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 Ca²⁺-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.

Introduction

Continuation of life depends on the reproductive success of individual species. In this context, the fertilization ability of the male gametes, i.e., sperm cells, is important. The spermatozoa of oviparous fishes become motile after being discharged into the aqueous environment while, in viviparous and ovoviviparous fishes, their sperms acquire motility after being discharged into the female genital tract.^1-3 In either case, sperm cells have to locate the female gamete in order to ensure the fertilization. This requires complex tactic yet efficient molecular signaling events that guide the sperm cells movement towards the egg.⁴ Ion concentrations, osmolarity and pH of the media into which they are released are crucial for initiation of sperm motility.^5-7 The regulatory mechanisms involved in these functions and the molecules involved in such movements are not well established. So far, it has been established that Ca_²⁺-signaling play important role in such events.^8-12 Though in general this suggests the importance of different Ca²⁺ channels in the context of sperm functions, the molecular identities and nature of the different Ca²⁺ channels involved in such functions are not well understood. Different complex Ca²⁺ signaling events in general are required for the proper functioning and survival of the sperm cells. However, the nature and spatio-temporal requirements of the Ca²⁺ signaling is variable and depends largely on several internal and external factors.^13,14 In addition to the internal factors, the Ca²⁺-signaling experienced by the sperm cells is directly modulated by the environment into which the sperms are released, thus affecting their survival and fertilizing ability. Sperm cells exhibit extreme selectivity and sensitivity towards proper environment, which promotes their survival, whereas slight changes in the environment is detrimental for these cells. For example, sperm cells demonstrate extreme sensitivity for minute changes in pH, temperature, osmolarity of the media and presence of very low level of elements etc.^7-15 The Ca²⁺ channels present in the sperm cells play an important role in these processes.¹⁶ The Ca²⁺ channels present in the sperm cells are potential molecular targets, which respond to several stimuli in order to modulate the intracellular Ca²⁺ levels.¹⁶ Ca²⁺-signaling also plays an important role in several sperm cell-specific functions such as acrosomal reaction, capacitation, hyper motility etc.¹⁷ Notably, the Ca²⁺-signaling experienced by the sperm cells is variable and this variability often correlates with species differences, morphology of the sperm cells and the environment where the sperms are released.^4,18

Recently, a number of studies have demonstrated that Transient Receptor Potential (TRP) family of channels present in neurons can conduct Ca²⁺ influx in response to different physical factors such as temperature, osmotic pressure, pH and light.¹⁹ In addition, these channels can detect a battery of signaling molecules and thus contribute to the chemosensory processes relevant for neuronal guidance and contact formation.²⁰ As TRP channels act as molecular sensors of physical and chemical factors and are able to conduct Ca²⁺-influx, we explored if thermosensitive TRP channels, especially the members of TRPV subfamily are present in the sperm cells of early vertebrates. In that context, we explored sperm cells from fresh water teleost fish species, namely Labeo rohita (Common name Rahu). Here we report the presence and functional role of TRPV1 in the fish sperm cells.

Results

In order to understand if fish sperm cells contain thermosensitive TRPV channels, we performed western blot analysis of freshly collected sperm cells with a TRPV1-specific antibody (Sigma Aldrich) (Fig. 1A). A band specific for TRPV1 at around 95 KDa, the expected size of the TRPV1 was noticed. This indicates the endogenous expression of TRPV1 in fish sperm cells. FACS analysis was performed to determine the number of fish sperm cells expressing TRPV1 channels. It was noted that in an individual sample, the antibody recognizing N-terminus of TRPV1 (Sigma Aldrich) reacts with nearly 20% sperm cells compared with unstained cells (Fig. 1B). To further confirm that the sperm cells indeed express TRPV1 endogenously, another antibody recognizing the C-terminus of TRPV1 (Alomone) was used in presence as well as in absence of a specific blocking peptide (Alomone). Nearly 13% of the sperm cells are detectable with this antibody. However, when the same antibody (Alomone) was used along with a specific blocking peptide, only 1.82% cells were detected (Fig. 1C). The mean fluorescence intensity also reduces in presence of this blocking peptide. The comparable immunoreactivities obtained by these 2 different antibodies and effective reduction in the respective immunoreacitivity due to the presence of a blocking peptide show the specificity of the antibodies used and hence strongly support the endogenous expression of TRPV1 in these sperm cells. The FACS analysis also indicates that the endogenous expression of TRPV1 in fish sperm cells is not uniform and a large number of cells express TRPV1 below detection limit or these cells do not express TRPV1 at all. In several cases, time-dependent changes in the protein profiles in sperm cells have been co-related with the sperm functions. Therefore, we compared the endogenous expression of TRPV1 in beginning and after 1 h of incubation in 37°C temperature. To get an estimation of the percentage of cells expressing TRPV1, sperm cells from 3 individual fishes in 2 different time points (0 h and 1 h) were analyzed by FACS. Data showed that only 20–40% cells express TRPV1 in the beginning (p = 0.01183; unstained 0 h vs. 0 h sample stained for TRPV1). Interestingly, the expression level reduces slightly with time. After 1 h, approximately 20% cells show detectable TRPV1 indicating a possible time-dependent decay of TRPV1 (p = 0.0002417; unstained 0 h vs. 1 h sample stained for TRPV1) (Fig. 1D). However, the difference between 0 h and 1 h time points become non-significant (p value = 0.2147). To confirm if TRPV1 really decays with time, the mean fluorescence intensity (MFI) values were measured and compared. This reduction in the number of TRPV1-positive cells also correlates with the reduction in the mean fluorescence intensity of TRPV1 when freshly isolated samples were compared with 1-h-old samples (Fig. 1E). Reduction in the MFI-values after 1 h becomes highly significant (p value = 0.000005621; 0 h vs. 1 h sample stained for TRPV1). This indicates that though the number of sperms expressing TRPV1 does not significantly decrease over time, the expression levels may drop down with time.

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.

To visualize the expression pattern of TRPV1 channels in fish sperms, we performed immunolocalization followed by confocal microscopic analysis of freshly collected sperm cells. When probed with TRPV1 specific antibody (Alomone), we noted the presence of TRPV1 in the sperm cells (Fig. 2A, upper panel). This immunoreactivity was abolished when we used a specific blocking peptide (Alomone) suggesting the immunoreactivity was indeed specific for TRPV1 (Fig. 2A, lower panel). Notably, TRPV1 specific immunoreactivity was observed primarily in the head and neck regions. Some faint staining was detected in the tail regions too (Fig. 2B). To confirm further, we used another antibody specific for TRPV1 (Sigma Aldrich). The second antibody also reveals similar pattern of immunoreactivity in the sperm cells (Fig. 2C). In agreement with the FACS data, many of the TRPV1 containing cells reveal differences in the immunoreactivity, both in terms of amount as well as their exact localization. Taken together, these results suggest that fish sperm cells contain TRPV1 endogenously.

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.

Next, we tested the importance of TRPV1 in the sperm functions. For that purpose, we tested the sperm motility and a series of motility experiments were performed. Immediately after coming in contact with water, the initial sperm motility is generally high (approximately 90% or higher percentage of the cells reveal motility in most cases), but it turned out to be variable in individual samples. The initial motility in general reduces with respect to the storage duration also, i.e., the duration of milt stored in 4–8°C after collection. Based on a series of experiments, a general trend becomes prominent (Fig. 3). We observed that in control conditions (i.e., no drugs added to the water), these cells move fast for a very short duration (2–5 min only) and afterwards majority of these cells become absolutely static and/or start floating (Video 1). In contrast, presence of NADA (50 μM added at the beginning) causes majority of the cells to move for a prolonged time (even more than 60 min in some experiments) (Video 2). In the same notion presence of 5′I-RTX (50 μM) results in reduction of sperm movement in the early points (Video 3). Similarly, presence of higher concentration of NADA (1 mM added at the beginning) also reveals higher motility, especially in the early time points (data not shown). We also observed that addition of NADA (1mM) after 6–8 min (at a point when sperm cells do not move at all in control conditions) restores the cell movement further (Video 4). This NADA-induced movement sustained for a prolonged time and we noted movement till 75 min or more. Addition of Ruthenium-Red (1 mM), a general inhibitor of TRP channels results in sharp decline of sperm movement, even in the NADA-treated samples (Video 4). Taken together, these results strongly suggest a functional role of TRPV1 in regulating fish sperm motility.

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.

Next, we attempted to reconstruct the TRPV1 phylogenetic history, especially with respect to fish lineages. For that purpose, we compiled a list of TRPV1 orthologs (Table 1) by confirming orthology with help of a standard method of orthology assessment using bi-directional BLAST, assisted by BLAST2GO tool21 with E-value lower than 1e-6. Bayesian phylogeny constructs a posterior distribution for a parameter using a phylogenetic tree and a model of evolution, based on the prior for that parameter and the likelihood of the data composed by the multiple alignments. Using this Bayesian phylogenetic method, we reconstructed phylogenetic history of TRPV1 within different vertebrate genomes (Fig. 4) from TRPV1 sequences listed in Table 1. Tetrapods have single copy of TRPV1 gene. In contrast, fishes demonstrate copy number variation in TRPV1/2 genes with three copies in Atlantic cod, Gadus morhua (GmoTRPV1/2a-c) and two copies TRPV1/2a-b in the following 3 fishes: Takifugu rubripes (TruTRPV1/2a-b), Tetraodon nigroviridis (TniTRPV1/2a-b) and Oreochromis niloticus (OniTRPV1/2ab). However, some fishes have single copy only, such as Danio rerio (DreTRPV1/2), Latimeria chalumnae (LchTRPV1/2), Xiphophorus maculatus (XmaTRPV1/2), Gasterosteus aculeatus (GacTRPV1/2) and Oryzias latipes (OlaTRPV1/2). This suggests that after separation of tetrapods and fishes, some of these teleost fishes had duplication of TRPV1 gene. Absence of duplicated TRPV1/2 genes in Danio rerio and Latimeria chalumnae, corroborates with the timing of origin of copy number variation which happens within 320 MY after separation of D. rerio from other teleost fishes.²²

Table 1. List of TRPV1 sequences collected from Ensembl (Release 69, October 2012) and GenBank databases.

Sequence name	Species	Accession id	Protein length	E‐value	Mean similarity (%)
HsaTRPV1	Homo sapiens	ENSP00000382661	839	0	97.00
MmuTRPV1	Mus_musculus	ENSMUSP00000099585	839	0	94.95
RnoTRPV1	Rattus norvegicus	ENSRNOP00000026493	838	0	94.65
GgaTRPV1	Gallus gallus	ENSGALP00000007393	843	0	81.45
MgaTRPV1	Meleagris gallopavo	ENSMGAP00000006764	843	0	81.55
TguTRPV1	Taeniopygia guttata	ENSTGUP00000007211	844	0	82.20
XtrTRPV1	Xenopus tropicalis	ENSXETP00000012743	838	0	77.55
DreTRPV1/2	Danio rerio	ENSXETP00000012743	819	0	70.30
LchTRPV1/2	Latimeria chalumnae	ENSLACP00000005090*	363	6.73E‐136	73.75
TruTRPV1/2a	Takifugu rubripes	ENSTRUT00000001736	653	0	72.45
TruTRPV1/2b	Takifugu rubripes	ENSTRUP00000011132	684	0	70.20
TniTRPV1/2a	Tetraodon nigroviridis	ENSTNIP00000011373	690	0	70.45
TniTRPV1/2b	Tetraodon nigroviridis	ENSTNIP00000009206	683	0	70.50
OniTRPV1/2a	Oreochromis niloticus	ENSONIP00000016114	775	0	72.95%
OniTRPV1/2b	Oreochromis niloticus	ENSONIP00000001735	733	0	67.15
GmoTRPV1/2a	Gadus morhua	ENSGMOP00000015245	705	0	67.95
GmoTRPV1/2b	Gadus morhua	ENSGMOP00000014152	661	0	69.70
GmoTRPV1/2c	Gadus morhua	ENSGMOP00000002023	651	5.76E‐159	60.65
XmaTRPV1/2	Xiphophorus maculatus	ENSXMAP00000005454	787	0	64.05
GacTRPV1/2	Gasterosteus aculeatus	ENSGACP00000026761	739	0	71.90
OlaTRPV1/2	Oryzias latipes	ENSORLP00000014367	698	0	70.30
SfoTRPV1/2	Salvelinus fontinalis	EV390862.1*$	141	6.22E‐35	69.95
OmyTRPV1/2	Oncorhynchus mykis	BX884280.3*$	171	8.84E‐106	78.65
IpuTRPV1/2	Ictalurus punctatus	CF262177.1*$	336	5.73E‐137	69.75
SsaTRPV1/2	Salmo salar	ACI34236.1$	804	0	72.30
CIN‐homolog	Ciona intestinalis	XP_002130280$	1150	0	61.40

Orthology of TRPV1 genes are confirmed by bidirectional BLASTP using BLAST2GO tool with E-value lower than 1^e-6.25 Resulting E-value and mean similarity are shown. ENS, Ensembl; ^$GenBank; *Partial

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 V3.2.1.⁵⁸ 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.

Taken together, our work strongly suggests for the sperm-specific expression of the thermosensitive TRPV1 in fresh water living fish (Labeo rohita). Such findings may have implications in cryopreservation of fish sperm cells and artificial breeding of common food carps as well as conservation of rare species.

Discussion

Conservation of different domains and motifs in ion channels often correlates with the prime function of respective ion channels. In fact, the conservation of different domains and motifs as well as tissue and cell-specific expression of ion channels are crucial for the survival of any species as these aspects are indicative of the selection pressures, and thus, function of the ion channels throughout evolution. Recently, we have reported that TRPV1 is a highly conserved protein throughout the evolution.²³ Endogenous expression and involvement of TRPV1 in the sperm cells from different species is in full agreement with its conservation at the protein as well as in the genome level.

In this work, we have explored the physical and functional presence of thermo-sensitive channel, namely TRPV1 in the sperm cells from teleost fish and demonstrate the endogenous presence as well as importance of TRPV1 in the sperm cells of Labeo rohita. The motility experiments with the fish milt also shed light on the general nature of fish sperm motility. The fish milt that we used is mainly a complex suspension of biologically active but uncharacterized lipids. In absence of water, the sperms remain static and start moving vigorously only after encountering with water, though their movement lasts for a very short duration. Though the exact molecular mechanism is poorly understood, we noted that absolutely metal/ion-free water is extremely lethal to the fish sperm cells as all cells die in double distilled water, whereas in waters with trace of ions (such as pond water or tap water) is suitable. This is in accordance with the requirement of other ions and thus ion channels in the fish sperm motility. This result also fits well with the previous reports demonstrating the endogenous presence of TRP channels in sperm cells from diverse organisms. For example, endogenous expression of TRPV1 in boar spermatozoa has been linked with endocannabinoid and anandamide signaling, which have implications in capacitation and acrosomal reaction.^24,25 In case of human also, endogenous expression of TRPV1 has been detected in sperm and linked with sperm functions. In human sperm cells, both hot and cold-sensitive TRP ion channels are present and regulate several functions related to motility and fertility.^26-28 In boar sperm cells, TRPV1 function is important for fertilization.^29-31 In mouse sperm cells, TRPP2 is present at the anterior sperm head region and it is essential for the zona pellucida induced Ca²⁺entry into mouse sperm and subsequent acrosomal exocytosis during fertilization.³² In mouse sperm cells, TRPM8 is involved in the detection of temperature changes and is important for acrosome reaction regulation.^33,34 In the same context, involvement of TRPV6 in male fertility and sterility has been demonstrated.^35,36 Involvement of TRP channels in reproduction may have more important roles and is not limited just to sperm cell survival and movement required for fertilization. TRP channels may even have roles in the spermatogenesis as well, an important aspect which might be evolutionary conserved as well.^37-39 While these reports confirm the endogenous expression and function of TRP channels in the sperm cells of only higher vertebrates, presence of TRP channels in the sperm of any aquatic animals and/or lower vertebrates have not been shown yet. In this work we demonstrated that presence of NADA generally induces a sustained sperm movement (at least for Labeo rohita). Interestingly, this NADA-induced sperm movement can be blocked to some extent by 5′-IRTX, a well-established antagonist of mammalian TRPV1. Also we noted that further application of Ruthenium-Red (1 mM) in NADA-treated samples results in sharp decline of the sperm movements suggesting that NADA-induced sperm movements are due to the involvement of TRP channels mainly. However, we noted that application of capsaicin (a very specific agonist of TRPV1 in several species, mainly in mammals) did not alter the motility fish sperms and cells remain mostly non-responsive to capsaicin (data not shown). Though the relative responsiveness of NADA, 5′IRTX and capsaicin to fish sperm indicates the involvement of TRPV1 in fish motility, the species-specificity of these compounds and involvement of other TRP channels cannot be ruled out completely.

Altogether, our results confirm the endogenous expression and functional importance of TRPV1 in the sperms of lower vertebrates, at least in teleost fishes. TRPV1 is present in various teleost fishes with copy numbers ranging from 1–3 (Table 1; Fig. 4). The copy number variation of TRPV1 gene in teleost fishes originated by duplication events after separation of fishes from last common ancestor of tetrapods and fishes is evident from synteny analysis of TRPV genes.⁴⁰ These duplication events are not older than 320 MY, as it happened after separation of D. rerio from other teleost fishes.²²

Recently, presence of TRP channels in the sperm cells have been detected in some lower eukaryotic organisms too. For example, in C. elegans, TRP channels are required for sperm-egg interactions during fertilization.⁴¹ Polycystin channels localize to cilia and activate Drosophila sperm cells by mediating Ca²⁺-influx.^42,43 In Drosophila, a polycystin-2 homolog channel localized in flagella and is required for male fertility.⁴⁴ Our results are in full agreement with the previous reports, which demonstrated that temperature is an important regulatory factor for maturation and motility in fish sperm cells.^45-47 Indeed it is known that carp sperms take more time for acquiring motility when incubated in cold water (15°C) vs. warm water (20°C).⁴⁷ This thermo sensitivity of sperms could be due to presence of thermosensitive TRP channels in the sperms.

In this work, we demonstrated that a fraction of fish sperm cells express TRPV1 and it localizes mainly in the neck region of the cells. In some cases, TRPV1 localizes in head and tail region also. The differential level of expression and localization of TRPV1 may be a correlative of differential response of sperm cells in response to temperature and other factors. The other functional roles of TRPV1 in fish sperms need to be explored in details.

Materials and Methods

Collection of fish sperm

Male broods of Labeo rohita were collected from the experimental pond of Physiology Division, Central Institute of Freshwater Aquaculture (CIFA). Milters were induced with “Ovaprim” at the rate of 0.2–0.3 ml/kg body weight of the fish during peak breeding season (in the time of early August). Milters were stripped into separate plastic tubes held over ice. Extreme care was taken to avoid contamination of water, blood, urine, feces, etc. After that, milt samples were processed for further analysis.

SDS PAGE and western blot analysis

Freshly collected milt was diluted in 1× PBS and quickly centrifuged at 8000 RPM for 5 min in 25°C. After that the pellet fraction was diluted in 1× PBS supplemented with protease inhibitor cocktail (Sigma Aldrich) and sonicated (pulse rate 50Hz for 5 min, 5 s intervals) in ice. 5× Laemmli gel sample buffer was added directly to the sonicated samples. The samples were boiled and subsequently analyzed by 10% sodium dodecyl sulfate PAGE (SDS-PAGE) according to Laemmli.⁴⁸ Due to the high lipid and DNA content, the samples were separated by SDS-PAGE for around 12 h in a mini-gel (BioRad). The proteins were electrophoretically transferred to PVDF membrane (Milipore) according to the procedure described elsewhere.⁴⁹ After blocking for 1 h in TBST (20 mM Tris [pH 7.4], 0.9% (w/v) NaCl, and 0.1% (v/v) Tween 20) containing 5% (w/v) dry skim milk, the membranes were incubated with mouse monoclonal antibody directed against C-terminus of TRPV1 (Dilution 1:200; Sigma Aldrich) for overnight. After extensive washing in TBST, the membranes were incubated with horse-radish peroxidase-conjugated secondary antibody raised against mouse (GE Healthcare) for 1 h at room temperature (25°C). Again, the membranes were extensively washed in TBST and bands were visualized on chemi-doc (BioRad) by enhanced chemiluminescence according to the manufacturer’s instructions (Thermo scientific).

Immunofluorescence analysis and microscopy

For immunocytochemical analysis, immediately after collection, sperm cells were diluted in PBS and fixed with paraformaldehyde (PFA) (final concentration 2%). After fixing the cells with PFA, the cells were permeabilized with 0.1% Triton X-100 in PBS (5 min). Subsequently, the cells were blocked with 5% bovine serum albumin for 1 h. The primary antibodies were used at the following dilutions: mouse monoclonal anti-TRPV1 (1: 200, Sigma Aldrich), rabbit polyclonal anti-TRPV1 (1: 200, Alomone Lab). In some experiments, blocking peptide [EDAEVFKDSMVPGEK, corresponding to amino acid residues 824–838 of rat TRPV1 (Accession O35433)] was used to confirm the specificity of the immunoreactivity. The blocking peptides were used at 1:1 dilution. The blocking peptides were used against rabbit polyclonal anti-TRPV1 (Alomone Lab).

All primary antibodies were incubated for overnight at 4°C in PBST buffer (PBS supplemented with 0.1% Tween-20). AlexaFluor-488 labeled anti rabbit or anti mouse antibodies (Molecular probes) were used as secondary antibodies and were used at 1:1000 dilutions. All images were taken on a confocal laser-scanning microscope (LSM-780, Zeiss) with a 63X-objective and analyzed with the Zeiss LSM image examiner software and Adobe Photoshop.

Motility assay for fish sperm

Freshly collected water free neat milt from Rohu fishes were collected from the pond and the milt from different individual fishes were brought to lab within 30–40 min. Temperature of the milt was strictly maintained at 15–20°C and subsequently the milt was stored at 4–8°C freezer for a 96 h during which all the motility experiments were done. A spot of 4 µL water was made on microscopic slide (Globe Scientific, 1304) and then a small quantity (0.1–0.2 μl) of Rohu milt was added to the spot. Immediately after adding the milt to the water, a coverslip (Fisher Scientific) was gently placed above the water spot and the sperm movement was visualized/captured by using Olympus (BX51) microscope. The approximate percentage of cell motility was estimated as described previously.^50-53 The movements of rohu spermatozoa were recorded as movies (400-500 frames/minute) for 1 minute. The original movies were processed using Movie-maker software. For modulation of TRPV1, specific activator (NADA, Sigma Aldrich) or inhibitors such as (5′I-RTX, Sigma Aldrich) or RutheniumRed (Sigma Aldrich) at different concentrations was added to the water and these solutions were used for spotting. In some experiments aimed to add activators or inhibitors after a fixed time, the drug containing water solution (5×) was made and 1 µL of this 5× solution was added from the side of the coverslip (after the required time on a control sample). Due to the capillary action, the drug solution added to the side goes inside quickly. For this assay, we used normal tap water as double distilled water turned out to be extremely lethal for the cells.

FACS analysis

Expression of thermo-sensitive TRPV1 channel in fish sperm cells was assessed by Flow cytometry. Single cell suspensions of fish sperm cells were made in presence and absence of TRPV1 specific blocking peptide (EDAEVFKDSMVPGEK, corresponding to amino acid residues 824–838 of rat TRPV1 [Accession O35433]) and used for flow cytometry on a FACS Calibur instrument (BD Biosciences). The cells were washed, blocked (with 5% bovine serum albumin) and incubated with rabbit polyclonal anti-TRPV1 antibodies (1:200, Alomone Lab) or mouse monoclonal anti-TRPV1 (1: 200, Sigma Aldrich) for overnight followed by washing by PBST buffer (PBS supplemented with 0.1% Tween-20). The cells were then incubated with AlexaFluor-488 labeled anti-rabbit or anti-mouse antibodies (1:1000 dilution, Molecular probes) for 1 h. After washing, the cells were re-suspended in PBS supplemented with 2% BSA, 0.1% sodium azide. Unstained cells were used as a negative control. The labeled cells were washed, detected by Flow Cytometry using a FACS Calibur and analyzed by Cell Quest Pro software (BD Biosciences). Around 10,000 cells were analyzed for each sample.

Sequence collection and phylogenetic analyses

TRPV1 sequences were collected using Ensembl⁵⁴ from following species: Latimeria chalumnae, Takifugu rubripes, Tetraodon nigroviridis, Oreochromis niloticus, Gadus morhua, Danio rerio, Gallus gallus, Meleagris gallopavo, Taeniopygia guttata, Xenopus tropicalis, Mus musculus, Rattus norvegicus and Homo sapiens. Furthermore, TRPV1 homologs were collected using GenBank from following species: Salvelinus fontinalis, Oncorhynchus tshawytscha, Oncorhynchus mykiss, Ictalurus punctatus, Salmo salar and Ciona intestinalis. All these sequences were scanned by BLAST tool⁵⁵ with E-value lower than 1e-6. Collected sequences were annotated and orthologous status of these sequences were confirmed by using bi-directional blast with the help of BLAST2GO software²¹ with E-value lower than 1e-6. Result of this analysis is summarized in Table 1. Protein sequence alignment of collected TRPV1 was performed using MUSCLE software.^56,57 Phylogenetic tree was constructed using this protein alignment by the well-accepted and most powerful Bayesian approach (5 runs, until average standard deviation of split frequencies was lower than 0.0098, 25% burn-in-period, WAG matrix-based model in the MrBayes version V3.2.1.^58,59

Statistical test

The FACS data for 3 individual fishes (n = 3) were imported to “R” software for statistical analysis and graphical representation. Using R, box-plots were generated to represent the percentage of TRPV1 positive cells and their mean fluorescence intensities in arbit unit (au). The Annova test was done for each set of data to check the reliability and significance of the data points. P-values < 0.05 were considered as statistically significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Material

Supplemental material be found here: http://www.landesbioscience.com/journals/channels/article/25793