Temperature-dependent contractility of rat tunica dartos muscle: Contribution of cold, menthol-sensitive TRPM8

Highlights

• •Tunica dartos smooth muscle (TDSM), which is responsible for scrotal thermoregulation, responds to cooling by enhancement of basal tension, and increase of the amplitude and duration of synaptically-evoked contractions.
• •TDSM cold-induced tension, but not synaptically-evoked contractions are sensitive to the blockers of TRPM8 cold/menthol receptor.
• •TRPM8 is localized to the TDSM cells' endoplasmic reticulum and its activation by cold causes intracellular calcium mobilization.
• •Chemical castration of male rats consequent to pharmacological blockade of androgen receptors eliminates the effects of cold on TDSM tension, but not on synaptically-evoked contractions, and abolishes TRPM8 protein expression.
• •TRPM8 is an important determinant of cold-dependent contractility of the tunica dartos muscle and scrotal thermoregulation which is crucial for maintaining normal spermatogenesis and male fertility.

Abstract

Tunica dartos smooth muscle (TDSM) lies beneath the scrotal skin, and its contraction leads to scrotum wrinkling upon cooling. However, neither the nature of TDSM cold-sensitivity nor the underlying molecular sensors are well understood. Here we have investigated the role of cold/menthol-sensitive TRPM8 channel in TDSM temperature-dependent contractility. The contraction of isolated male rat TDSM strips was studied by tensiometry. TRPM8 expression was assayed by RT-PCR and fluorescence immunochemistry. Isolated TDSM strips responded to cooling from 33 °C to 20 °C by enhancement of basal tension, and increase of the amplitude and duration of electric field stimulated (EFS) contractions. The effects of cold on basal tension, but not on EFS-contractions, could be 80% inhibited by TRPM8 blockers, capsazepine and BCTC [N-(4‑tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide], and could be partially mimicked by menthol. RT-PCR and immunolabeling showed TRPM8 mRNA and protein expression in TDSM cells with protein labelling being predominantly localized to intracellular compartments. Chemical castration of male rats consequent to the treatment with androgen receptor blocker, flutamide, led to the abrogation of cold effects on TDSM basal tension, but not on EFS-contractions, and to the disappearance of TRPM8 protein expression. We conclude that TRPM8 is involved in the maintenance of basal cold-induced TDSM tonus, but not in sympathetic nerve-mediated contractility, by acting as endoplasmic reticulum Ca²⁺ release channel whose expression in TDSM cells requires the presence of a functional androgen receptor. Thus, TRPM8 plays a crucial role in scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.

Abbreviations

α-ARs

α-adrenoceptors;

AR

androgen receptor;

CTP

cholera toxin subunit B;

EFS

electric field stimulation;

ER

endoplasmic reticulum;

PM

plasma membrane;

TDSM

tunica dartos smooth muscle;

TRPM8

Transient Receptor Potential Melastatin 8.

1. Introduction

Contractility of the smooth muscle of the male reproductive system such as vas deferens forming ejaculatory ducts and tunica dartos constituting the wall of the scrotum is very sensitive to temperature changes. However, neither the nature of such sensitivity nor the underlying molecular sensors are well understood.

The muscle wall of the scrotum, the dartos muscle (tunica dartos), represents the thin layer of smooth muscle tissue tightly connected with the scrotum's skin. By its origin, this is a typical superficial muscle with its fibre bundles interwoven with fibroelastic connective tissue to form a mesh-like structure penetrated by blood vessels [1]. Tunica dartos acts to maintain the temperature of the scrotum within the optimum required for spermatogenesis (usually 2–3 °C below body temperature) [1,2]. Upon cooling it contracts to wrinkle the scrotal skin and shorten the scrotal length. The former reduces the surface area available for heat loss, whilst the latter reduces the heat exchange within the pampiniform plexus, a network of veins surrounding the spermatic artery [3]. Heating induces opposite thermoregulatory effects by causing dartos muscle relaxation. In humans, spermatogenesis is most efficient at 34 °C with scrotal hyperthermia considered to represent a risk factor for male infertility [4,5]. However, neither the nature nor the localization of molecular sensors involved in tunica dartos thermosensitivity is well understood.

Tunica dartos smooth muscle (TDSM) is innervated by sympathetic fibres from the genital branch of the genitofemoral nerve. It contracts to noradrenaline via α-adrenoceptors (α-ARs) [6,7]. It has been shown that in rat dartos muscle contractile responses to electric field stimulation (EFS), noradrenaline, thromboxane A₂ receptor agonist, U46619, or high-K⁺ all exhibit temperature-dependent decrease with Q₁₀ of around 5 (within 30 °C to 40 °C) [7]. The results suggested that these effects are not Ca²⁺-dependent, but are rather explained by smooth muscle myosin phosphatase activity, having a Q₁₀ of about 5.3 [7,8]. Further studies have shown that explants of rat TDSM can contract in response to cooling per se in the absence of autonomic neural input or exogenous agonists [9]. This contraction could be potentiated by the presence of the overlying scrotal skin, prompting the conclusion that the skin may release noradrenaline or some other agent with α-adrenergic activity during cooling [9].

The presence of cooling-induced contraction of the isolated TDSM [9] suggests a possible involvement of some molecular sensors of ambient temperatures from the family of Transient Receptor Potential (TRP) family of ion channels, which were shown to contribute to such sensitivity in some types of smooth muscle [10], [11], [12], [13]. In the present study, we decided to revisit the problem of temperature sensitivity of the tunica dartos muscle with particular emphasis on the possible involvement of cold/menthol-sensitive TRPM8 channel. Our data show that TRPM8 is indeed involved in cold-induced TDSM contraction by acting as endoplasmic reticulum (ER) Ca²⁺ release channel whose expression in TDSM cells is androgen-regulated, requiring the presence of a functional androgen receptor.

2. Methods

2.1. Tunica dartos strips preparation and contraction measurements

All animal protocols complied with EU Directive 2010/63/EU for animal experiments (http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm). The experimental protocol described herein was approved by the Bogomoletz Institute of Physiology (BIPh) Bioethics Committee (Permission No 2/17 from 05.09.2017). Animals (rats) used in this study were bred, housed and maintained in the specialized animal facility (vivarium) of BIPh. Every effort was made to minimize the animal's suffering. Male Wistar rats weighing 200–250 g were exposed to a rising concentration of CO₂, and death was confirmed by subsequent decapitation. The scrotum was excised together with testes and cremaster muscle, and placed in a preparation chamber filled with Krebs solution (in mM): 120.4 NaCl, 5.9 KCl, 1.2 MgCl₂, 1.2 NaH₂PO₄, 1.8 CaCl₂, 15.5 NaHCO₃, 11.5 glucose (рН 7.4). The scrotum was spread-out and pinned to the rubber bottom of the preparation chamber with the scrotal skin facing downwards. Under stereo microscopic control, the tissue was dissected into two halves along the midline, the testes and cremaster muscle were removed, and the strips of dartos muscle around 10 mm long and 2 mm wide with no skin attached were cut from each half either at right angles or parallel to the midline. Control experiments showed no difference in the contractility of such strips apparently due to the isotropic meshwork arrangement of the dartos smooth muscle.

Chemical castration of male rats was performed with flutamide, a selective antagonist of the androgen receptor (AR), competing with androgens like testosterone and dihydrotestosterone for binding to ARs in androgen-sensitive tissues. By doing so, flutamide prevents the physiological effects of male hormones. The tablet form of flutamide (Flutafarm®, Farmac JSC, Ukraine) mashed in water (0.5% m/v) was administered to laboratory male rats daily for 30 days in the dosage of 25 mg/kg via oral route by gavage. Control animals were given a placebo (sugar pills) by the same route.

For contraction measurements, the strip was placed in an experimental chamber (volume 0.5 ml) continuously superfused (gravity-driven flow rate 2 ml/min) with preheated (to 33 °C, physiological temperature of the scrotum) oxygenated (95% O₂ and 5% CO₂) experimental Krebs solution with one end of the strip fixed still and another end attached using ligature to the capacitive force sensor [14]. Before commencing the measurements the strip was allowed to equilibrate in normal Krebs solution under the basal tension of 1 mN for 1 hour. Electric field stimulation (EFS) was performed via Ag/AgCl electrodes placed on opposite sides of the strip with a 2-second-long train of pulses (pulse duration 0.5 ms, amplitude 100 V, frequency 10 Hz) delivered at intervals 1 or 3 min (depending on conditions needed) to ensure complete restoration of basal tone. Such parameters of EFS failed to produce TDSM strip contraction in the presence of catecholamines release inhibitor, guanethidine (1 μM), suggesting that it effectively activates only nerve-mediated response. The experimental chamber and solution inlet manifold were water jacketed. The temperature of the perfusion solution was changed by switching water jackets between two heating circulator baths, one set at a control temperature of 33 °C and another one at a test temperature of 20 °C. Recording of contractile activity was made using pCLAMP software and DigiData 1200 to the computer and in parallel on a pen recorder.

Menthol (TRPM8 agonist, Sigma-Aldrich), capsazepine (TRPM8 antagonist, Sigma-Aldrich) and BCTC (N-(4‑tert-butylphenyl)−4-(3-chloropyridin-2-yl)piperazine-1-carboxamide, TRPM8 antagonist, Sigma-Aldrich) were dissolved in ethanol and M8-B (N-(2-aminoethyl)-N-[[3‑methoxy-4-(phenylmethoxy)phenyl]methyl]−2-thiophenecarboxamide hydrochloride, selective TRPM8 antagonist, Tocris) – in DMSO as 10 mM stocks, and were added to the experimental Krebs solution in the desired concentration. Control experiments indicated that ethanol or DMSO in the concentration up to 0.1% did not produce any effect on the TDSM strip's contractility.

2.2. Immunochemistry and confocal microscopy

For immunolabeling isolated rat TDSM cells were used. Cells isolation followed a two-step procedure. TDSM tissue was chopped into small pieces and incubated for 25 min at 37 °С in 1 ml of the first-step enzymatic Ca²⁺-free Krebs solution supplemented with BSA (1 mg), DL-Dithiothreitol (1 mg) and papain (1 ml) (all from Sigma-Aldrich). After this tissue was triturated with a Pasteur pipette for 2 min and centrifuged for 4 min at 1000 rpm with the supernatant removed and replaced with 1 ml of the second-step enzymatic Ca²⁺-free Krebs solution containing BSA (1 mg), collagenase type II (0.5 mg) and trypsin inhibitor (0.5 mg) (all from Sigma-Aldrich). After 20 min incubation at 37 °С, the remaining undigested pieces were pipetted again to obtain a homogenous suspension. To remove the digestion solution, the suspension was centrifuged for 4 min at 1000 rpm, and the pellet was dispersed in normal Krebs. Aliquots of the cells were then plated on glass coverslips and incubated for 24 h at +37 °C to allow attachment.

The attached cells were washed with a 0.1 M phosphate buffer solution (PBS) and incubated with Cholera toxin subunit B (CTxB) FITC conjugate membrane marker (Sigma-Aldrich C1655, 1:2000) for 15 min at room temperature. After three times wash in 0.1 M PBS for 10 min and subsequent incubation in 5% goat serum or 1% bovine albumin to block non-specific binding sites live cells were treated with ER-tracker Blue-White DPX (1 µM) (Invitrogen, E12353) for 20 min at +37 °C and then again washed three times for 10 min in 0.1 M PBS. Both CTxB FITC conjugate and ER-tracker Blue-White DPX were diluted in 0.1 M PBS with 5% goat serum (PBGS solution).

CTxB- and ER-tracker-treated cells were fixed with 4% formaldehyde for 30 min at room temperature. Fixed cells were permeabilized with 0.2% Triton X-100 (PBGS with 0.2% Triton X-100) for 2 h at room temperature. Permeabilized cells were labelled with the specific primary anti-TRPM8 antibody (Novusbio, NBP1–97,311, 1:100) for 48 h at +4 °C and then with secondary Alexa Fluor® 647 antibody (Invitrogen, A32733 goat anti-rabbit, 1:1000) for 2 h at room temperature. Both primary anti-TRPM8 and secondary Alexa Fluor® 647 antibodies were diluted in PBGS.

The used anti-TRPM8 antibody was generated against an epitope sequence located in the internal portion of the human TRPM8 protein (between residues 250–300), and displays human, mouse and rat reactivity. Thus, wild-type HEK293 cells were used as a negative control for TRPM8 expression with immunostaining performed according to the same protocol as for rat TDSM cells.

Immunostained preparations were dried at room temperature and covered with an antifade mounting solution (ProLong Glass Antifade Mountant, Thermo Fisher Scientific). Confocal fluorescent images were acquired on Olympus FV1000 FluoView confocal laser scanning biological microscope equipped with Multiline Argon (457/488/515), Helium-Neon (543) lasers and UV 405 nm laser diode system. Slides were examined at 40x magnification (Olympus UApo/340 40x/1.15na water objective). FITC fluorescence was excited with a 488 nm, and the fluorescent signal was recorded at >510 nm. ALEXA fluorescence was excited at 543 nm, and the fluorescent signal was recorded at >668 nm wavelength, ER-tracker Blue-White DPX was excited at 405 nm, and emission was recorded at 430 nm. Images were analysed using the Olympus FV1000 software.

2.3. RT-PCR

Total RNA was isolated from rat TDSM tissue using RNAzol® RT (Sigma-Aldrich). The concentration and purity of RNA samples were assessed based on the ratio of absorbence at 260 nm and 280 nm with a NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific). Only the samples with A260/A280 > 1.6 were used in the experiments. cDNA synthesis was done using an "MMLV-reverse transcriptase kit", oligo-dT₁₈ Primer (50 pM per reaction) and random hexamer primer (50 pM per reaction) (all reagents from Thermo Fisher Scientific).

Specific primers were created with the help of NCBI/Primer-BLAST and tested with FastPCR 4.0.13 (PCR Team) software for the “Primer quality” parameter to exceed 70. To avoid genomic DNA amplification, primers were designed to be complementary to the ends of adjacent exons. As a housekeeping gene, β-actin, Actb, an element of the cytoskeleton that is expressed in all cells of an organism under all conditions was selected. The sequences of the primers were: TRPM8 (Trpm8) 5′- CCCCCACCTCCTCACGGTCA-3′ (F), 5′- AGGTCGGCAGACTCCCAGCG-3′ (R); β-actin (Actb) 5′-CTGTGTGGATTGGTGGCTCT-3′ (F), 5′-GCTCAGTAACAGTCCGCCTA-3′ (R). RT-PCR was performed using a one-step amplification kit (Astravir Technology, Ukraine) according to the manufacturer's instructions. The reaction mixtures of a total volume of 20 μl contained 5 μl of total RNA from the samples. The random hexamer-primed reverse transcription and cDNA amplification were performed on a T-CY thermocycler (CreaCon Technologies, Netherlands). The PCR temperature cycling conditions were as follows: reverse transcription for 20 min at 50 °C, initial denaturation and “hot start” of Taq polymerase for 12 min at 95 °C, then 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. Visualization of amplicons generated by RT-PCR was performed after separation on a 2.5% agarose gel electrophoresis with ethidium bromide staining on transilluminator TFP-M/WL (Vilber, France).

2.4. Data analysis and statistics

Each functional experiment was performed on 6–10 TDSM strips from at least 3 animals of the same group, and the changes in basal tension, overall amplitude and duration (at 75% relaxation from maximal amplitude) of EFS-evoked contractions (EFS-contractions) in response to cooling and bath application of studied compound(s) were measured, averaged and expressed as mean±SD with the number of strips indicated by “n”. Statistical comparison of the data was made by pair-sample t-test with P-values presented for each case. For comparison between control and flutamide-treated groups of animals amplitudes of the contractions were normalized to TDSM strip weight.

3. Results

3.1. Temperature-dependence of basal tension and EFS-contractions of the rat tunica dartos

Rat tunica dartos smooth muscle (TDSM) strips responded to the cooling of Krebs bath solution from 33°С to 20 °С by significantly increasing both basal tension and the amplitude of EFS-contractions (Fig. 1). Lowering of bath temperature also notably prolonged the duration of EFS-contraction. The onset of temperature effects became clearly discernible on cooling to 28 °C and gradually increased on further cooling up to 20 °C. Heating the bath solution back to 33 °C produced basal tension relaxation and return of EFS-contractions amplitude and duration back to the pre-cooling values, suggesting complete reversibility of the effects.

Fig. 1.

Rat TDSM strips reversibly respond to cooling by enhancement of basal tension, and increase of EFS-contractions amplitude and duration. A: Representative recording of TDSM strip contractility in response to cooling of bath solution from 33 °C to 20 °C and reheating back to 33 °C; temperature changes are shown below the recording; dashed line – basal tension at 33 °C; upward spikes – EFS-contractions. B: Comparison of EFS-contractions (marked by asterisks in panel A) at 33 °C (left) and 20 °C (right) at an expanded time scale.

While the cooling-induced enhancement of basal tension can be potentially linked to the action of putative temperature sensors in the TDSM cells, including myosin phosphatase [7], the observed effects of cooling on EFS-contractions, which involve synaptic action of noradrenaline, may also depend on other mechanisms, taking place at colder temperatures among which slowing of noradrenaline reuptake, which is the most common route of neurotransmitter removal from the synaptic cleft [15] or stimulation of plasma membrane α-ARs translocation [16,17].

3.2. Effects of TRPM8 agonists and antagonists

Cold/menthol-sensitive TRPM8 channel acts as the primary receptor of cooling in the peripheral nervous system [18,19,20]. However, it is also expressed in some other tissues not directly exposed to the ambient temperatures, including the smooth muscle ones [11,21,22] in which case it can be also activated by endogenous ligands such as androgens and lysophospholipids [23,24]. Moreover, TRPM8 can be localized not only in the plasma membrane but also in the membrane of the endoplasmic reticulum (ER), acting as a Ca²⁺ release channel [21,25].

To answer the question of whether or not TRPM8 is involved in cold-evoked TDSM tension we first checked for its sensitivity to the two TRPM8 antagonists, capsazepine (10 µM) and BCTC (10 µM). Fig. 2A shows that the application of capsazepine on top of the contraction caused by bath cooling from 33 °С to 20 °С produced strong relaxation almost to the pre-20 °С level without essentially affecting either the amplitude or duration of EFS-contractions. Such an effect of capsazepine could be reproduced by BCTC. Furthermore, pre-exposure of the TDSM strip to either capsazepine or BCTC largely prevented the development of tension in response to bath cooling by 76±8% (n = 7) and 79±6% (n = 7), respectively, still not affecting parameters of EFS-contraction (Fig. 2B-C).

Fig. 2.

TRPM8 antagonists suppress cooling-induced tension but do not influence EFS-contractions. A: Representative recording of TDSM strip contractility in response to stepwise cooling of the bath solution from 33 °C to 20 °C and application of capsazepine (10 µM, shown by a horizontal line on top). A: Representative recording of TDSM strip contractility in response to ramp-cooling from 33 °C to 20 °C. B: Same as in A, but before and on the 30-th min of the same TDSM strip exposure to BCTC (10 µM). Temperature change protocols in A-B are shown below the recordings; dashed lines – basal tensions at 33 °C; note, a strong decrease of cooling-induced tension by capsazepine and BCTC and lack of their effects on EFS-contractions. C: Quantification of capsazepine (CAP) and BCTC effects on the amplitude of cooling-evoked tension; n = 10 from 3 animals; box – 25th and 75th percentiles, "□" – mean, line – median, whiskers – min and max, " × " – 1st and 99th percentiles; "***" P<2.5 × 10⁻¹¹ (capsazepine) and P<9.7 × 10⁻¹³ (BCTC).

Despite robustly reacting to the cooling, TDSM strips responded to the TRPM8 agonist menthol (100 µM) applied at 33 °С by a contraction which generally had a smaller amplitude, was transient in time and was accompanied by a brief periodic relaxation event (Fig. 3A). Moreover, pre-exposure of TDSM strips to menthol concentration-dependently reduced the amplitude of cooling-induced tension as compared to menthol-free conditions by 37.4 ± 8.2% (n = 7) and by 69.4 ± 9.7% (n = 7) in the presence of 100 µM and 200 µM of menthol, respectively (Fig. 3B).

Fig. 3.

TRPM8 agonist, menthol, produces transient enhancement of basal tension and antagonizes the effect of cooling. A: Representative recording of TDSM strip contraction in response to the application of menthol (100 μM, shown on top); note, the transient nature of menthol-induced contraction and appearance on its background of brief periodic relaxation events (marked by "*"). B: Representative recordings of the same TDSM strip contraction in response to cooling administered under control conditions and on the background of 100 μM and 200 μM menthol; cooling and menthol application protocols are shown on top; note, progressive reduction of cooling-induced contraction with TDSM strip pre-exposure to the increasing menthol concentration; line breaks correspond to 30 min.

Thus, experiments with TRPM8 inhibitors and menthol support the notion that cooling-evoked enhancement of TDSM basal tension is most likely linked to the activation of the TRPM8 channel expressed in TDSM cells. On the contrary, the insensitivity of cooling-evoked changes of EFS-contractions to TRPM8 inhibitors suggests their independence of TRPM8 and their likely association with alterations in noradrenaline reuptake at colder temperatures.

3.3. Expression and function of TRPM8 cold receptor in rat TDSM

Expression of TRPM8 on mRNA and protein levels was demonstrated in several types of smooth muscle, including gastric [26], colonic [10], vascular [12,13,22,27], and vas deference [11]. In view of functional evidence of TRPM8 involvement in temperature-dependent TDSM contractility, we next asked whether TRPM8 is expressed in this muscle as well. Our RT-PCR experiments have clearly shown the presence of TRPM8 mRNA in rat TDSM (Fig. 4A). Moreover, the brightness of TRPM8 amplicons in gels from TDSM tissues appeared almost identical to those from prostate, which is known to be a rich source of TRPM8 [28,29] and was therefore used as the positive control.

Fig. 4.

TRPM8 expression in rat tunica dartos. A: RT-PCR gels obtained from TDSM and prostate (to serve as TRPM8-positive control) from four animals (#1–4) showing the presence of Trpm8 amplicons of the expected size in both tissues; β-actin-coding Actb gene was used as a positive control (PC) while for negative control (NC) the total RNA was substituted with TE buffer. B: Z-stack laser-scanning confocal images of isolated TDSM cell co-stained with fluorescently labelled plasma membrane marker CTxB (green) and anti-TRPM8 antibody (red); note, predominant localization of the clusters of TRMP8 staining within the perimeter of the cell with no superimposition of red and green colours (to yield yellow); calibration bars 10 μm. C: Transmitted light (left, TM) and confocal images (middle and right) of HEK293 cells (used as a negative control for TRPM8 protein) stained with fluorescently labelled CTxB (green) and anti-TRPM8 antibody (red); note, absence of red colour on the right (anti-TRPM8) image, indicating lack of TRPM8 protein expression; calibration bars 10 μm. D: TDSM cells triple labelling with CTxB (green), anti-TRPM8 antibody (red) and ER-tracker (blue); note, the presence of magenta colour inside the cell, which corresponds to the superimposition of red and blue with basically no yellow colour on the merged image, indicating TRPM8 protein localization in the ER; calibration bars 10 μm.

To make sure that TRPM8 is specifically expressed in TDSM cells and if so to get insight into its subcellular localization we proceeded to immunocytochemical detection of TRPM8 protein using specific antibodies along with plasma membrane (PM) labelling by cholera toxin subunit B (CTxB). CTxB binds to GM1 gangliosides, characteristic complex lipids present in the external layer of plasma membranes, making it a reliable plasma membrane marker [30]. Since the anti-TRPM8 antibody used in our study display both human and rat reactivity, we have used wild-type HEK-293 cells as TRPM8-negative control. Fig. 4B presents the series of Z-stack confocal laser scanning microscopy images of representative TDSM cell stained with anti-TRPM8 (red) antibody and CTxB (green) (see also Supplementary File 1 showing animated 3D reconstruction from Z-stack images of another TDSM cell). These images show the distribution of the spots of red colour inside the TDSM cell perimeter with basically no yellow colour corresponding to the superimposition of the red and green present. This suggests that the TRPM8 protein (red) is predominantly localized in the form of isolated clusters in the intracellular compartments with very little or no presence in the PM (green). The specificity of TRPM8 staining was confirmed in HEK-293 cells which showed no anti-TRPM8 (red) reactivity (Fig. 4C). Thus, a contractile response associated with TRPM8 activation must result from TRPM8-mediated calcium release from intracellular compartments, most likely endoplasmic reticulum Ca²⁺ store. This conclusion was further supported with TDSM cells triple labelling wherein in addition to CTxB (green) and anti-TRPM8 antibody (red) they were stained with ER-tracker (blue) (Fig. 4D). Merging the respective images provided a magenta colour inside the cell, which corresponds to the superimposition of red and blue with basically no yellow colour present (Fig. 4D).

To obtain functional evidence on the presence of TRPM8-mediated Ca²⁺ mobilization in TDSM cells we have proceeded to measure cold-evoked tension in TDSM strips in nominally Ca²⁺-free extracellular solutions. Fig. 5A shows that the removal of extracellular Ca²⁺ caused cessation of the TDSM strip's EFS-contractions and the decrease of basal tone, consistent with the effective elimination of the processes dependent on the entry of extracellular Ca²⁺. Despite this, the amplitude of the tension in response to cooling decreased only marginally (Fig. 5A), suggesting the involvement of intracellularly mobilized Ca²⁺ in cold-induced contractile response. Moreover, if after switching to a nominally Ca²⁺-free solution TDSM strip was pre-exposed to TRPM8 inhibitor M8-B (10 μM), subsequent application of cooling failed to evoke a contractile response (Fig. 5B), thereby providing strong evidence that mobilization of intracellular Ca²⁺ occurred via TRPM8 expressed in the ER membrane.

Fig. 5.

TDSM cooling-induced tension is preserved in a Ca²⁺-free external solution and is sensitive to TRPM8 inhibitors. A, B: Representative recordings of TDSM strip contractility in response to cooling in the presence and in the absence of extracellular calcium ([Ca²⁺]_o). Changes in the bath solution temperature (t °C, between 33 °C and 20 °C) and [Ca²⁺]_o (between 2 mM and 0 mM) are depicted by thin and thick lines, respectively, above the recordings. Note, that at [Ca²⁺]_o = 0 cooling from 33 °C to 20 °C is still able to induce TDSM contraction, however, such contraction could be eliminated in the presence of TRPM8 inhibitor M8-B (10 μm, shown in B by thick line below the temperature marking). Upward spikes in A are EFS-contractions. Their cessation after switching to [Ca²⁺]_o = 0 indicates the effective elimination of the processes dependent on extracellular Ca²⁺.

3.4. Antiandrogens abolish cooling-evoked TDSM contraction

Male prostate and scrotal skin are known to contain the highest concentration of total androgens [31], whereas the decline of androgen levels in elderly men among other consequences results in the scrotum sagging in part due to loss of strength of the dartos muscles and in cessation of scrotum reactions to temperature changes. Furthermore, expression of the TRPM8 cold receptor-channel in prostate apical epithelial and smooth muscle cells was shown to be androgen-regulated, requiring the presence of a functional androgen receptor (AR) [21].

To test whether or not functional AR is necessary to support cold-evoked, TRPM8-mediated TDSM contractility we proceeded to the experiments on animals treated for 30 days with flutamide, a selective AR antagonist competing with androgens (i.e. testosterone and dihydrotestosterone (DHT)) for binding to AR in androgen-sensitive tissues. Figs. 5A-C show that TDSM strips from flutamide-treated rats responded to the cooling from 33 °С to 20 °С by a significantly smaller increase of basal tension compared to the control, whereas changes in EFS-contractions amplitude and duration remained not much different from the control. Quantification of these effects revealed that the amplitude of cooling-induced basal tension in flutamide-treated animals constituted only 25±5% (n = 8) of that in control animals (Fig. 6C), whereas both amplitude and duration of EFS contractions showed no statistically significant differences (Fig. 6D, E). Thus, only temperature-dependent regulation of TDSM basal tension remains androgen-sensitive, whilst cold-induced changes in EFS-contractions, whose characteristics are determined by the processes of neuro-muscular synaptic transmission, do not demonstrate explicit signs of androgen sensitivity.

Fig. 6.

Chemical castration of male rats with selective AR antagonist, flutamide, suppresses TDSM cold-induced tension and TRPM8 protein expression. A: Representative recording of TDSM strip contractility from a flutamide-treated rat in response to cooling of bath solution from 33 °C to 20 °C and reheating back to 33 °C; temperature changes are shown below the recording; dashed line – basal tension at 33 °C; upward spikes – EFS-contractions; note, very small increase in basal tension in response to cooling. B: Comparison of EFS-contractions (marked by asterisks in panel A) at 33 °C (left) and 20 °C (right) at an expanded time scale. C: Quantification of cooling-evoked increase of basal tension in TDSM strips from control (blue) and flutamide-treated (cyan) male rats; n = 10 from 3 animals; box – 25th and 75th percentiles, "□" – mean, line – median, whiskers – min and max, " × " – 1st and 99th percentiles; "***" P<5.4 × 10⁻⁹. D: Comparison of normalized EFS-contractions of TDSM from control (top) and flutamide-treated (bottom) rats at 33 °C (red) and 20 °C (blue); CD₇₅ denotes contraction duration at 75% relaxation. E: Quantification of the amplitude (top box chart) and duration (CD₇₅, bottom box chart) of EFS-contractions (EFSC) at 33 °C (red) and 20 °C (blue) in control and flutamide-treated male rats; n = 10 from 3 animals for each condition; same designations as in panel C; P-values between 33 °C and 20 °C are shown on graphs; note, no significant difference in EFSC parameters between control and flutamide-treated groups. F: Representative transmitted light (TM, top) and confocal images (middle and bottom) of TDSM cell from flutamide-treated male rat stained with fluorescently labelled CTxB (green) and anti-TRPM8 antibody (red); note, the absence of the red colour on the bottom (anti-TRPM8) image, indicating lack of TRPM8 protein expression; calibration bars 10 μm.

In view of the fact that cold-induced TDSM tension involves TRPM8 activation, whose expression was shown to be androgen-regulated [21], we next asked whether the decrease of such tension in flutamide-treated animals is associated with reduced TRPM8 levels. Fig. 6F shows that consistent with such possibility immunostaining of TDSM cells from flutamide-treated rats with anti-TRPM8 antibodies did not provide a positive signal, suggesting that the expression of TRPM8 protein is basically lost. This allowed us to conclude that functional AR is imperative for maintaining TDSM cold sensitivity.

4. Discussion

Our study shows that the temperature dependence of tunica dartos smooth muscle whose primary function is to regulate the temperature of the scrotum to ensure proper spermatogenesis is largely determined by Ca²⁺-permeable TRPM8 cold/menthol receptor-channel expressed in muscle cells. In fact, there are only two studies specifically designed to determine the mechanisms of TDSM thermosensitivity. In the first one, it was concluded that myosin phosphatase is the major contributor to heat-induced TDSM relaxation [7]. Indeed, smooth muscle myosin phosphatase is characterized by a very high Q10 value of around 5 (5.2 ± 5.3). Thus, it was reasoned that since the contractile state of smooth muscle is determined by the degree of myosin phosphorylation, enhancement of phosphatase activity at increasing temperatures would promote myosin dephosphorylation and smooth muscle relaxation. Experiments with phosphatases inhibitor, calyculin-A, which greatly reduced heat-induced TDSM relaxation, supported this notion [7]. In another study, it was shown that explants (i.e. strips) of TDSM from the rat scrotum contract in response to cooling and that the tension developed during cooling was potentiated by the presence of the overlying skin [9]. It was hypothesized that the potentiation is caused by some soluble agent released from the skin and affecting the underlying muscle, although neither the nature of this agent nor the mechanism of cold-evoked contraction was identified.

The sensitivity of TDSM cold-evoked contraction to TRPM8 antagonists and the possibility of activating contraction by TRPM8 agonist, menthol, provide strong arguments for the involvement of TRPM8-mediated Ca²⁺ signalling. The difference in the kinetics and amplitude of TDSM contraction in response to cold (i.e. slowly developing, sustained, large amplitude) and menthol (i.e. rapidly developing, transient, small amplitude) to our opinion is explained by the fact that cold-induced, TRPM8-mediated Ca²⁺ signalling is complemented by simultaneous inhibition of smooth muscle myosin phosphatase [7], whereas in the event of TRPM8 activation by menthol at constant high temperature such inhibition is obviously absent. Despite being a chemical TRPM8 agonist, menthol is also known to exert a number of non-specific actions, among which is the inhibition of l-type voltage-gated calcium channels [12,32] which can further contribute to different contractile responses as compared to cold.

Our data indicate that TRPM8 in TDSM most likely acts not as a classical plasma membrane Ca²⁺-permeable ion channel, but as endoplasmic reticulum (ER) Ca²⁺ release channel. The possibility of TRPM8 localization and function in the ER membrane was first demonstrated in prostate cancer cells in which TRPM8-mediated Ca²⁺ release from the ER could activate store-operated Ca²⁺ entry [25] and signalling events important for prostate carcinogenesis [33]. Targeting of classical TRPM8 channel and its truncated isoforms to intracellular compartments and their participation in Ca²⁺ release from the sarco-endoplasmic reticulum have been also demonstrated in epidermis, lung, vasculature and some of the visceral smooth muscle [11,12,[34], [35], [36]] with cold-induced TRPM8-mediated Ca²⁺ signalling supporting the processes as diverse as respiratory sensitivity to cold air, epidermis adaptation to low temperatures, cold-dependent vasoconstriction and hypertension, cold-dependent smooth muscle contractility. Possible coupling of TRPM8-mediated Ca²⁺ signalling to the activation of Ca²⁺-dependent potassium channels may also cause spasmolytic effects in gastric smooth muscle [10] and contribute to vasodilation [13]. Our data indicate that in TDSM ER-localized TRPM8 plays important role in scrotal thermoregulation by causing TDSM contraction upon cooling to wrinkle the scrotal skin and shorten the scrotal length. Simultaneous cold-induced TRPM8-mediated Ca²⁺ release and cold-induced inhibition of smooth muscle myosin phosphatase shown before [7] apparently provide for the persistency and durability of the cooling effect.

It is well known that as men age their scrotum starts to sag and lose the ability to react to temperature changes. Our data show that like in the prostate [21], expression of TRPM8 in TDSM is androgen-regulated, requiring the presence of a functional androgen receptor. In view of the fact that androgen levels decrease as men get older, our data provide a molecular mechanism for these phenomena. Besides, in addition to a genomic action, testosterone may directly activate the TRPM8 channel at a low picomolar range [23] further contributing to the maintenance of the scrotal tonus during a male's reproductive period and loss thereof with age.

In conclusion, this study provides strong evidence of the functional role of TRPM8 in cooling-evoked contractility of the tunica dartos muscle and scrotal thermoregulation which is important for maintaining normal spermatogenesis and male fertility.

Funding

This work was supported by the National Academy of Sciences of Ukraine and the National Research Foundation of Ukraine grant 2020.02/0189.

Availability of data and material

All data are contained within the manuscript.

CRediT authorship contribution statement

Igor B. Philyppov: Conceptualization, Methodology, Investigation, Formal analysis, Writing – review & editing. Ganna V. Sotkis: Investigation, Formal analysis, Resources, Writing – review & editing. Bizhan R. Sharopov: Investigation, Formal analysis, Writing – review & editing. Anastasiia O. Danshyna: Investigation, Formal analysis, Writing – review & editing. Semen I. Yelyashov: Investigation, Formal analysis, Writing – review & editing. Valeri G. Naidenov: Investigation, Formal analysis, Writing – review & editing. Olga P. Lyubanova: Resources, Data curation, Writing – review & editing. Yaroslav M. Shuba: Conceptualization, Methodology, Formal analysis, Writing – original draft, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare no conflict of interest.

Data availability

• Data will be made available on request.