title: Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans.
abstract: OBJECTIVE
  Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro?
  
  
  CONCLUSIONS
  In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro.
  
  
  BACKGROUND
  Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm.
  
  
  METHODS
  Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h.
  
  
  METHODS
  We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers.
  
  
  RESULTS
  Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP₃β- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function.
  
  
  CONCLUSIONS
  This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive.
  
  
  CONCLUSIONS
  Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.
authors.affiliation: Unit of Reproductive Physiology,  Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013,  China.
authors.forename: Kun
authors.initials: K
authors.lastname: Li
authors.forename: Ya
authors.initials: Y
authors.lastname: Ni
authors.forename: Yi
authors.initials: Y
authors.lastname: He
authors.forename: Wen-Ying
authors.initials: WY
authors.lastname: Chen
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Lu
authors.forename: C Yan
authors.initials: CY
authors.lastname: Cheng
authors.forename: Ren-Shan
authors.initials: RS
authors.lastname: Ge
authors.forename: Qi-Xian
authors.initials: QX
authors.lastname: Shi
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D000067956:Adenylyl Cyclase Inhibitors; D000328:Adult; D000818:Animals; D018118:Chloride Channels / Q000037:antagonists & inhibitors*; D002712:Chlorides / Q000378:metabolism; D003272:Contraceptive Agents, Male / Q000009:adverse effects / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006224:Cricetinae; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000378:metabolism; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005306:Fertilization / Q000187:drug effects*; D006801:Humans; D006834:Hydrazines / Q000009:adverse effects / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007191:Indazoles / Q000009:adverse effects / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D049990:Membrane Transport Modulators / Q000009:adverse effects / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013075:Sperm Capacitation / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D000072216:Zona Pellucida Glycoproteins
chemical_list: C432482:1-(2,4-dichlorobenzyl)indazole-3-carbohydrazide; D000067956:Adenylyl Cyclase Inhibitors; D018118:Chloride Channels; D002712:Chlorides; D003272:Contraceptive Agents, Male; D004527:Egg Proteins; D004791:Enzyme Inhibitors; D006834:Hydrazines; D007191:Indazoles; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D010726:Phosphodiesterase Inhibitors; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000072216:Zona Pellucida Glycoproteins; D000242:Cyclic AMP