p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.
OBJECTIVE
Is the environmental endocrine disruptor p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters?


CONCLUSIONS
p,p'-DDE promoted Ca(2+) flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization.


BACKGROUND
p,p'-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known.


METHODS
Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p'-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p'-DDE incubation periods were also performed in order to monitor sperm rapid Ca(2+) responses. All experiments were repeated on a minimum of five sperm samples from different individuals.


METHODS
All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively.


RESULTS
p,p'-DDE rapidly increased [Ca(2+)]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca(2+) medium demonstrated that extracellular Ca(2+) influx was responsible for this Ca(2+) increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p'-DDE-induced [Ca(2+)]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p'-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p'-DDE concentrations, suggesting that [Ca(2+)]i rise may cause premature acrosome reaction (P < 0.05).


CONCLUSIONS
This is an in vitro study, and caution must be taken when extrapolating the results.


CONCLUSIONS
A novel non-genomic p,p'-DDE mechanism specific to sperm is shown in this study. p,p'-DDE was able to induce [Ca(2+)]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors.


BACKGROUND
The study was supported by both the Portuguese National Science Foundation (FCT; PEst-C/SAU/LA0001/2011) and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare.
Biology of Reproduction and Stem Cell Group, CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3001-401, Portugal.
Renata S
RS
Tavares
Steven
S
Mansell
Christopher L R
CL
Barratt
Stuart M
SM
Wilson
Stephen J
SJ
Publicover
João
J
Ramalho-Santos
D001562:Benzimidazoles / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003521:Cyclopropanes; D003633:Dichlorodiphenyl Dichloroethylene / Q000633:toxicity*; D052244:Endocrine Disruptors / Q000633:toxicity*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D020748:Mibefradil / Q000494:pharmacology; D009281:Naphthalenes; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
D001562:Benzimidazoles; C438823:CATSPER1 protein, human; D015220:Calcium Channels; D003521:Cyclopropanes; D052244:Endocrine Disruptors; D009281:Naphthalenes; C484287:NNC 55-0396; D020748:Mibefradil; D003633:Dichlorodiphenyl Dichloroethylene; D002118:Calcium
Ca2+ influx; CatSper; acrosome reaction; intracellular Ca2+ concentration; p,p′-DDE