fdfd09e8bb9331f62653226ff3172de8 biology_a2021v10n11p1105.pdf 31b6d673d487280a662e668e0a6726cd4c5cafdb biology_a2021v10n11p1105.pdf 3f9f12f35bf86f3d7a3856f011a4de6c0a7c08cd3c8f14a1167778f7af2a812c biology_a2021v10n11p1105.pdf Title: Exogenous Albumin Is Crucial for Pig Sperm to Elicit In Vitro Capacitation Whereas Bicarbonate Only Modulates Its Efficiency Subject: This work sought to address whether the presence of exogenous bicarbonate is required for pig sperm to elicit in vitro capacitation and further progesterone-induced acrosome exocytosis. For this purpose, sperm were either incubated in a standard in vitro capacitation medium or a similar medium with different concentrations of bicarbonate (either 0 mM, 5 mM, 15 mM or 38 mM) and BSA (either 0 mg/mL or 5 mg/mL). The achievement of in vitro capacitation and progesterone-induced acrosomal exocytosis was tested through the analysis of sperm motility, plasma membrane integrity and lipid disorder, acrosome exocytosis, intracellular calcium levels, mitochondria membrane potential, O2 consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3) and protein kinase A (PKA). While sperm incubated in media without BSA or BSA/bicarbonate, they did not achieve in vitro capacitation; those incubated in media with BSA achieved the capacitated status under any bicarbonate concentration, even when bicarbonate was absent. Moreover, there were differences related to the concentration of bicarbonate, since sperm incubated in media with BSA and with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in achieving in vitro capacitation than those incubated in the presence of BSA and 15 mM or 38 mM bicarbonate. Additionally, at the end of the experiment, sperm incubated in the presence of BSA and 38 mM bicarbonate showed significantly (p < 0.05) lower values of motility and plasma membrane integrity than those incubated in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is instrumental for pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome exocytosis. Furthermore, while exogenous bicarbonate does not seem to be essential to launch sperm capacitation, it does modulate its efficiency. Keywords: sperm capacitation; pig; bicarbonate; bovine serum albumin (BSA); glycogen synthase kinase (GSK3); protein kinase A (PKA) Author: Bruna Resende Chaves, Ana Paula Pinoti Pavaneli, Olga Blanco-Prieto, Elisabeth Pinart, Sergi Bonet, Márcio Gilberto Zangeronimo, Joan E. Rodríguez-Gil and Marc Yeste Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Oct 26 12:30:47 2021 CEST ModDate: Tue Oct 26 12:39:19 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 24 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3344911 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- GPMQUR+VnURWPalladioL Type 1 Custom yes yes no 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 15 0 RRQADO+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 THUDAG+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 YJUTPD+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 SSNIUI+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 GLCFZC+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 40 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 62 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes 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CNNBCO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 302 0 CNNBCK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 316 0 CNNBCL+PalatinoLinotype CID TrueType Identity-H yes yes yes 319 0 CNNBCM+PalatinoLinotype TrueType WinAnsi yes yes no 325 0 CNNBCO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 328 0 CNNBCK+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 340 0 CNNBCL+PalatinoLinotype CID TrueType Identity-H yes yes yes 343 0 CNNBCM+PalatinoLinotype TrueType WinAnsi yes yes no 349 0 CNNBCO+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 352 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-02-12 02:25:37 CET RepresentationInformation: biology_a2021v10n11p1105.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-02-11 11:13:28 CET Size: 3344911 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 668 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Reagents Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Semen Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: In Vitro Capacitation and Progesterone-Induced Acrosome Exocytosis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Sperm Motility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Flow Cytometry Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: Evaluation of Sperm Membrane Integrity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Evaluation of Sperm Membrane Lipid Disorder Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Evaluation of Acrosome Integrity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Evaluation of Intracellular Calcium Levels Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Evaluation of Mitochondrial Membrane Potential Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Immunoblotting of Tyr-P-GSK3 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Immunoblotting of DARPP-32 (PKA) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Determination of O2 Consumption Rate Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Statistical Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Children: Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Plasma Membrane Integrity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Membrane Lipid Disorder Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Acrosome Integrity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Intracellular Calcium Levels Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Mitochondrial Membrane Potential Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Sperm Motility Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on DARPP-32 Phosphorylation Levels Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Effects of Different Concentrations of Bicarbonate and BSA on Tyrosine Phosphorylation Levels of GSK3 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Effects of Bicarbonate and BSA on O2 Consumption Rate Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Info: Title: Exogenous Albumin Is Crucial for Pig Sperm to Elicit In Vitro Capacitation Whereas Bicarbonate Only Modulates Its Efficiency Author: Bruna Resende Chaves, Ana Paula Pinoti Pavaneli, Olga Blanco-Prieto, Elisabeth Pinart, Sergi Bonet, Márcio Gilberto Zangeronimo, Joan E. Rodríguez-Gil and Marc Yeste Subject: This work sought to address whether the presence of exogenous bicarbonate is required for pig sperm to elicit in vitro capacitation and further progesterone-induced acrosome exocytosis. For this purpose, sperm were either incubated in a standard in vitro capacitation medium or a similar medium with different concentrations of bicarbonate (either 0 mM, 5 mM, 15 mM or 38 mM) and BSA (either 0 mg/mL or 5 mg/mL). The achievement of in vitro capacitation and progesterone-induced acrosomal exocytosis was tested through the analysis of sperm motility, plasma membrane integrity and lipid disorder, acrosome exocytosis, intracellular calcium levels, mitochondria membrane potential, O2 consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3) and protein kinase A (PKA). While sperm incubated in media without BSA or BSA/bicarbonate, they did not achieve in vitro capacitation; those incubated in media with BSA achieved the capacitated status under any bicarbonate concentration, even when bicarbonate was absent. Moreover, there were differences related to the concentration of bicarbonate, since sperm incubated in media with BSA and with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in achieving in vitro capacitation than those incubated in the presence of BSA and 15 mM or 38 mM bicarbonate. Additionally, at the end of the experiment, sperm incubated in the presence of BSA and 38 mM bicarbonate showed significantly (p < 0.05) lower values of motility and plasma membrane integrity than those incubated in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is instrumental for pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome exocytosis. Furthermore, while exogenous bicarbonate does not seem to be essential to launch sperm capacitation, it does modulate its efficiency. 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