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 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: 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,* 1166 Introduction 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 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-Aldrich, Saint Louis, MO, USA. The antiphosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology (Lake Placid, NY, USA) and superoxide dismutase (SOD) assay kit was from Fluka. All other reagents were from Sigma-Aldrich. Human sperm Freshly ejaculated human semen was obtained by masturbation from healthy and fertile volunteers, collected directly into a sterile plastic tube and transported immediately into the laboratory. Prior informed consent was obtained from the donors for this study. The samples were allowed to liquefy at 378C for 45 min. Semen characteristics and analysis were performed according to the normal criteria as per World Health Organization guidelines (WHO manual, 1992). Sperm count and motility analysis were performed manually as well as in a Computer Automated Semen Analyzer (CASA) system using a small drop of liquefied semen placed on a ‘Makler’ counting chamber (Sefý Medica, Hafia, Israel) pre-warmed to 378C. Semen samples with .65 million/ml sperm count, .70% motility and normal sperm morphology were used. This study was approved by the Institutional Ethics Committee. Assessment of spermicidal activity Spermicidal potential of new spermicides and N-9 was assessed by the modified Sander–Cramer assay (Sander and Cramer, 1941) as detailed earlier (Gupta et al., 2005; Jain et al., 2007, 2009). Briefly, the test compounds were dissolved in a minimum volume of dimethylsulfoxide and diluted with physiological saline (0.85% NaCl) to make a 1.0% (10 mg/ml) solution (DSE compounds were dissolved directly in saline). The solutions were further diluted serially with saline. A spermicidal test was performed with each dilution starting from 1.0% until the minimum effective concentration (MEC) was arrived at. Human semen (0.05 ml) was added to 0.25 ml of spermicidal compound solution and vortexed at low speed for 10 s. A drop was immediately placed on a microscope slide, covered with a cover glass and examined under a phase contrast microscope. The result was scored positive if 100% spermatozoa became immotile within 20 s and remained immotile even after dilution with excess phosphate-buffered saline (PBS), for another 60 min. The MEC was determined in three individual semen samples from different donors. Scanning electron microscopy of human sperm Human sperm were treated with spermicide solutions at MEC for 60 min, washed twice with PBS and fixed in 1% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer for 4 h at 48C (D’Cruz et al., 1998). Sperm were washed in cacodylate buffer, placed on poly-L-lysine (0.1%)-coated glass chips and allowed to adhere for 1 h at room temperature. Adhered sperm samples were post-fixed in 1% OsO4 for 1 h at room temperature and subsequently dehydrated through an ascending series of ethanol, critical point dried and coated with Au– Pd (80:20) using a sputter coater (Polaron E5000). All samples were examined in an FEI XL 30 (Philips FEI, The Netherlands) scanning electron microscope (SEM) at an accelerating voltage of 30 KV. At least 150 cells were scanned in each sample for intactness of the sperm surface. Transmission electron microsco (...truncated)