Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF.
Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF.
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DOI：
10.1093/humrep/dew056
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发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
Da Silva SM
中科院分区：
文献类型：
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作者：
Brown SG;Publicover SJ;Mansell SA;Lishko PV;Williams HL;Ramalingam M;Wilson SM;Barratt CL;Sutton KA;Da Silva SM
接受IVF和ICSI患者的精子中是否存在明显的外（K+）传导和静息膜电位（Vm）异常，如果存在，它们对受精成功的功能影响是什么？向外传导可忽略（≈5%的患者）或向内传导增强（≈4%的患者），两者都导致Vm去极化，与IVF后的低受精率相关。精子特异性钾通道敲除小鼠不育且精子功能缺陷，表明这些通道对生育至关重要。这些观察结果表明，人类精子中K+通道的功能障碍可能是男性生育能力低下的重要原因。然而，人们对人类精子中K+通道的性质以及K+通道缺陷的发生率和功能后果知之甚少。精子来自2013年5月至2015年12月期间在医院辅助生殖技术诊所就诊的健康志愿研究捐赠者和低生育能力的IVF和ICSI患者。总共有40名IVF患者，41名ICSI患者和26名正常精子捐赠者参与了这项研究。使用电生理学（全细胞贴片夹紧）检查样品。当发现异常电生理特征时，进一步检查精子是否有黄体酮引起的Ca2+内流，如果有足够的样本，精子是否能渗透到粘性介质中。通过全外显子组测序，特异性评估钾钙激活通道亚家族M α 1 （KCNMA1）、钾钙激活通道亚家族U成员1 （KCNU1）和富含亮氨酸重复序列52 （LRRC52）基因以及其他与K+信号相关的基因。在IVF患者中，通过与受精率的比较来评估电生理异常的功能意义。膜片钳电生理用于评估体外（K+）电导和静息膜电位（Vm），信号/运动测定用于评估IVF和ICSI患者样本精子的功能特征。体外受精（IVF）和ICSI患者精子的Vm平均值和外膜导度与相同条件下制备的对照（供体）精子的Vm平均值和外膜导度无显著差异，但个体间差异显著（P< 0.02），存在大量异常值（>25%）。特别是，在约10%的患者（7/81）中，我们观察到向外电导可忽略（4例）或向内电流增强（3例），两者都导致Vm去极化。体外受精患者的临床资料分析显示，去极化Vm（≥0 mV）与低受精率显著相关（P= 0.012）。电生理异常（电导和Vm）的精子对孕酮反应正常，[Ca2+]i升高，黏性介质渗透，表明精子阳离子通道（CatSper）通道功能保留。由于实际、技术、伦理和后勤方面的原因，我们无法从导性异常的男性身上获得足够的额外精液样本来确定导性缺陷的原因。全外显子组测序仅适用于两名有电导缺陷的男性。这些数据大大增加了对离子通道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。钾通道电导（Gm）和/或Vm调控受损在人类精子中既常见又复杂，重要的是当细胞Vm完全去极化时，与受精能力受损有关。大部分数据由MRC项目资助（#MR/K013343/1, MR/012492/1）获得。额外的资金由NHS Tayside、TENOVUS、首席科学家办公室NRS奖学金和阿伯泰大学提供。作者声明不存在利益冲突。不适用。
Are significant abnormalities in outward (K+) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K+ channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K+ channels in human spermatozoa or the incidence and functional consequences of K+ channel defects. Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca2+ influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K+ signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. Patch clamp electrophysiology was used to assess outward (K+) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca2+]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized. The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest. Not applicable.