title: Acrosome reaction and Ca²⁺ imaging in single human spermatozoa: new regulatory roles of [Ca²⁺]i.
abstract: The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca²⁺ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca²⁺ indicator. These AR and intracellular Ca²⁺ ([Ca²⁺]i) recordings revealed that the presence of [Ca²⁺]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca²⁺]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca²⁺]i rise ~2 min before AR. Our findings uncover new AR features related to [Ca²⁺]i.
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México.
authors.forename: Claudia
authors.initials: C
authors.lastname: Sánchez-Cárdenas
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México.
authors.forename: Martha Rocio
authors.initials: MR
authors.lastname: Servín-Vences
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México.
authors.forename: Omar
authors.initials: O
authors.lastname: José
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México.
authors.forename: Claudia Lydia
authors.initials: CL
authors.lastname: Treviño
authors.affiliation: Departamento de Neurociencia Cognitiva, Instituto de Fisiología Celular, UNAM, Circuito exterior s/n, Ciudad Universitaria, México DF.
authors.forename: Arturo
authors.initials: A
authors.lastname: Hernández-Cruz
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, México darszon@ibt.unam.mx.
authors.forename: Alberto
authors.initials: A
authors.lastname: Darszon
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D000814:Aniline Compounds / Q000737:chemistry; D061207:Calcium Ionophores / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D022081:Cell Surface Extensions / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008855:Microscopy, Electron, Scanning; D011374:Progesterone / Q000378:metabolism; D011726:Pyridinium Compounds / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D055101:Semen Analysis / Q000379:methods*; D059010:Single-Cell Analysis; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D061207:Calcium Ionophores; C092350:FM 4-64; C409648:Fluo 4; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D014966:Xanthenes; D011374:Progesterone; D015759:Ionomycin
keywords: FM4-64; acrosome reaction; calcium; progesterone; sperm
references.citation: Biol Reprod. 1989 Oct;41(4):635-41
references.pmid: 2695176
references.citation: Fertil Steril. 2012 Jun;97(6):1309-15
references.pmid: 22494923
references.citation: Biol Reprod. 2009 Aug;81(2):396-405
references.pmid: 19369646
references.citation: Dev Biol. 1994 Sep;165(1):152-64
references.pmid: 8088433
references.citation: Histochem Cell Biol. 2010 Aug;134(2):103-13
references.pmid: 20577884
references.citation: Dev Biol. 2007 Feb 1;302(1):324-32
references.pmid: 17054937
references.citation: Nature. 1996 Apr 11;380(6574):531-4
references.pmid: 8606773
references.citation: Biotechniques. 2006 Aug;41(2):191-7
references.pmid: 16925021
references.citation: Trends Neurosci. 2001 Nov;24(11):637-43
references.pmid: 11672807
references.citation: J Androl. 1991 Sep-Oct;12(5):323-30
references.pmid: 1765568
references.citation: Physiol Rev. 2011 Oct;91(4):1305-55
references.pmid: 22013213
references.citation: Biochem J. 2004 Mar 15;378(Pt 3):827-32
references.pmid: 14606954
references.citation: Biol Reprod. 2006 Dec;75(6):933-9
references.pmid: 16957023
references.citation: Biochem J. 2012 Dec 1;448(2):189-200
references.pmid: 22943284
references.citation: Nature. 2011 Mar 17;471(7338):387-91
references.pmid: 21412339
references.citation: J Cell Sci. 2008 Jul 1;121(Pt 13):2130-5
references.pmid: 18522990
references.citation: Dev Biol. 1997 Feb 1;182(1):67-75
references.pmid: 9073447
references.citation: Methods Mol Biol. 2013;927:321-48
references.pmid: 22992926
references.citation: Anat Rec. 1988 Mar;220(3):267-80
references.pmid: 3364752
references.citation: Exp Anim. 2010;59(1):105-7
references.pmid: 20224175
references.citation: Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12765-9
references.pmid: 7809118
references.citation: J Cell Physiol. 2009 Sep;220(3):611-20
references.pmid: 19373867
references.citation: Skelet Muscle. 2011 May 04;1(1):18
references.pmid: 21798098
references.citation: Int J Dev Biol. 2008;52(5-6):665-76
references.pmid: 18649280
references.citation: Physiology (Bethesda). 2010 Jun;25(3):165-75
references.pmid: 20551230
references.citation: Nature. 2011 Mar 17;471(7338):382-6
references.pmid: 21412338
references.citation: J Cell Biol. 1995 Aug;130(4):857-69
references.pmid: 7642703
references.citation: Int J Androl. 2012 Oct;35(5):633-44
references.pmid: 22775762
references.citation: Proc Natl Acad Sci U S A. 1993 May 15;90(10):4660-4
references.pmid: 8506314
references.citation: J Biol Chem. 2004 Oct 29;279(44):46315-25
references.pmid: 15322137
references.citation: Int J Dev Biol. 2008;52(5-6):503-10
references.pmid: 18649263
references.citation: J Exp Biol. 1964 Sep;41:603-8
references.pmid: 14221043
references.citation: Biol Reprod. 2012 Oct 18;87(4):92
references.pmid: 22914313
references.citation: Nature. 1998 Apr 2;392(6675):497-501
references.pmid: 9548254
references.citation: Nat Protoc. 2006;1(6):2916-21
references.pmid: 17406552
references.citation: PLoS Biol. 2005 Oct;3(10):e323
references.pmid: 16131227
references.citation: IUBMB Life. 2007 Apr-May;59(4-5):286-92
references.pmid: 17505967
references.citation: Reproduction. 2012 Jul;144(1):101-9
references.pmid: 22580372
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):4892-6
references.pmid: 21383182
references.citation: Dev Biol. 2000 Jun 15;222(2):326-35
references.pmid: 10837122
references.citation: Asian J Androl. 2011 May;13(3):395-405
references.pmid: 21540868
references.citation: Mol Hum Reprod. 1996 Apr;2(4):225-32
references.pmid: 9238684
references.citation: Hum Reprod Update. 2008 Nov-Dec;14(6):647-57
references.pmid: 18653675