Specific loss of CatSper function is sufficient to compromise fertilizing capacity of human spermatozoa.
Specific loss of CatSper function is sufficient to compromise fertilizing capacity of human spermatozoa.
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DOI：
10.1093/humrep/dev243
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发表时间：
2015-12
期刊：
影响因子：
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通讯作者：
Martins da Silva S
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文献类型：
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作者：
Williams HL;Mansell S;Alasmari W;Brown SG;Wilson SM;Sutton KA;Miller MR;Lishko PV;Barratt CL;Publicover SJ;Martins da Silva S
在精子浓度和活力正常的IVF患者中，是否存在明显的CatSper功能异常？如果存在，它们对受精成功的功能意义是什么？几乎没有CatSper电流的精子对黄体酮激活CatSper没有反应，体外受精失败。在人类精子中，黄体酮诱导的Ca2+内流是由精子特异性Ca2+通道CatSper介导的。亚理想的Ca2+内流与精液参数异常的男性显著相关，并且在男性中更为普遍，并且与受精能力降低有关。然而，CatSper电流的异常只能直接使用电生理学来评估。只有一个报告的CatSper缺陷的人没有显示黄体酮增强CatSper电流。CatSper 2基因异常存在，但没有关于[Ca2+]i对黄体酮激活CatSper的反应的信息。此外，精液样本显示明显异常（少弱无畸形精子症），精子中存在多个次优功能反应。因此，不能断定CatSper功能受损单独导致不孕症或CatSper阻断是避孕的潜在安全目标。精子来自2013年1月至2014年12月期间在医院辅助生殖技术诊所就诊的捐赠者和低生育能力试管婴儿患者。共有134名试管婴儿患者，28名正常精子捐赠者和10名因试管婴儿受精失败/低受精史而被召回的患者参加了这项研究。首先使用孕酮诱导的Ca2+内流筛选样品，如果细胞数量足够，还通过过度激活和渗透到粘性介质中来评估样品。用正常精子供体反应幅度分布的99%置信区间来定义有缺陷的Ca2+对黄体酮的反应。样品显示有缺陷的Ca2+反应进一步检查，以表征潜在的CatSper异常。对于钙信号持续且强烈失败的男性，使用电生理学（贴片夹紧）直接评估CatSper功能，并获得血液样本进行遗传分析。共有101/102（99%）试管婴儿患者和22/23（96%）供体表现出正常的Ca2+反应。平均（±SD）标准化峰值反应在供体和体外受精患者之间没有差异（2.57±0.68 [n = 34]来自23个不同的供体]和2.66±0.68 [n = 102 IVF患者]，P = 0.63）。在回忆的患者中，9/10（90%）显示正常的Ca2+反应。三名男性最初被确定为Ca2+内流缺陷。然而，只有一个（患者1）在重复精液样本中有缺陷反应。患者1的精子电生理实验显示几乎没有CatSper电流，外显子筛选显示在CatSper复合物的编码区没有突变。当用黄体酮刺激精子时，粘性介质的渗透没有增加，重要的是体外受精失败。一个关键的限制涉及到一个特定的功能参数（由孕酮诱导的Ca2+内流）在新鲜的精子样本中工作，从捐赠者和患者的有限的生存能力。因此，由于实际、技术和后勤原因，一些男性（约22%的试管婴儿患者）无法进行筛查。因此，黄体酮诱导的显著Ca2+异常的发生率可能高于这里观察到的1%。此外，我们使用了一个严格的Ca2+内流缺陷的定义，这样只有实质性的异常被选择为进一步的研究。此外，电生理仅对一名患者进行了稳健且可重复的缺钙反应。该患者的CatSper电流可忽略不计，但Ca2+内流正常或低于正常的男性可能存在更细微的异常（例如电流存在但明显较小）。这些数据大大增加了对CatSper在人类精子功能中的作用及其对男性生育能力的影响的理解。值得注意的是，这些发现提供了第一个直接证据，证明CatSper是人类男性避孕的合适和特定目标。最初的资金来自NHS Tayside，不孕症研究信托基金，TENOVUS，首席科学家办公室NRS奖学金，威康信托基金，阿伯泰大学。大部分数据是通过MRC项目拨款（# 4190）获得的。作者声明不存在利益冲突。不适用。
Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success? Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF. In human spermatozoa, Ca2+ influx induced by progesterone is mediated by CatSper, a sperm-specific Ca2+ channel. A suboptimal Ca2+ influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca2+]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception. Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study. Samples were primarily screened using the Ca2+ influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca2+ response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca2+ response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis. A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca2+ response. The mean (±SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca2+ response. Three men were initially identified with a defective Ca2+ influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF. A key limitation relates to working with a specific functional parameter (Ca2+ influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼22% of IVF patients) could not be screened. As such the incidence of significant Ca2+ abnormalities induced by progesterone may be higher than the ∼1% observed here. Additionally, we used a strict definition of a defective Ca2+ influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca2+ influx. These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception. Initial funding was from NHS Tayside, Infertility Research Trust, TENOVUS, Chief Scientist Office NRS Fellowship, the Wellcome Trust, University of Abertay. The majority of the data were obtained using funding from a MRC project grant (# 4190). The authors declare that there is no conflict of interest. Not applicable.