Functional attenuation of human sperm by novel, non-surfactant spermicides: precise targeting of membrane physiology without affecting structure

Human Reproduction, Vol.25, No.5 pp. 1165– 1176, 2010 Advanced Access publication on February 22, 2010 doi:10.1093/humrep/deq036 ORIGINAL ARTICLE Fertility control Functional attenuation of human sperm by novel, non-surfactant spermicides: precise targeting of membrane physiology without affecting structure Rajeev K. Jain 1,†, Ashish Jain 1,†, Rajeev Kumar 1, Vikas Verma 1, Jagdamba P. Maikhuri 1, Vishnu L. Sharma 2, Kalyan Mitra 3, Sanjay Batra 2, and Gopal Gupta 1,* 1 Division of Endocrinology, Central Drug Research Institute (CSIR), Lucknow 226 001, India 2Division of Medicinal Chemistry, Central Drug Research Institute (CSIR), Lucknow 226 001, India 3Electron Microscopy Unit, Central Drug Research Institute (CSIR), Lucknow 226 001, India *Correspondence address. E-mail: Submitted on October 4, 2009; resubmitted on January 20, 2010; accepted on January 25, 2010 background: We have attempted to identify structural, physiological and other targets on human sperm vulnerable to the spermicidal action of two novel series of non-detergent molecules, reported to irreversibly immobilize human sperm in ,30 s, apparently without disrupting plasma membrane. methods: Three sperm samples were studied. Scanning and transmission electron microscopy were used to assess structural aberrations of sperm membrane; plasma membrane potential and intracellular pH measurements (fluorometric) were used to detect changes in sperm physiology; reactive oxygen species (ROS, fluorometric) and superoxide dismutase activity (colorimetric) were indicators of oxidative stress; and sperm dynein ATPase activity demonstrated alterations in motor energy potential, in response to spermicide treatment. Post-ejaculation tyrosine phosphorylation of human sperm proteins (immunoblotting) was a marker for functional integrity. results: Disulfide esters of carbothioic acid (DSE compounds) caused complete sperm attenuation at 0.002% concentration with hyper-polarization of sperm membrane potential (P , 0.001), intracellular alkalinization (P , 0.01), ROS generation (P , 0.05) and no apparent effect on sperm (n ¼ 150) membrane structure. Isoxazolecarbaldehyde compounds required 0.03% for spermicidal action and caused disrupted outer acrosomal membrane structure, depolarization of membrane potential (P , 0.001), intracellular acidification (P , 0.01) and ROS generation (P , 0.01). Detergent [nonoxynol-9 (N-9)] action was sustainable at 0.05% and involved complete breakdown of structural and physiological membrane integrity with ROS generation (P , 0.001). All spermicides caused functional attenuation of sperm without inhibiting motor energetics. Unlike N-9, DSE-37 (vaginal dose, 200 mg) completely inhibited pregnancy in rats and vaginal epithelium was unchanged (24 h,10 mg). conclusions: The study reveals a unique mechanism of action for DSE spermicides. DSE-37 holds promise as a safe vaginal contraceptive. CDRI Communication No. 7545. Key words: sperm / contraception / disulfide esters of carbothioic acid / non-surfactant spermicide / nonoxynol-9 † These authors contributed equally. & The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 1166 Introduction Spermicides capable of killing 100% human sperm almost instantaneously at physiological concentrations in vitro are likely to provide adequate pregnancy protection in vivo. However, the molecules designed for such activity often also target other cells in vagina, such as the cervico-vaginal epithelia and lactobacilli that are crucial in maintaining the natural barrier to invasion by pathogens, especially the human immunodeficiency virus (HIV). Nonoxynol-9 (N-9) is one such molecule that kills sperm, bacteria (including lactobacillus), virus and cervico-vaginal cells etc. in vitro (Ojha et al., 2003; Beer et al., 2006; Dover et al., 2007) by its strong surfactant action. Under in vivo conditions N-9 induces a proinflammatory response in vagina (Stafford et al., 1998; Patton et al., 1999) resulting in increased (rather than decreased) incidence of sexually transmitted disease and HIV infections in users. Hence molecules with specific action are required that can act by precisely targeting the sperm cells in vagina while remaining practically inert to other cell types at spermicidal concentration. Our enduring efforts to design, synthesize and evaluate novel molecules with a specific, mechanism-based action on sperm cells resulted in several series of potent non-detergent structures (Srivastava et al., 1999; Kumaria et al., 2002; Maikhuri et al., 2003; Kumar et al., 2006; Kiran Kumar et al., 2006, 2008; Dwivedi et al., 2007). Recently we reported two novel series of non-detergent spermicidal compounds, the disulfide esters (DSE) of carbothioic acid (Jain et al., 2007, 2009) and the isoxazolecarbaldehydes (ISX, Gupta et al., 2005): while the former displayed extremely potent spermicidal action by killing 100% human sperm at just 4% of the concentration required by N-9 in vitro, the latter presented a rare combination of acrosin inhibition and spermicidal action in the same structure. As per the strict criteria of spermicidal action, only those compounds were selected that irreversibly immobilized 100% sperm in ,30 s, in vitro. Though the potent and rapid action of detergent spermicides, such as N-9, is explicable by their ability to destabilize the cell membrane, it is difficult to offer a plausible explanation for a similar action of non-detergent spermicides, especially when the action is much more potent and at extremely low concentrations, as in case of DSE molecules. Even the most potent metabolic inhibitors such as cyanide fail to exhibit spermicidal action equivalent to N-9, in vitro (Hong et al., 1983). The spermatozoon is a terminally differentiated cell with a virtually dormant nucleus (Zheng et al., 2008) where signaling through cell membrane controls most of the vital cellular events. Rapid action of spermicides is mostly an event mediated through cell surface by alterations in membrane structure and/or physiology. In the present communication, we have made an attempt to identify some of the possible targets in the sperm membrane and cytosol that play a significant role in sperm motility and viability, and therefore might be crucial for the spermicidal action of these new non-surfactant molecules. Materials and Methods New spermicidal compounds and reagents The synthesis and characterization of test compounds, the DSE of carbothioic acid (DSE-36 and DSE-37) and the ISXs (ISX-1c, ISX-1d, ISX-1g, ISX-1h and ISX-1i), have been detailed earlier (Gupta et al., 2005; Jain Jain et al. et al., 2009). Fluorescent dyes bis(1,3-dibarbituric acid)-trimethine oxanol (DiBAC4), 20 ,70 -bis(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl (BCECF-AM) ester and 20 ,70 -dichlorofluorescein diacetate (DCFDA) were purchased from Sigma-Ald (...truncated)