目的:Na+/H+交换剂(NHEs)是否以及如何调节人精子的生理功能?
结论:NHE介导的鞭毛细胞内pH(pHi)稳态促进了pH敏感性的激活,精子特异性Ca2+通道(CatSper)和精子特异性K+通道(KSper),随后调节精子活力,过度激活,鞭毛酪氨酸磷酸化,和孕酮(P4)诱导的顶体反应。
背景:精子pHi碱化是获得精子受精能力的必要先决条件。不同的精子功能受到特定pHi调节机制的严格控制。NHE被建议调节精子H+外排。
方法:这是一项实验室研究,使用了超过50名精子捐献者的样本,为期1年。为了评估NHE对人类精子功能的作用,5-(N,N-二甲基)-阿米洛利(DMA),一种高度选择性的NHE抑制剂,被利用。使用不同的单个精子样品或细胞重复所有实验至少五次。
方法:利用pH荧光指示剂pHrodoRed-AM,我们检测到人类精子中单细胞pHi值的改变。通过全细胞膜片钳技术记录了人精子中CatSper和KSper的电流。测量了载有Fluo4-AM的人精子的种群和单细胞Ca2浓度([Ca2]i)的变化。在将精子装载3,3'-二丙基硫二碳花青碘化物和2'后,通过多模平板读数器定量检查膜电位(Vm)和种群pHi,7\'-双-(2-羧乙基)-5-(和-6)-羧基荧光素,乙酰氧基甲酯,分别。精子运动参数通过计算机辅助精液分析系统进行评估。通过免疫荧光测定酪氨酸磷酸化,通过Pisumsativum凝集素-FITC染色评估精子顶体反应。
结果:DMA诱导的NHEs抑制使人类精子鞭毛pHi从7.20±0.04严重酸化至6.38±0.12(平均值±SEM),DMA对顶体pHi的影响不明显(从5.90±0.13到5.57±0.12,平均值±SEM)。全细胞膜片钳记录显示,NHE抑制可显着抑制碱化诱导的CatSper和KSper活化。因此,在存在DMA的情况下,检测到[Ca2]i稳态和Vm维持的损害。在获能过程中,用DMA预处理2小时有效降低精子pHi,进而降低精子活力和动力学参数。精子获能相关功能,包括过度激活,酪氨酸磷酸化,和P4诱导的顶体反应,也受到NHE抑制的损害。
方法:不适用。
结论:这是一项体外研究。当将这些结果外推到体内应用时,应谨慎行事。
结论:这项研究表明,NHE是人类CatSper和KSper的重要生理调节因子,这对人类精子生育来说是不可或缺的,提示NHEs功能异常可能是男性不育发病的潜在机制。
■这项工作得到了国家自然科学基金(32271167和81871202归X.Z.)的支持,江苏省创新创业人才计划(JSSCR20211543至X.Z.),江苏省社会发展项目(编号:BE2022765到X.Z.),南通市社会民生工程(编号:MS22022087到X.Z.),和江苏省自然科学基金(BK20220608至H.K.)。作者没有竞争利益可声明。
OBJECTIVE: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm?
CONCLUSIONS: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction.
BACKGROUND: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux.
METHODS: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells.
METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3\'-dipropylthiadicarbocyanine iodide and 2\',7\'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining.
RESULTS: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition.
METHODS: N/A.
CONCLUSIONS: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications.
CONCLUSIONS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility.
UNASSIGNED: This work was supported by the National Natural Science Foundation of
China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.