The role of reactive oxygen species (ROS) (Aitken and Drevet, 2020) and calcium in sperm function is well established (Jimenez-Gonzalez et al., 2006) where physiological level drives important sperm functions such as capacitation, acrosome reaction, fertilization, and augmented level results in DNA fragmentation, change in membrane fluidity, apoptosis and failed fertilization. With the silencing of transcriptional and translational machinery in spermatozoa, maintenance of ion homeostasis particularly calcium and redox balance in a spatiotemporal manner is essential to mediate successful fertilization (Suarez, 2008; Beltrán et al., 2016; Puga Molina et al., 2018). Although data on spermatozoa in general and with respect to humans are unavailable, bi-directional crosstalk between Ca²⁺-signaling and ROS modulates physiological processes as a part of a regulatory circuit is reported (Görlach et al., 2015). High levels of ROS increase Ca²⁺-influx into cells by modulating the activity of versatile Ca²⁺ channels, pumps, and exchangers. Subsequently, Ca²⁺-overload in cells activates ROS-generating enzymes augmenting free radical production (Görlach et al., 2015). At physiological levels, such reciprocal interaction generally functions as a compensatory mechanism to maintain the redox balance and Ca²⁺-homeostasis of the cell. However, at pathological levels of either Ca²⁺ and/or ROS, the entire feedback cycle would essentially get disturbed thereby potentially damaging the cell, and ultimately triggering apoptosis (Feno et al., 2019). Thus, it could be surmised that ROS regulates the function of the intracellular calcium channels, which in turn control ROS levels. With regard to spermatozoa, studies have only speculated indirect activation of calcium channels in sperm by ROS via activation of kinases and subsequent phosphorylation of the channel. However, several ion channels can be directly activated by ROS to modulate the pathophysiological conditions. One such versatile yet less explored redox-sensitive ion channel in human sperm is the transient receptor potential vanilloid 1 (TRPV1) channel (Francavilla et al., 2009; Lewis et al., 2012; De Toni et al., 2016). TRPV1 is a multimodal calcium conducting ion channel belonging to the mammalian TRP channel family, whose activity in regulating sperm capacitation and acrosomal reaction has been demonstrated in different species (Maccarrone et al., 2005; Francavilla et al., 2009; Bernabò et al., 2010; Cobellis et al., 2010; Catanzaro et al., 2011; Gervasi et al., 2011; Lewis et al., 2012; Osycka-Salut et al., 2012; De Toni et al., 2016; Kumar et al., 2019). However, most of the studies of TRP in human spermatozoa have been undertaken with fertile and/or normozoospermic individuals, with few data as yet available on the activity and expression of TRP in spermatozoa of males with known or idiopathic infertility. Recently, Zhu et al. (2018) reported reduced expression of TRPC5 in patients with varicocele-associated asthenozoospermia.

Albeit, the physiological relevance of ROS being demonstrated as a regulatory factor for sperm capacitation and acrosome reaction (Oehninger et al., 1995; Hsu et al., 1999; Rivlin et al., 2004), the underlying factors involved in the ROS-mediated events need further analysis. The bi-directional modulation of TRPV1 and ROS in mediating cell injury and/or death has been reported in other systems (Choi et al., 2014; Ogawa et al., 2016). Due to the paucity of information on the functioning of TRP channels under oxidative stress conditions in spermatozoa, it is explored in the present study by combining in vivo, in vitro, and in silico approaches. The experiments were designed in three phases with regard to the expression of functional TRPV1 channels in human spermatozoa. In the first phase, TRPV1 expression was correlated with pregnancy outcome both under natural conditions and assisted reproduction, while in the second phase, it was studied in conditions where spermatozoa are subjected to oxidative predominances such as semen with high ROS, varicocele, or spermatozoa with a morphological anomaly or maturation arrest. In addition, varicocele is a unique condition where abnormal distension of the pampiniform venous plexus that circumvents the venous drainage from the testes result in hypoxic and hyperthermic conditions, leading to oxidative stress (Samanta et al., 2018). In the third phase, a novel approach was undertaken to study the effect of hydrogen peroxide (H₂O₂) in presence of TRPV1 modulators in the context of acrosomal reaction and Ca²⁺-influx in sperm. The effect of channel modulators on sperm functions in oxidative stress conditions further implied the relevance of TRPV1 in determining sperm quality and fertility potential. Finally, in silico analysis of the disease and function networks affected by TRPV1 in conjunction with curated differentially expressed proteins (DEPs) known to be associated with pregnancy outcome post assisted reproductive technology (ART) was carried out to give a better understanding of the role of TRPV1 in spermatozoa.

The current study is targeted to observe the effect of differential expression of the heat-sensitive TRP channel TRPV1 in sperm on influencing fertility outcome via natural conception or conception with ART as well as in hypoxic and hyperthermic conditions (varicocele) with elevated ROS level. The selection of the respective TRP channel is based on its involvement with thermosensitivity and as oxidative stress signal transducers (Mori et al., 2016; Ogawa et al., 2016; Castillo, et al., 2018). Both conditions have been demonstrated to influence sperm quality and fecundity potential (Rao et al., 2016; Aitken and Drevet, 2020). Therefore, we further probed into the function of the channel in response to ROS (H₂O₂) exposure in the presence and absence of agonist and antagonist to validate its functional significance in human spermatozoa.

TRPV1 activity regulating sperm capacitation and acrosomal reaction has been well substantiated in different species (Maccarrone et al., 2005; Francavilla et al., 2009; Bernabò et al., 2010; Cobellis et al., 2010; Catanzaro et al., 2011; Gervasi et al., 2011; Lewis et al., 2012; Osycka-Salut et al., 2012; De Toni et al., 2016; Kumar et al., 2019). TRPV1 has been the most explored TRP channel in human sperm (Francavilla et al., 2009; Lewis et al., 2012; De Toni et al., 2016; Martínez-León et al., 2019) with functionality correlated to fertility, barring only one report (Mundt et al., 2018). The authors (Mundt et al., 2018) used TRPV1 agonist capsaicin which is 1,000–10,000 times less potent than RTX (Maggi et al., 1990) used in the present study and did not have an antagonist assay or peptide specificity assay. Therefore, they might have missed the localization of the channel in human sperm. The peptide control presented in this work regarding the used TRPV1 antibody is convincing and is further corroborated by the most potent TRPV1 agonist RTX and antagonist iRTX assay.

In the current study, the expression of the TRPV1 channel was probed in spermatozoa of male partners with successful pregnancy outcomes (fertile group) in comparison to those with unsuccessful pregnancy outcomes (infertile group) via natural/assisted reproduction in the phase I experiment for finding out the role of TRPV1 in imparting fertility potential to the spermatozoa. This is a pilot study in this field, which demonstrated that abnormal expression of the TRPV1 channel negatively affects the sperm quality as well as diminishes the fecundity potential of the sperm. Lewis et al. (2012) have demonstrated that functional loss of TRPV1 in spermatozoa of infertile men would eventually lead to fertilization failure due to abnormal capacitation and acrosomal reaction. Nevertheless, it is pertinent to mention here that knock-out models of TRPV1 animals are rarely sterile (Caterina et al., 2000; Colburn et al., 2007). Since TRPV1 channel activation is controlled by very diverse processes and, in many cases, exhibits complex polymodal properties, an analogy cannot be drawn between TRPV1 null mice and humans. Furthermore, natural selection always traded for the coexistence of redundant mechanisms, particularly for reproduction as a safety strategy wherein the failure of one of them does not paralyze the whole process (Bernabò et al., 2014). Hence, it may be the reason for the maintenance of fertility in TRPV1 KO mice where in absence of one ion channel (TRPV1) maintenance of fertility is taken care of by other redundant ion channels such as CatSper responsible for rheotaxisis under natural conditions (Miki and Clapham, 2013). It will not be out of context to mention here that in the knockout model system of another relatively close family member TRPV4, impairment of sperm’s migration ability, motility, and hyperactivation in response to thermotaxis was observed (Hamano et al., 2016) which may be responsible for decreased fertility. Similarly, binding of cannabinoids like anandamide and 2-arachibonoylglycerol to the TRPV1 and cannabinoids receptors (CB1 and CB2) in spermatozoa of fertile men was virtually absent for TRPV1 in spermatozoa of infertile men translating into a loss of function (capacitation, acrosomal reaction, and fertilization) in these patients (Lewis et al., 2012). However, the mRNA and protein levels remained stable in spermatozoa of the infertile patients. It is not inevitable to notice such changes in spermatozoa having limited capacity for transcription and translation. However, the functionality can be attributed to either translocation of the channel protein or its post-translational modification. The differential expression of TRPV1 in naturally fertile (NC+) and sub-fertile (IVF+ and ICSI+) may be attributed to its role in sperm motility and acrosomal reaction. In the case of IVF+ patients, where placing the spermatozoa in close proximity to the oocyte resulted in successful fertilization and embryo development, the role of TRPV1 may be attributed to its perfect acrosomal reaction and defective motility. In fact, in these patients, TRPV1 was predominantly located in the head implying its role in acrosomal reaction, but it is a significantly lower expression in the mid-piece and tail may be responsible for its decline motility making them incompetent under the natural condition to reach the egg up in the female reproductive tract (i.e., fallopian tube). Similarly, in the case of ICSI+ patients, its localization was predominantly in the mid-piece while very poor in the head region implying its lack of potency in acrosomal reaction; hence, natural fertilization was circumvented by intracytoplasmic injection into the oocyte. Therefore, the expression of TRPV1 in human spermatozoa may be a predictive factor for ART outcome. Within the female genital tract, sperm undergoes guided migration from the sperm deposition site to the fertilization site, which involves several tactic cues such as thermotaxis and chemotaxis. Remarkably, De Toni et al. (2016) observed that lower expression of TRPV1 impairs the ability of spermatozoa to migrate along a temperature gradient. The regulatory role of TRPV1 for the timely occurrence of acrosomal reaction was seen in other mammalian sperms (Bernabò et al., 2010; Kumar et al., 2019). Taking boar as the model system, it was explained that during capacitation, relocalization of TRPV1 occurs from post-acrosomal to the anterior region of the sperm head (Bernabò et al., 2008). TRPV1 was found to migrate to lipid microdomains of plasma membrane following bicarbonate-induced membrane reorganization, which might mediate the relocalization (Barbonetti et al., 2010). It was later demonstrated that exposure to TRPV1 agonist caused actin depolymerization in sperm, leading to loss of acrosomal integrity (Bernabò et al., 2010). Moreover, endocannabinoids preferentially anandamide have been shown to regulate the proper initiation and progression of capacitation and acrosomal reaction in sperm by the activating TRPV1 receptor in the female reproductive tract (Francavilla et al., 2009; Bernabò et al., 2010; Catanzaro et al., 2011; Gervasi et al., 2011; Kumar et al., 2019). Disruption of the above signaling cascades modulating motility, capacitation, and acrosomal reaction in vivo, possibly due to down-regulation of TRPV1 in these subfertile males would explain their need to undertake assisted techniques for successful fertilization in vitro.

Likewise, the differential expression of the TRPV1 channel has been reported to regulate the cellular response to oxidative stress and participate in stress-induced cellular damage (Bai and Lipski, 2010); nevertheless, a report on human spermatozoa is unavailable. The reduced expression of TRPV1 in spermatozoa of infertile patient samples with high ROS (ROS+) was reported for the first time. Lower expression of TRPV1 in these samples (ROS+) as compared to samples with low ROS (NC+), implied the possible involvement of TRPV1 in ROS signaling in sperm. This was further corroborated with the other major infertility condition varicocele. Spermatozoa are rich in polyunsaturated fatty acids which are more prone to systemic ROS attack resulting in a change in membrane fluidity (Agarwal et al., 2015; Aitken and Drevet, 2020) which might be responsible for the declined localization of the TRPV1 channel in spermatozoa membrane microdomain (Ramal-Sanchez et al., 2021) in these patients. In addition, estrogen-sensitive tissues like testis in TRPV1 KO mice are more susceptible to oxidative stress (Mizrak and Van Dissel-Emiliani, 2008). The lower levels of TRPV1 in spermatozoa of idiopathic males with high ROS could be attributed to irreversible oxidative modification of the channel and subsequent protein degradation as spermatozoa have limited capacity for transcription and translation. Moreover, Mizrak and Van Dissel-Emiliani (2008) have demonstrated the protective effect of TRPV1 in combating heat stress in male germ cells. It can be speculated that activation of TRPV1 might initiate downstream signaling pathways essential for germ cell survival under heat-stress conditions. Similarly, hypoxic activation of TRPV1 is also proposed to have physiological inference (Kim et al., 2012). Testicular and spermatozoal damage in unilateral varicocele patients has been shown to be due to hyperthermia and hypoxia-induced oxidative stress (Swain et al., 2020). Downregulation of TRPV1 in spermatozoa of these men would have aggravated the prevailing hyperthermic- and hypoxic-induced oxidative stress condition or vice versa.

In mature spermatozoa, TRPV1 was reported to be present in both head, mid-piece, and tail regions (De Toni et al., 2016; Kumar et al., 2019). Intracellular localization of TRPV1 was also proposed in spermatozoa (Maccarrone et al., 2005). We observed a similar expression pattern in the mature spermatozoa fraction. Immature spermatozoa having much lesser density (isolated from the 45% and 90% percoll gradient junction) than the mature ones (isolated as pellet penetrating 95% percoll) have a lower nucleus to cytoplasmic ratio. The retention of cytoplasm in these immature sperm cells due to aborted spermiogenesis may be responsible for the differential localization in subcellular cytoplasmic organelles increasing its intensity when measured by ICC. Similar results were also obtained with respect to abnormal spermatozoa with retention of residual cytoplasm. Previously, the co-expression of the channel in testicular tissues and their crosstalk has been proposed to regulate sperm development (Stein et al., 2004). Although TRPV1 mRNA and protein have been detected in spermatogonia, spermatocytes, and spermatids in mouse testis, spermatocytes were shown to have the highest expression of TRPV1 (Grimaldi et al., 2009). Therefore, its higher localization in immature cells is highly likely as it represents a partial developmental stage. Mizrak and van Dissel-Emiliani, have demonstrated TRPV1 knock-out models to be more susceptible to hyperthermia-induced testicular damage and consequent dysfunctional sperm development (Mizrak and Van Dissel-Emiliani, 2008). When the expression of TRPV1 in immature spermatozoa was studied by FACS, a completely reversed trend showing a declined expression with respect to mature sperms was observed. This may be due to the fact that during ICC preparation the spermatozoa are exposed to TritonX 100 making them permeable to the antibody for detection on intracellular proteins, while only the surface channels were detected in FACS.

TRPV1 is the only TRP channel reported so far to get activated within the redox potential range between −2000 mV and −3000 mV, making it one of the ideal redox sensors in cells (Mori et al., 2016). Oxidants and electrophiles generally have a redox potential threshold within this range. Redox sensing ability of TRPV1 has been corroborated in different cellular systems, where the channel is reported to get activated by ROS (Mukherjea et al., 2008; Yang et al., 2013; Johnstone et al., 2019). The co-localization of TRPV1 with NADPH oxidase 4 (NOX4) also speculated the intricate modulation of the channel function by intracellular H₂O₂ levels (Lin et al., 2015). Furthermore, the heat sensitivity of TRPV1 is ameliorated in dysfunctional redox conditions, indicating the modulation of the thermosensitive role of the channel in oxidative stress conditions (Vyklicky et al., 2002; Susankova et al., 2006). This is particularly important in the context of sperm migration in the female reproductive tract post-ejaculation. The involvement of multiple factors in synchronizing the timely occurrence of acrosomal reactions in mammalian sperm is undeniable. The cardinal role played by a minimal amount of ROS in this regard is pivotal and well documented. However, the identity and regulatory activity of key ion channels that act as downstream mediators complementing the physiological actions of ROS is not fully understood. The evidence obtained here is one such preliminary and novel study undertaken, demonstrating TRPV1 as a possible redox sensor and modulator in spermatozoa. The current findings suggest that H₂O₂ would initiate, regulate and synchronize key sperm functions by directly acting on TRPV1 as the downstream mediator. Exposure of spermatozoa to lower concentrations of H₂O₂ (1–10 µM) has been shown to regulate hyperactivated motility and acrosomal reaction (Oehninger et al., 1995). In other cellular models, TRPV1 channel activity due to the administration of agonist capsaicin was reported to be potentiated in presence of H₂O₂ (Demirdaş et al., 2017; Lam et al., 2018), while the effect of H₂O₂ could be reversed by treatment with TRPV1 antagonist capsazepine (Özdemir et al., 2016). However, we observed that the effect of H₂O₂ in inducing acrosomal reaction was attenuated in presence of TRPV1 antagonist 5′I-RTX implying the association of TRPV1 in mediating the H₂O₂ effect in spermatozoa. On the other hand, no significant potentiation of effect on the exposure of RTX to H₂O₂-treated sperm samples was noticed which could be due to the oxidation of thiol groups in RTX, compromising its binding to TRPV1. In fact, RTX binding to TRPV1 is reported to be thiol dependent and needs free SH groups for binding (Szallasi and Blumberg, 1993). In contrast, H₂O₂ mediates signaling by oxidizing the thiol groups on the redox-regulated proteins (Gutscher et al., 2009). In presence of H₂O₂, ligand binding would have been altered which has masked the potentiated effect of RTX in presence of H₂O₂ on sperm function, as observed here. Nevertheless, an increment in the percentage of sperm with acrosomal reaction was observed in both the treatment groups: H₂O₂ and H₂O₂ + RTX. The augmented Ca²⁺ influx due to channel activation by H₂O₂, both in the presence or absence of RTX, could be attributed to the major factor facilitating the aforementioned effect on the acrosomal reaction. An increase in intracellular calcium levels in response to TRPV1 channel activation have been previously demonstrated in human, boar, and bull sperm (Bernabò et al., 2010; De Toni et al., 2016; Gervasi et al., 2016). In concordance with these, a significant increment in Ca²⁺ was obtained for the two treatment groups, namely, H₂O₂ and H₂O₂ + RTX as compared to untreated groups, which was attenuated by incubating with iRTX. For the first time, we show that TRPV1 could be attributed to being one such calcium channel targeted by H₂O₂ to regulate Ca²⁺-dependent key sperm functions, especially acrosomal reaction. However, one interesting finding is the increase in [Ca²⁺]i in the spermatozoa of the iRTX-incubated (negative control) sample in absence of any stimulator till 15 min; thereafter, as expected, a sharp decline in [Ca²⁺]i was seen. It is generally believed and expected that activation of TRPV1 will increase intracellular Ca²⁺ and inhibition of TRPV1 will reduce the cytoplasmic Ca²⁺. TRPV1 is present in the endoplasmic reticulum (ER) and a number of reports suggest that this channel is involved in ER-mediated Ca²⁺-buffering (Haustrate et al., 2020). The sperm cell is an ER-free cellular system; hence, it could be possible that inhibition of TRPV1 may impair Ca²⁺-buffering leading to a rise in cellular Ca²⁺. Furthermore, iRTX binds to the TRPV1 at the same pocket as RTX, but with lesser affinity (Wahl et al., 2001). Therefore, the former may take a longer time to stabilize its interaction with the TRPV1 channel. Considering the sperm “lipidome” as a unique one and structurally much different than the somatic cells, it might be possible that TRPV1 in sperm has different pharmacological kinetics with respect to iRTX. Notably, previously, we reported increased motility of fish sperm in response to iRTX than in the control condition (Majhi et al., 2013).

Bibliographical studies have documented TRPV1 to be involved in various sperm functions ranging from acrosomal reactions, calcium trafficking, and fertilization; however, the contribution of the ion channel in determining the fecundity potential of sperm beyond fertilization awaits further exploration. The canonical pathways and networks related to sperm fertility, affected by these differentially expressed TRP channels, were further explored to understand their relevance to sperm physiology. As far as practicable, all the major female partners of the couples with confounding female factor abnormalities were eliminated in the current study, implying the restricted involvement of male factors in influencing the fertility outcome. Albeit the detailed correlation between TRP channel expression and prediction of pregnancy outcome remain indescribable, a pertinent explanation could be derived from the IPA results. Analysis of the crosstalk between TRPV1 with candidate proteins validated to influence fertility outcome showed that networks associated with cell death and survival, cellular compromise, hematological disease, and embryonic development were primarily affected by these TRP channels. The key biological disease and functions affected by TRPV1 in conjunction with the reported fertility proteins were apoptosis, cell movement, cellular homeostasis, genital tract cancer, and synthesis of nitric oxide (an important contributor to oxidative stress). The association of TRPV1 with tumorigenesis could be extrapolated to the regulation of cell division, especially during early embryonic development. Studies have demonstrated a great deal of similarity with regard to gene expression, epigenetic regulation, protein profiling, signaling pathways, and immunological aspects between embryogenesis and tumorigenesis (Ma et al., 2010; Manzo, 2019). Interestingly, TRPV1 activation has been demonstrated to stimulate notch and hedgehog/Wnt signaling as downstream pathways (Stock et al., 2014). Later is one of the key pathways characterized to be common between early embryogenesis and cancer (Kelleher et al., 2006).

The results of the present study surmise the role of TRPV1 in sperm function post ejaculation, particularly with respect to acrosome reaction and motility and at least partly toward Ca⁺² homeostasis. The expression profile of TRPV1 may serve as an additional prognostic marker in ART outcome as well as a redox sensor in oxidative stress-mediated sperm dysfunction. However, further molecular studies involving miRNA-mediated TRPV1 regulation in spermatozoa may shed some light on its role. Because spermatozoa despite having limited transcriptional and translational activities are reported to be regulated by miRNA, particularly under asthenozoospermic, oligoasthenozoospermic, and/or tetrazoospermic patients compared to normozoospermic men, while several recent studies also report the regulation of TRPV1 by miRNAs (Ramal-Sanchez et al., 2021). This pilot study on the role of TRPV1 in sperm opens an arena for deciphering the scope of TRPV1 ligands and the interaction and binding sites, particularly in understanding sperm function (Fixemer et al., 2003; Puntambekar et al., 2005; Yang et al., 2018).