Singh, VijayKumar, Akshay2020-12-222020-12-222018-06-23https://krishikosh.egranth.ac.in/handle/1/5810157098Functional characterisation of TRPV1 channel in bull spermatozoaCalcium influx in flagellated cell like spermatozoa plays critical role in regulation of sperm motility, capacitation, hyperactive motility and acrosome reaction. Calcium influx is primarily mediated by Catsper channels and transient receptor potential (TRP) channels. TRP Vanilloid 1 is considered as most versatile thermosensitive, pH sensitive and chemosensitive ion channel found in spermatozoa. Little information is available with contrasting reports regarding the role of TRPV1 in regulation of sperm function. Molecular and functional characterisation of TRPV1 was carried out in spermatozoa of Hariana bulls. Sixty four ejaculates were collected from four bulls and were used for series of experiments. Immunoblotting and immunocytochemistry were employed for the molecular characterisation of TRPV1. Immunoblotting identified a single band of 104 kDa corresponding to TRPV1 in Hariana bull spermatozoa. Positive immune-reactivity was seen in acrosomal, preacrosomal, post acrosomal and flagellar regions corresponding to TRPV1. Functional study was carried out using TRPV1 blocker namely Capsazepine (Cp) @ 10µM and one activator was used namely Anandamide (AEA) @ 0.3µM. In the study, three groups were used namely, control (100 µL of sperm dilution medium (SDM) containing 1×106 cells), vehicle (3µL) and drug (Cp, AEA and their combinations). Different time of incubations was used depending on the experiments. Blocking of TRPV1 resulted in significant (P<0.05) reduction in progressive sperm motility as compared to the control; and with activation using AEA, PSM was decreased significantly (P< 0.05) till 1h and after that PSM was sustained as compared to control. However, both during blocking and activation of TRPV1, per cent spermatozoa showing hyperactive motility was increased (20-30%) (P< 0.05). Evaluation of Cp and AEA treated spermatozoa stained with CTC revealed significant (P< 0.05) increase in B-pattern of spermatozoa indicating induction of capacitation. Spermatozoa treated with different pH gradients showed significant (P< 0.05) reduction in motility as compared to control both with and without drugs modulating TRPV1. Functions of TRPV1 were found to be mediated through cAMP and PKA pathway in the induction of hypermotility in sperm cells as evident from inhibition of sAC and PKA. Both L- and T- type of calcium channels were found to be associated with TRPV1 function as evident from their respective blocking and its effect on PSM. Blocking as well as activation of TRPV1 showed significant (P< 0.05) reduction in sperm livability, per cent spermatozoa having intact membrane, per cent spermatozoa having intact acrosome, per cent spermatozoa showing high mitochondrial transmembrane potential indicating the involvement of TRPV1 in the process of regulation of sperm functional dynamics. From the study, it was concluded that TRPV1 channels are found in bull spermatozoa and are pH dependent. These channels mediate number of sperm functions like hyper motility, capacitation and acrosome reaction through complex interacting pathways through calcium and pH dependent mechanisms. 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 regulationEnglishThesis