Functional and molecular characterization of voltage gated sodium channel Nav1.8 in bull spermatozoa.
The aim of our study was to characterize the voltage gated sodium channel Nav1.8 in bull spermatozoa. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the nonsignificant variations between different ejaculates. Functional characterization was undertaken using A-803467, a selective blocker of Nav1.8, and veratridine as an opener of the voltage gated sodium channels while molecular characterization was done using western blotting and indirect immunofluorescence assays. In vitro capacitation was induced using heparin, and to study the functional involvement of Nav1.8 in regulation of capacitation induced hyper sperm motility, A-803467 was used. Selective blocking of NaV1.8 by A-803467 at 6 and 8 μM concentration significantly (P < 0.05) decreased the forward progressive sperm motility in a time-dependent manner, while, blocking at higher concentrations (10 and 15 μM) resulted in fast forward motility in spermatozoa after 2 h of incubation and it was observed up to 3 h. Treatment of sperm cells with veratridine (6, 8, 10, 15, 20 μM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 4 h. However, hyperactive motility was induced by veratridine at higher concentrations (10 and 15 μM) after 2 h of incubation. In vitro capacitated spermatozoa treated with A-803467 revealed significant (P < 0.05) reduction in forward progressive motility after 2 h of incubation. Both A-803467 and veratridine altered the percentage of spermatozoa showing high mitochondrial transmembrane potential in concentration- and time-dependent manner. High concentrations (10 and 15 μM) of A-803467 and veratridine resulted in bent neck condition in spermatozoa along with significant (P < 0.05) reduction in membrane integrity (HOST negative). Immunoblot revealed the presence of a single protein band of 260 kDa molecular weight along with positive immunoreactivity (IR) in head, neck, middle piece and tail of the spermatozoa. Strongest IR was observed in the neck and middle piece whereas weak IR was observed in tail and acrosomal region of the spermatozoa. Results of our present study evidently revealed the presence of voltage gated sodium channel Nav1.8 in bull spermatozoa and its functional involvement in regulation of spermatozoa dynamics in terms of motility, membrane integrity, acrosome integrity, capacitation and mitochondrial transmembrane potential. Further studies are warranted to unravel their mechanistic pathways and/or their interaction with other ion channels in regulating sperm dynamics.
College of Biotechnology, UP Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.
Dharmendra Singh
DS
Chauhan
Department of Veterinary Physiology, College of Veterinary Science & Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India. Electronic address: dilip_swain@yahoo.com.
Dilip Kumar
DK
Swain
Department of Veterinary Gynecology & 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.
Nadeem
N
Shah
Department of Veterinary Gynecology & 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.
Hanuman Prasad
HP
Yadav
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science & Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.
Udayraj P
UP
Nakade
Department of Veterinary Gynecology & 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.
Vijay Kumar
VK
Singh
Department of Veterinary Biochemistry, College of Veterinary Science & Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.
Rajesh
R
Nigam
Department of Veterinary Physiology, College of Veterinary Science & Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.
Sarvajeet
S
Yadav
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science & Animal Husbandry, U.P. Pandit Deendayal Upadhayaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, 281001, Uttar Pradesh, India.
Satish Kumar
SK
Garg
D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D005663:Furans / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology; D062559:NAV1.8 Voltage-Gated Sodium Channel / Q000378:metabolism*; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism*; D014701:Veratridine / Q000494:pharmacology
C520740:A 803467; D000814:Aniline Compounds; D005663:Furans; D062559:NAV1.8 Voltage-Gated Sodium Channel; D014701:Veratridine
A-803467; Bull; Immunolocalization; Na(v) 1.8; Sperm motility; Veratridine