title: Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa.
abstract: Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions?
  
  
  
  DES promoted Ca2+flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+signaling, tyrosine phosphorylation and sperm functions.
  
  
  
  DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown.
  
  
  
  Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples.
  
  
  
  Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin-nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay.
  
  
  
  DES exposure rapidly increased human sperm [Ca2+]idose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]iwas derived from extracellular Ca2+influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction.
  
  
  
  N/A.
  
  
  
  Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications.
  
  
  
  The present study revealed that DES interfered with progesterone-stimulated Ca2+signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function.
  
  
  
  National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Qian-Xing
authors.initials: QX
authors.lastname: Zou
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Peng
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Reproductive Medical Center, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi 330006, PR China.
authors.forename: Hou-Yang
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: Cheng
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Yuan-Qiao
authors.initials: YQ
authors.lastname: He
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Shi-Qi
authors.initials: SQ
authors.lastname: Weng
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Hua-Feng
authors.initials: HF
authors.lastname: Wang
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China.
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Zheng
authors.affiliation: Institute of Life Science and School of Life Science, Nanchang University, Nanchang, Jiangxi 330031, PR China luotao@ncu.edu.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Luo
mesh_terms: D020101:Acrosome Reaction / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D004054:Diethylstilbestrol / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004968:Estrogens, Non-Steroidal / Q000494:pharmacology*; D006801:Humans; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D011374:Progesterone / Q000494:pharmacology*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism
chemical_list: C438823:CATSPER1 protein, human; D015220:Calcium Channels; D004968:Estrogens, Non-Steroidal; D011374:Progesterone; D004054:Diethylstilbestrol; D002118:Calcium
keywords: CatSper; acrosome reaction; diethylstilbestrol; endocrine-disrupting chemical; human sperm; hyperactivation; intracellular calcium concentration; progesterone; tyrosine phosphorylation