Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans

Kun Li 2 Ya Ni 1 2 Yi He 1 Wen-Ying Chen 2 Jian-Xin Lu 1 C. Yan Cheng 0 Ren-Shan Ge 0 1 Qi-Xian Shi 1 2 0 The Population Council, Center for Biomedical Research , NY 10021 USA 1 Department of Laboratory Medicine, Wenzhou Medical College , Wenzhou, Zhejiang 325035 , China 2 Unit of Reproductive Physiology, Zhejiang Academy of Medical Sciences , Hangzhou, Zhejiang 310013 , China what is known already: Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoligerm 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 Cl2 ion transport function in the human sperm. study desigin, size and duration: 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 mM and/or other reagents under capacitating conditions for 0 - 5 h. participants/materials, setting, methods: We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 378C. 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 Cl2-deficient HTF medium as well as the various Cl2 channel blockers. main results and the role of chance: Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP3b- 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 capacitationassociated events only in the complete Cl2-HTF medium but not Cl2-deficient medium, illustrating the likely involvement of Cl2. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. limitations, reasons for caution: 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. - These authors contributed equally to this work. & The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 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. Introduction Earlier studies have shown that adjudin [1-(2,4-dichlorobenzyl)1H-indazole-3-carbohydrazide, formerly called AF-2364], an analog of lonidamine [1-(2,4-dichlorobenzyl) 1H-indazole-3-caboxylic acid], is a potential male contraceptive (Cheng et al., 2011). Adjudin exerts its effect by disrupting Sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization (apical ES, a testisspecific anchoring junction type), by targeting testin and actin filament bundles that disrupt the actin-based cytoskeleton in Sertoli cells (Mruk and Cheng, 2011), causing the release of spermatids prematurely from the seminiferous epithelium, thereby resulting in rat infertility (Cheng et al., 2001). However, the effects of adjudin on sperm fertilizing ability in vitro, in particular humans have yet to be evaluated. It is known that ejaculated human spermatozoa must spend a period of time in the female genital tract or in appropriate artificial medium in vitro in order to gain the capacity to fertilize an egg. This phenomenon is termed capacitation (Yanagimachi, 1994; Visconti et al., 1998, 2011; Florman and Ducibella, 2006). Capacitated spermatozoa acquire the ability to bind specifically to the zona pellucida (ZP) of the ovum and undergo the acrosome reaction (Liu and Baker, 1994). The acrosome reaction is an exocytosis event induced by progesterone or the ZP (Roldan et al., 1994; Yanagimachi, 1994). The acrosome reaction is crucial for successful sperm binding and penetration of the ZP, and is followed by plasma membrane fusion with the egg (Florman and Storey, 1982; Wassarman, 1987). The development of in vitro procedures for assessing the fertilizing capacity of human spermatozoa has been greatly enhanced due to wide-spread application of IVF for therapy of infertility (Yanagimachi et al., 1976; Steptoe and Edwards, 1978; Aitken et al., 1983; Trounson and Webb, 1984). It was reported that AF-2785 [1-(2,4-dichlorobenzyl)1H-indazole-3-acrylic acid], another analog of lonidamine, and a potent blocker of CFTR (cystic fibrosis transmembrane conductance regulator), plays an important role in male reproduction by impeding the secretion of chloride ions from the epithelial cells in the rat epididymis (Gong et al., 2000, 2002; Gong and Wong, 2000). It remains to be determined if adjudin inhibits the CFTR function leading to the reduction in sperm-fertilizing ability. CFTR acts as a cAMP-activated chloride (Cl2) channel, which plays an important role in male reproductive function (Wong, 1998). Besides CFTR Cl2 channels in the sperm, four structural Cl2 channel families have been identified to date: (i) Cl2-selective ion channels (Estevez et al., 2003); (ii) the GABAA receptor/Cl2 channels (Shi and Roldan, 1995); (iii) Ca2+-activated Cl2 channels (Orta et al., 2012) and (iv) Na+/K+/Cl2 co-transporter channels (Wertheimer et al., 2008). As with other Cl (...truncated)