Kumar Akshay, Mishra Abhishek Kumar, Singh Vijay, Yadav Sarvajeet, Saxena Atul, Garg Satish Kumar, Swain Dilip Kumar
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.
College of Biotechnology, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.
Theriogenology. 2019 Apr 1;128:207-217. doi: 10.1016/j.theriogenology.2019.01.029. Epub 2019 Feb 5.
In view of the limited information available on functional significance of TRPV1 in regulating sperm functions, present study was undertaken on bull spermatozoa. Sixty four ejaculates were collected from four Hariana bulls and were used for molecular and functional characterisation of TRPV1. Immunoblotting using TRPV1 specific antibody revealed the presence of a single band of 104 kDa corresponding to TRPV1 in Hariana bull spermatozoa. Indirect immuno fluorescence revealed positive immune-reactivity to TRPV1 at acrosomal, pre-acrosomal, post acrosomal and flagellar regions of spermatozoa. Based on the results of pilot study dose-response analysis, doses of anandamide (AEA; 0.3 μM) and capsazepine (Cp; 10 μM) were selected for further studies. Three groups of semen samples (control 100 μL diluted semen having 1 × 10 spermatozoa; anandamide (3 μL AEA+97 μL of diluted semen containing 1 × 10 spermatozoa and Cp (1 μL Cp+99 μL of diluted semen containing 1 × 10 spermatozoa) were used to study the functional involvement of TRPV1 in bull spermatozoa. Blocking of TRPV1 with Cp resulted in significant (P < 0.05) reduction in progressive sperm motility (PSM) as compared to control. With activation of TRPV1 using AEA also, PSM was significantly (P < 0.05) decreased till 1h and thereafter the PSM was sustained to the level as observed in control. However, both during blocking and activation of TRPV1, per cent spermatozoa showing hyperactive motility were significantly (P < 0.05) increased (20-30%) compared to the control. Treatment with both Cp and AEA revealed significant (P < 0.05) increase in B-pattern of spermatozoa in chlortetracycline hydrochloride (CTC) staining indicating induction of capacitation. Inhibition of soluble adenyl cyclase (sAC) with 99 nM KH7and protein kinase A (PKA) with 3 μM P9115 significantly (P < 0.05) decreased PSM both in the presence of Cp and AEA. Blocking as well as activation of TRPV1 showed significant (P < 0.05) reduction in sperm livability, intact membrane, intact acrosome, high mitochondrial transmembrane potential; hence indicating the involvement of TRPV1 in regulation of sperm functions in bulls. From the study-it was concluded that TRPV1 channels are found in bull spermatozoa and mediate number of sperm functions like motility, hypermotility, capacitation and acrosome reaction. Further studies are required to find out the possible relationship between TRPV1 channels and other channels in regulating spermatozoa function and possible mechanisms associated with TRPV1 activation as well as its role in sperm function regulation.
鉴于关于瞬时受体电位香草酸亚型1（TRPV1）在调节精子功能方面的功能意义的可用信息有限，本研究以公牛精子为对象展开。从四头哈里亚纳公牛采集了64份精液，用于TRPV1的分子和功能特性研究。使用TRPV1特异性抗体进行免疫印迹分析，结果显示在哈里亚纳公牛精子中出现了一条对应于TRPV1的104 kDa单条带。间接免疫荧光显示精子的顶体、顶体前、顶体后和鞭毛区域对TRPV1呈阳性免疫反应。基于初步研究剂量反应分析的结果，选择了花生四烯乙醇胺（AEA；0.3 μM）和辣椒平（Cp；10 μM）的剂量用于进一步研究。三组精液样本（对照组为100 μL稀释精液，含有1×10⁶精子；花生四烯乙醇胺组为3 μL AEA + 97 μL含1×10⁶精子的稀释精液；辣椒平组为1 μL Cp + 99 μL含1×10⁶精子的稀释精液）用于研究TRPV1在公牛精子中的功能作用。与对照组相比，用Cp阻断TRPV1导致精子前向运动率（PSM）显著（P < 0.05）降低。使用AEA激活TRPV1时，PSM在1小时内也显著（P < 0.05）下降，此后PSM维持在对照组观察到的水平。然而，在阻断和激活TRPV1的过程中，与对照组相比，表现出超激活运动的精子百分比均显著（P < 0.05）增加（20 - 30%）。用Cp和AEA处理均显示盐酸金霉素（CTC）染色中精子B型显著（P < 0.05）增加，表明诱导了精子获能。用99 nM KH7抑制可溶性腺苷酸环化酶（sAC）和用3 μM P9115抑制蛋白激酶A（PKA）在存在Cp和AEA的情况下均显著（P < 0.05）降低了PSM。阻断以及激活TRPV1均显示精子活力、完整膜、完整顶体、高线粒体跨膜电位显著（P < 0.05）降低；因此表明TRPV1参与了公牛精子功能的调节。从该研究得出结论，TRPV1通道存在于公牛精子中，并介导多种精子功能，如运动、超激活运动、获能和顶体反应。需要进一步研究以找出TRPV1通道与其他通道在调节精子功能方面的可能关系，以及与TRPV1激活相关的可能机制及其在精子功能调节中的作用。
Zotero 插件