Invitrogen (CA, USA)

Alexa Fluor 488-phalloidin, M199 medium, gentamicin and fungizone were purchased from Invitrogen (CA, USA). F-actin levels while no F-actin was recognized with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the rules of actin polymerization and motility. agglutinin-FITC (PSA-FITC), l–lysophosphatidylcholine (LPC) and bovine serum albumin (BSA) were acquired from Sigma-Aldrich (MO, USA). KT5720 was purchased from Tocris Bioscience (Bristol, UK). Latrunculin B (Lat B) was acquired from Cayman Chemical (MI, USA). Monoclonal antibody anti-MRP4 and anti-rabbit IgG coupled to Alexa Fluor 555 were from Cell Signaling Technology (MA, USA) and Abcam (Cambridge, UK) respectively. Alexa Imidazoleacetic acid Fluor 488-phalloidin, M199 medium, gentamicin and fungizone were purchased from Invitrogen (CA, USA). All other chemicals were of analytical grade and from standard sources. Sperm preparation Straws of freezing bovine semen (20C25??106 spermatozoa/ml) were kindly provided by Centro de Reproduccin Bovina San Antonio de Areco (CRB), Centro de Inseminacin Artificial La Elisa (CIALE) and Cooperativa de Inseminacin Artificial Venado Tuerto (CIAVT). Straws were thawed for 30?s inside a water bath at 38.5?C. Sperm were HDAC5 selected from the wool glass column method as previously explained59, and washed by centrifugation in BSA-free Tyrodes Albumin Lactate Pyruvate (sp-TALP). Finally, pellets were resuspended in BSA-free sp-TALP and assessed for motility and sperm concentration using a hemocytometer mounted on a microscope stage heated at 38.5?C. In vitro sperm capacitation Ten??106 spermatozoa/ml were incubated in non-capacitating (NC) medium (sp-TALP: 99?mM NaCl; 3.1?mM KCl; 0.4?mM Imidazoleacetic acid NaH2PO4; 0.4?mM MgCl2.6H2O; 21.6?mM sodium lactate; 10?mM HEPES; 2?mM CaCl2.H2O; 25?mM NaHCO3; 1?mM sodium piruvate; 50?mg/ml gentamycin; pH 7.37)60 or capacitating (CAP) medium (0.3% BSA and 40?mM NaHCO3 sp-TALP) at 38.5?C and 5% CO2 atmosphere for 15 or 45 min61. This CAP medium offers previously shown to be adequate to accomplish capacitation and cAMP extrusion13. In some experiments, cells were co-incubated with cAMP (10?nM), an MRP4 inhibitor (50?M MK571), PKA inhibitors (50?M H89; 100?nM KT5720 or 1?mM Rp cAMPS) or an F-actin assembly inhibitor (10?M Latrunculin B). The same cAMP and MK571 concentrations were used as with earlier works from our group13,15. Viability assay Spermatozoa were incubated in the presence or absence of MK571 (50?M) for 45?min. Then, samples were incubated with Hoechst 33,258 (2?g/ml, 5?min). Spermatozoa were fixed and examined having a Nikon Eclipse E200 (Tokyo, Japan) fluorescence microscope (magnification 1,000?) coupled to a DS-Fi1 Nikon photographic video camera. Live sperm were identified as those without a bright and homogeneous transmission in its head. At least 200 spermatozoa per condition were evaluated. Assessment of sperm capacitation Capacitation was assessed by different techniques: l–lysophosphatidylcholine (LPC)-induced acrosome reaction/agglutinin (PSA)-FITC staining, chlortetracycline (CTC) assay and evaluation of sperm launch from bovine oviductal epithelial cells (BOEC). The induction of the acrosome reaction was performed as previously explained13. Spermatozoa were incubated in NC or CAP conditions in the presence or absence of MK571 (50?M). After 45?min, samples were incubated or not for 15?min with LPC (5?M) to induce acrosomal reaction. Cell viability was assessed with Hoechst 33,258 (2?g/ml, 5?min incubation). Spermatozoa were fixed, permeabilized and stained with PSA-FITC in order to evaluate acrosomal reaction. At least 200 cells per condition were examined having a Nikon Eclipse E200 (Tokyo, Japan) fluorescence microscope (magnification 1,000?). Capacitation was estimated as the difference between the quantity of live and reacted spermatozoa in the presence of LPC and the number of live sperm spontaneously reacted. The CTC assay Imidazoleacetic acid was performed as previously detailed59. In a similar way, after 45?min spermatozoa were incubated with CTC (500?M) and examined having a fluorescence microscope. The percentage of cells having a capacitated pattern (also known as B pattern) was quantified62. Bovine oviductal epithelia cell cultures and sperm co-cultures As sperm plasma membranes are remodel during capacitation, spermatozoa detach from your oviductal epithelium. Consequently, we assessed sperm capacitation from the evaluation of sperm launch from oviductal cells in BOEC-sperm co-cultures treated with different conditions. Bovine oviducts were kindly Imidazoleacetic acid donated from Compa?a de Carniceros Sociedad Annima (COCARSA) slaughterhouse (Buenos Aires, Argentina). Oviductal epithelia cell cultures were prepared as explained previously63. Briefly, oviducts were collected, transferred at 4?C, cleaned of surrounding cells and washed three times in sterile PBS at 4?C. Then, oviducts were isolated, flushed with sterile PBS and.