p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.
p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.
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DOI：
10.1093/humrep/det372
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发表时间：
2013-12
期刊：
影响因子：
--
通讯作者：
Ramalho-Santos J
中科院分区：
文献类型：
--
作者：
Tavares RS;Mansell S;Barratt CL;Wilson SM;Publicover SJ;Ramalho-Santos J
环境内分泌干扰物p，p′-dichlorodiphenyldichloroethylene（p，p′-DDE）是否能引起人类精子的非基因组改变，从而影响精子功能参数？p，p′-DDE通过激活CatSper通道促进Ca 2+流入人类精子，即使在人类生殖液中发现的剂量下也是如此，最终损害了对受精重要的精子参数。p，p′-DDE可能促进非基因组作用，并与预先存在的信号通路直接相互作用，如在其他细胞类型中观察到的那样。然而，尽管在男性和女性生殖液中经常发现，但其对人类精子功能的影响尚不清楚。本研究包括来自健康个体的正常精子样本。将样品在体外37°C和5% CO2条件下暴露于几种浓度的p，p′-DDE 3天，以模拟体内雌性生殖道中假定的连续暴露于该毒物。缩短p，p′-DDE孵育时间以监测精子快速Ca 2+反应。所有的实验都是在来自不同个体的至少五个精子样本上重复的。所有健康个体均在伯明翰大学生物科学学院、邓迪大学医学研究所和科英布拉大学医院人类生殖服务中心招募。通过对负载俄勒冈州绿色BAPTA-1 AM的单精子进行成像来监测细胞内Ca 2+浓度（[Ca 2 +]i），并进行进一步的全细胞膜片钳记录以验证我们的结果。分别使用活/死精子活力试剂盒和顶体含量标记物PSA-FITC评估精子活力和顶体完整性。p，p′-DDE即使在极低剂量（1 pM和1 nM）下也能迅速增加[Ca 2 +]i（P < 0.05），反应幅度高达200%，但不影响精子活力，连续暴露于最高试验浓度3天后除外（P < 0.05）。此外，在低Ca ~（2+）培养基中进行的实验表明，细胞外Ca ~（2+）内流是导致这种Ca ~（2+）增加的原因（P < 0.01）。精子特异性CatSper通道抑制剂Mibefradil和NNC 55-0396可逆转p，p′-DDE诱导的[Ca 2 +]i升高，提示CatSper参与了这一过程（P < 0.05）。事实上，全细胞膜片钳记录通过监测CatSper电流增加>100%（P < 0.01）证实CatSper是p，p′-DDE作用的靶点。p，p′-DDE暴露2 d后，顶体完整性受到影响，提示[Ca 2 +]i升高可能导致顶体反应提前（P < 0.05）。这是一项体外研究，外推结果时必须谨慎。本研究发现了一种新的精子特异性非基因组p，p′-DDE机制。p，p′-DDE可通过CatSper的开放诱导人精子[Ca 2 +]i升高，从而影响男性生育力。CatSper激活的混杂性质可能使人类精子易于受到某些持久性内分泌干扰物的作用。该研究得到了葡萄牙国家科学基金会（FCT; PEst-C/SAU/LA 0001/2011）和英国威康信托基金（Grant #86470）的支持。SM得到了不孕症研究信托基金的支持。他是FCT博士奖学金的获得者（SFRH/BD/46002/2008）。所有作者都没有任何利益冲突需要声明。
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? p,p′-DDE promoted Ca2+ flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization. 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. 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 Ca2+ responses. All experiments were repeated on a minimum of five sperm samples from different individuals. 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 Ca2+ concentration ([Ca2+]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. p,p′-DDE rapidly increased [Ca2+]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 Ca2+ medium demonstrated that extracellular Ca2+ influx was responsible for this Ca2+ increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p′-DDE-induced [Ca2+]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 [Ca2+]i rise may cause premature acrosome reaction (P < 0.05). This is an in vitro study, and caution must be taken when extrapolating the results. A novel non-genomic p,p′-DDE mechanism specific to sperm is shown in this study. p,p′-DDE was able to induce [Ca2+]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. 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.
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DOI：
10.1530/rep-09-0134
发表时间：
2009-09
期刊：
Reproduction (Cambridge, England)
影响因子：
--
作者：
Costello S;Michelangeli F;Nash K;Lefievre L;Morris J;Machado-Oliveira G;Barratt C;Kirkman-Brown J;Publicover S
通讯作者：
Publicover S
影响因子：
--
作者：
Campagna, Celine;Guillemette, Christine;Bailey, Janice L.
通讯作者：
Bailey, Janice L.
影响因子：
6.1
作者：
Hauser, R;Singh, NP;Altshul, L
通讯作者：
Altshul, L
DOI：
10.1073/pnas.2536658100
发表时间：
2003-12-09
影响因子：
11.1
作者：
Carlson, AE;Westenbroek, RE;Babcock, DF
通讯作者：
Babcock, DF
影响因子：
3.8
作者：
Loeffler, IK;Peterson, RE
通讯作者：
Peterson, RE