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牛精子中电压门控质子通道(Hv1)的功能见解。

Functional insights into voltage gated proton channel (Hv1) in bull spermatozoa.

机构信息

College of Biotechnology, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.

Department of Veterinary Gynaecology & Obstetrics, College of Veterinary Science & Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.

出版信息

Theriogenology. 2019 Sep 15;136:118-130. doi: 10.1016/j.theriogenology.2019.06.015. Epub 2019 Jun 17.

Abstract

Acid extrusion and intracellular alkalisation are the key events during sperm capacitation and these are mediated through proton gated channels (Hv1). Role of Hv1 in regulating sperm motility, capacitation and acrosome reaction has been documented in human spermatozoa; but no such data is available in bull spermatozoa; therefore, the present study was undertaken in Hariana bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls to investigate the functional involvement of Hv1 in regulation of sperm motility, capacitation and acrosome reaction in bull spermatozoa. Immunoblotting revealed the presence of a single band of 31.8 kDa corresponding to Hv1 in Hariana bull spermatozoa and immunoflourescence confirmed the positive immune-reactivity at principal piece of spermatozoa for Hv1. Functional study was carried out using 200 μM 2-Guanidinobenzimidazole (2-Guanidinobenzimidazole,selective Hv1 blocker) and 1 mM zinc chloride (potent Hv1 blocker), and 0.3 μM Anandamide (AEA), an activator of Hv1. Blocking of Hv1 resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM). Activation of Hv1 with AEA also resulted in significant (P < 0.05) reduction in PSM till 1 h and thereafter, the PSM was restored and reduction was almost similar to that in control. However, during activation and inactivation of Hv1, per cent spermatozoa showing hyperactive motility were found to be markedly increased (20-30%) compared to 10-20% in control. 2-Guanidinobenzimidazole, zinc and AEA treated spermatozoa revealed significant (P < 0.05) increase in B-pattern of spermatozoa indicating induction of capacitation. Downstream signalling of Hv1 activation or inactivation seemed to be mediated through cAMP and PKA pathways. Catsper channels were found to be intimately associated with Hv1 function in regulating sperm motility. Blocking as well as activation of Hv1 resulted in significant (P < 0.05) reduction in sperm livability, spermatozoa having intact membrane, intact acrosome, and high mitochondrial transmembrane potential (MTP). Our findings evidently suggest that Hv1 channels are present in bull spermatozoa and these regulate sperm functions like hypermotility, capacitation and acrosome reaction through complex interplay between different pathways involving cAMP, PKC, and Catsper. Further studies are required to find out the possible relationship between Hv1 channels and other channels in regulating spermatozoa functions.

摘要

酸挤出和细胞内碱化是精子获能过程中的关键事件,这些事件是通过质子门控通道(Hv1)介导的。在人类精子中,已经记录了 Hv1 在调节精子运动、获能和顶体反应中的作用;但是在公牛精子中没有这样的数据;因此,本研究在哈里亚纳公牛精子中进行。从 4 头哈里亚纳公牛中收集了 64 份精液,以研究 Hv1 在调节公牛精子运动、获能和顶体反应中的功能作用。免疫印迹显示哈里亚纳公牛精子中存在 31.8 kDa 的单一带,对应于 Hv1,免疫荧光证实了精子主段 Hv1 的阳性免疫反应。功能研究使用 200 μM 2-胍基苯并咪唑(2-胍基苯并咪唑,选择性 Hv1 阻断剂)和 1 mM 氯化锌(强 Hv1 阻断剂)和 0.3 μM 花生四烯酸酰胺(AEA),一种 Hv1 激活剂。阻断 Hv1 导致精子运动能力显著降低(P<0.05)。用 AEA 激活 Hv1 也导致 PSM 在 1 小时内显著降低(P<0.05),此后 PSM 得到恢复,减少几乎与对照相似。然而,在 Hv1 的激活和失活期间,显示超活跃运动的精子百分比发现与对照相比明显增加(20-30%),而对照为 10-20%。2-胍基苯并咪唑、锌和 AEA 处理的精子显示出显著的(P<0.05)B 型精子模式增加,表明诱导获能。Hv1 激活或失活的下游信号似乎是通过 cAMP 和 PKA 途径介导的。Catsper 通道被发现与 Hv1 调节精子运动的功能密切相关。阻断和激活 Hv1 导致精子活力显著降低(P<0.05),精子存活率、完整膜、完整顶体和高线粒体跨膜电位(MTP)降低。我们的发现显然表明,Hv1 通道存在于公牛精子中,这些通道通过涉及 cAMP、PKC 和 Catsper 的不同途径之间的复杂相互作用,调节精子功能,如超活力、获能和顶体反应。需要进一步研究以发现 Hv1 通道与调节精子功能的其他通道之间的可能关系。

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