Differential expression and localization of thermosensitive Transient Receptor Potential Vanilloid (TRPV) channels in the mature sperm of white pekin duck (Anas platyrhynchos)

Sperm cells have the ability of precise chemotactic and thermotactic movement which is crucial for fertilization, yet the key molecules involved in the detection of different chemical and physical stimuli and guide the sperm cells for proper navigation are not known. This aspect is more complex as each species have their own reproductive identity. Never-the-less, Ca2+-signaling and thus a series of Ca2+-channels seem to coordinate in order to regulate different functions mediated by sperm cells. However, such aspects are controlled by different Ca2+ channels and have species-specific differences. In this work we explored if TRPV channels are endogenously expressed in the mature spermatozoa obtained from avian species. We have used the sperm cells of white pekin duck (Anas platyrhynchos) as a representative avian species to explore the endogenous expression and localization of different TRPV channels. Western blot analysis (WB), flow cytometry, confocal imaging and super resolution imaging was performed for the characterization. Our results strongly suggest the expression and distinct localization of different TRPV channels in the sperm cells. All these TRPV channels are mainly absent in the head region. Only TRPV3 and TRPV4 are sparsely present in the neck region enriched with mitochondria. All these channels (TRPV1-6) are present in the tail region. The differential localization of TRPVs in duck sperm indicate their respective functions relevant in fertilization process of avian sperm. These findings may also have commercial importance in poultry production, cryopreservation of sperm as well as conservation of endangered species through artificial insemination.

the mature spermatozoa obtained from avian species. We have used the sperm cells of white pekin 23 duck (Anas platyrhynchos) as a representative avian species to explore the endogenous expression 24 and localization of different TRPV channels. Western blot analysis (WB), flow cytometry, 25 confocal imaging and super resolution imaging was performed for the characterization. Our results 26 strongly suggest the expression and distinct localization of different TRPV channels in the sperm 27 cells. All these TRPV channels are mainly absent in the head region. Only TRPV3 and TRPV4 are 28 sparsely present in the neck region enriched with mitochondria. All these channels (TRPV1-6) are 29 present in the tail region. The differential localization of TRPVs in duck sperm indicate their 30 respective functions relevant in fertilization process of avian sperm. These findings may also have 31 commercial importance in poultry production, cryopreservation of sperm as well as conservation 32 of endangered species through artificial insemination. swim up procedure across a temperature gradient of 35°C to 38°C were observed to show better 66 fertilizing ability [5]. This precise thermosensitivity also accords well with the existence of 67 temperature gradient between oviduct isthmus and ampulla [2]. 68 Timing of each of these responses is critical and premature or inappropriate activation of 69 these events can lead to failure in fertilization. Since sperm cells are mostly transcriptionally and 70 translationally inactive, all cellular activities within it are carried out by the pool of proteins that 71 are generally inherited during differentiation of spermatozoa and these proteins are responsible to 72 regulate sperm functions via secondary messengers like intracellular Ca 2+ [6][7][8]. In ejaculated 73 sperm cells, intracellular Ca 2+ -influences motility, chemotaxis, capacitation, hyperactivation and 74 acrosome reaction [9-13]. The Ca 2+ -signaling is initiated and maintained by Ca 2+ -permeable ion 75 channels present in the sperm. Besides regulating calcium signaling, these channels also sense the 76 chemical cues and thermal environment of the sperm and modulate the sperm in accordance with 77 the stimuli. 78 The ability to respond minute differences in temperature suggests that mature sperms are 79 equipped with the specialized thermo-responsive machineries at the molecular level. Such  (TRPV1) and consists of 6 members TRPV1, TRPV2, TRPV3, TRPV4,   91   TRPV5 and TRPV6. While TRPV1, TRPV2, TRPV3, TRPV4 are non-selective for cations and   92 are thermosensitive in nature, TRPV5 and TRPV6 are relatively more selective for Ca 2+ ion and 93 not thermosensitive in nature. Efforts by various groups has led to increasing knowledge 94 about their localization and functional relevance of specific TRP channels in various cell types, 95 including sperm cells. 96 Indeed, heat sensitive TRPV1 has been shown to be functional in fish, mice, boar, bull and 97 human sperm [16][17][18][19][20]. TRPV1 has been shown to be important for progesterone-induced sperm 98 oocyte fusion and TRPV1 activator anandamide can capacitate bull sperm [19,21]. TRPV1 has 99 also been demonstrated to mediate thermotaxis of human sperm [20]. Interestingly TRPV4 has 100 been also shown to mediate thermotaxis in mouse sperm and initiate the initial depolarization event 101 for capacitation in human sperm [22][23]. TRPV4 has been reported by us to be present in all 102 vertebrate sperm [24]. TRPV2, TRPV3, TRPV5 and TRPV6 haven't been reported in the sperm 103 of any species so far. In spite of several reports, TRP channels, including TRPV channels have 104 remained relatively unexplored in avian sperm. Previously we have described the unique 105 morphology of mature duck sperm and presence of different modified tubulin in such cells [25].

106
Since endogenous expression and localization of these channels can be correlated to sperm 107 function, therefore, in this work we explored the presence of TRPV channels in avian (duck 108 sperm). We noted distinct pattern of localization of the thermosensitive TRPV1, TRPV2, TRPV3, 109 TRPV4 channels and highly calcium-selective TRPV5, TRPV6 channels along the duck sperm.   analysis as described earlier [25]. For immunostaining, the fixed sperm were washed with PBS 137 thrice by centrifugation at 800 g for 5 minutes each time. The sperm were then permeabilized with 138 0.1% TritonX-100 (Sigma-Aldrich) in PBS for 5 minutes, followed by blocking with 5% Bovine 139 Serum Albumin (BSA, Sigma Aldrich) for 1 hour. All above procedures were performed at room

186
Western blotting was performed as described by us previously, but with minor 187 modifications [16,24]. Freshly collected duck sperm was diluted in 1XPBS and centrifuged at 800 In other cases, we have used two antibodies to detect the same channel, to establish the presence 218 of these channels at the protein level in duck sperm. Using an antibody, directed against the C-219 terminus of TRPV1 (Alomone Labs), we detected a ~95kDa band and most of the signal was 220 blocked upon pre-incubating the antibody with its antigenic peptide, confirming specificity of the 221 antibody used (Fig. 1A). Western blot analysis (using Ab1, that detects TRPV2 C-terminus,

222
Alomone Labs) revealed a ~86kDa band for TRPV2 and majority of the signal was blocked upon 223 pre-incubating the antibody with its antigenic peptide, confirming specificity of the antibody used 224 (Fig. 1B). Two prominent bands at ~75kDa and ~65kDa for TRPV3 were detected by western blot 225 analysis (using two different antibodies raised against the C-terminus of TRPV3 (Ab1: Alomone 226 Labs) and another antibody against N-terminus: Ab3 from Sigma Aldrich) (Fig. 1C). The presence against N-terminus (Ab3 from Sigma Aldrich) (Fig. 1D). Western blot analysis using two different 235 antibodies raised against the C-terminus of TRPV5 (Ab1: Alomone Labs and Ab2: Sigma-Aldrich) 236 revealed a prominent band at ~85kDa and a faint band at ~100kDa (Fig. 1E). Western blot analysis 237 (using Ab1: against the C-terminus of TRPV6, Alomone Labs) revealed a prominent band at 238 ~85kDa and that could be blocked by its antigenic peptide (Fig. 1F). This indicated that these 239 TRPV channels are endogenously expressed in mature sperm of duck.

240
Notably, the difference in band size and predicted molecular weight of the TRPV channels 241 could arise from species or individual specific differences, post-translational modifications, partial 242 degradation of the proteins or presence of splice variants [28]. However, we have consistently 243 obtained prominent bands near the predicted molecular weight for all channels tested. We cannot 244 rule out the possibility of some extent of non-specific binding associated with these antibodies, 245 which make it challenging to solely rely on western blotting. We have therefore characterized are positive for TRPV4 (Fig. 2). The mean fluorescence intensity (MFI) values also show nearly sperm. However, TRPV1 density was highest at the head (Fig. 3A, B, D). Interestingly, TRPV1 is 294 exclusively absent in the mitochondrial region (neck region) of sperm (indicated by white arrows).

295
SIM-based super resolution imaging (using Ab1) revealed a punctate distribution of TRPV1 296 throughout the sperm (Fig. 3C).   Using two different antibodies raised against the C-terminus of TRPV2 (Ab1 and Ab2) we 308 found that TRPV2 is primarily present at the tail of duck sperm (Fig. 4A, B, D). Confocal imaging 309 of MitoTracker red labelled duck sperm confirm the specific absence of TRPV2 in the 310 mitochondrial region (neck region) (Fig. 4A).

320
Confocal microscopy using two different TRPV3 antibodies (Ab1 and Ab2) we found that 321 TRPV3 is primarily present at the head and tail of duck sperm (Fig. 5A, B, D), while very low 322 levels of TRPV3 is present at the mitochondrial region (neck region) (Fig. 5A). SIM-based super 323 resolution imaging (using Ab1) revealed a more prominent punctate distribution of TRPV3 at the 324 tail of sperm (Fig. 5C).  Confocal microscopy using two different antibodies against the C-terminus of TRPV4 we 334 found that TRPV4 is very scarce in the head, prominent at the mitochondrial region (seen by its 335 colocalization with MitoTracker Red, indicated by white arrows) and at the tail of duck sperm 336 (Fig. 6A, B, D). SIM-based super resolution imaging (using Ab1) revealed punctate distribution 337 of TRPV4 throughout the sperm (Fig. 6C).  Confocal microscopy using two different antibodies against the C-terminus of TRPV5 we 347 found that TRPV5 is exclusively present at the tail (indicated by white arrows), with highest 348 density immediately after the mitochondrial region and intensity gradually decreasing towards the 349 tapering end of the tail (Fig. 7A, B, D). TRPV5 is absent at the head and neck region. SIM-based 350 super resolution imaging (using Ab1) revealed punctate distribution of TRPV5 at the tail of sperm 351 (Fig. 7C).

360
We detected the presence of TRPV6 throughout the sperm using confocal microscopy via 361 two different antibodies against the C-terminus of TRPV6 (Ab1 and Ab2) (Fig. 8A, B, D). SIM-362 based super resolution imaging (using Ab1) revealed punctate distribution of TRPV6 throughout 363 the sperm (Fig. 8C). Taken together, our results confirm the endogenous expression and 364 differential localization of TRPVs in duck sperm.

391
We have previously shown the presence of TRPV1 in the neck and tail region of fish sperm 392 [16]. Pharmacological modulation of TRPV1 alters fish sperm motility and their ability to swim 393 increases from 2 minutes to 90 minutes [16]. In this work, we have observed that TRPV1 is 394 abundant at the neck and tail regions, indicating that TRPV1 could also regulate duck sperm 395 motility. TRPV1 has also been shown to regulate thermotaxis of human sperm and this feature 396 could be conserved in other warm blooded vertebrates like birds too [20]. TRPV1 has also been 397 shown to regulate capacitation of other relevant species, at least for bull and boar sperm [18][19]. as sperm from TRPV4 -/animals (TRPV4 knock-out) didn't show this thermo-sensitivity [22].

419
Recently, TRPV4 has been confirmed as a temperature-sensitive ion channel of human sperm, and 420 TRPV4 activation has been demonstrated to trigger the initial membrane depolarization in sperm, 421 facilitating CatSper and Hv1 activation and, subsequently resulting in sperm hyperactivation, a 422 phenomenon essential for sperm-egg fusion [23]. Based on these rent work, it is possible that in 423 duck too, TRPV4 can regulate critical aspects of sperm physiology and can have a major impact 424 on fertility in duck.

425
TRPV5 and TRPV6 channels are not thermosensitive, but are highly Ca 2+ selective ion 426 channels. So far TRPV5 hasn't been reported in the sperm of any other species, except rat. In rat,

427
TRPV5 express in the spermatogenic cells and in spermatozoa [35]. In this work, we report that 428 TRPV5 has the highest intensity among all TRPV channels. TRPV5 expression is also very 429 specific, it starts with highest density immediately after the mitochondrial region and gradually 430 decreases towards the tapering end of the tail. Such prominent expression of TRPV5 in duck sperm 431 tail can be strongly correlated with the motility functions performed by the tail region.

432
Similarly, we demonstrate that TRPV6 is also present in the duck sperm. TRPV6 is 433 localized throughout the duck sperm, however among all TRPV channels tested in this work, the 434 expression of TRPV6 is quite low. Recent reports suggest that mutations rendering inactivation of 435 TRPV6 or excision of the pore-forming region and the complete cytosolic C terminus of TRPV6

436
(thus rendering it non-functional) leads to infertility in male mice [36][37]. Both these studies have and changes in such proteomes have huge importance in the context of fertility and sterility [40].

450
In this work, we demonstrate that at least one detectable form of TRPV1-TRPV6 is expressed in