Thermosensitive ion channel TRPV1 is endogenously expressed in the sperm of a fresh water teleost fish (Labeo rohita) and regulates sperm motility

Abstract

Sperm cells exhibit extremely high sensitivity in response to slight changes in temperature, osmotic pressure and/or presence of various chemical stimuli. In most cases throughout the evolution, these physico-chemical stimuli trigger Ca (2+)-signaling and subsequently alter structure, cellular function, motility and survival of the sperm cells. Few reports have recently demonstrated the presence of Transient Receptor Potential (TRP) channels in the sperm cells from higher eukaryotes, mainly from higher mammals. In this work, we have explored if the sperm cells from lower vertebrates can also have thermo-sensitive TRP channels. In this paper, we demonstrate the endogenous presence of one specific thermo-sensitive ion channel, namely Transient Receptor Potential Vanilloid family member sub type 1 (TRPV1) in the sperm cells collected from fresh water teleost fish, Labeo rohita. By using western blot analysis, fluorescence assisted cell sorting (FACS) and confocal microscopy; we confirm the presence of this non-selective cation channel. Activation of TRPV1 by an endogenous activator NADA significantly increases the quality as well as the duration of fish sperm movement. The sperm cell specific expression of TRPV1 matches well with our in silico sequence analysis. The results demonstrate that TRPV1 gene is conserved in various fishes, ranging from 1-3 in copy number, and it originated by fish-specific duplication events within the last 320 million years (MY). To the best of our knowledge, this is the first report demonstrating the presence of any thermo-sensitive TRP channels in the sperm cells of early vertebrates as well as of aquatic animals, which undergo external fertilization in fresh water. This observation may have implications in the aquaculture, breeding of several fresh water and marine fish species and cryopreservation of fish sperms.

Keywords: Ca2+ channels; Ca2+-signaling; Capsaicin; Labeo rohita; NADA; TRPV1; Vertebrate evolution; sperm cells; sperm motility; teleost fish.

Figure 1. Endogenous expression and immunodetection of TRPV1 channel in fish (Labeo rohita) sperm cells. (A) western blot analysis of sperm cells by a TRPV1-specific antibody. The arrow indicates a TRPV1-specific band at the position of 95 KDa. (B) Fluorescence activated cell sorting analysis of fish sperm cells by a TRPV1 specific antibody. A large number of cells react to an antibody recognizing the N-terminus of TRPV1 (Sigma Aldrich) (lower panel) when compared with the other unstained samples (upper panel). (C) Fluorescence activated cell sorting analysis of fish sperm cells by another antibody recognizing the C-terminus of TRPV1 (Alomone) and the respective blocking peptide. A large number of cells react to this TRPV1-specific antibody (lower panel) when compared with the other unstained samples (upper panel, left side) or peptide blocked sample (upper panel, right side). The comparative mean fluorescent intensity is provided in the lower panel (right side). (D) Not all sperm cells express TRPV1. This box-plot diagram represents the percentage of sperm cells that reveal TRPV1 staining. After 1 h, less number of cells remains positive for TRPV1. * = Significant, *** = highly significant, ns = non-significant. (E) Time-dependent decay of TRPV1 in sperm cells. This box-plot diagram reveals the mean fluorescence intensity (MFI) of TRPV1 present in the sperm population. The MFI-value for TRPV1 reduces after 1 h. *Significant, ***highly significant.

Figure 2. Immunolocalization of TRPV1 channel in Labeo rohita sperm cells. (A) Shown are the 3D confocal images of clustered sperm cells immunostained with TRPV1-specific antibody either in presence (lower panel) or in absence (upper panel) of a blocking peptide. (B) Confocal images depicting an enlarged area of a single sperm cell head region are shown here. The arrows indicate the regions that are enriched with TRPV1. Scale bars are indicated in the respective images. (C) Confocal image of sperm cells present in a cluster (upper panel), or an enlarged area of a single sperm cell (middle panel) or a single head region (lower panel) is shown here. The arrows indicate the regions that are enriched with TRPV1. Scale bars are indicated in the respective images.

Figure 3. Involvement of TRPV1 in fish sperm motility. The trend of a series of motility experiments with fish sperms are schematically represented here. (A) The percentage cell motility of the fish sperm in control conditions (red line and shaded regions) and in presence of NADA (50 μM, indicted by green line and shaded region) or 5′I-RTX (50 μM, indicated by blue line and shaded region) are shown. While the sperm movements in control conditions stop quickly, presence of NADA results in sperm motility for a prolonged time. (B) Application of NADA (1mM) in static sperm cells (when cells become static after initial movement), results in further stimulation and sustained movement of the cells for a prolonged time. Addition of Ruthenium-Red (1 mM) results in sharp decline of the motility. For details see Supplemental Movies.

Figure 4. Phylogenetic history of the TRPV1 gene. The Bayesian phylogenetic history demonstrates that there is a single copy of this gene is conserved across different vertebrates with some ray-finned fishes have 2 copies (TRPV1/2a-b). Bayesian phylogenetic tree of TRPV1 proteins from mammals (yellow), birds (cyan), and fishes was generated using Mrbayesversion V3.2.1. Fish-specific TRPV1/2b is marked in red shade. Putative TRPV like gene (GenBank id XP_002130280) from Ciona intestinalis served as the out-group in this Bayesian tree. Percentage posterior probabilities are shown at the node of the branches. Has, Homo sapiens; Mmu, Mus musculus; Rno, Rattus norvegicus; Gga, Gallus gallus; Mga, Meleagris gallopavo; Tgu, Taeniopygia guttata; Xtr, Xenopus tropicalis; Dre, Danio rerio; Lch, Latimeria chalumnae; Tru, Takifugu rubripes; Tni, Tetraodon nigroviridis; Oni, Oreochromis niloticus; Gmo, Gadus morhua; Xma, Xiphophorus maculates; Gac, Gasterosteus aculeatus; Ola, Oryzias latipes.