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, 11 (12), e0167622
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Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa

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Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa

Luca De Toni et al. PLoS One.

Abstract

The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31-37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression of TRPV1 in human testis.
A) Analysis of the expression of TRPV1 mRNA in human testis biopsies (Hum. testis) and ejaculated human spermatozoa (Hum. sperm) by RT-PCR. Data are reported as normalized relative quantification (nRQ). Relative expression was also reported as gel electrophoresis analysis of the target amplification product at 186 base pairs (TRPV1) and GAPDH as housekeeping gene (108 base pairs). B) Analysis of the protein expression of TRPV1 in human testis biopsies (Human testis) and ejaculated human spermatozoa (Human sperm) by western blot. The specificity of primary immunoreaction was assessed by co-incubation with the immunogen peptide (Peptide). Relative expression was reported as the ratio between the band density at ~90 kDa (TRPV1) and the band density of β-Actin as housekeeping (43 kDa). Images are representative of six independent experiments. Significance: * = P<0,05 vs human testis; ǂ = P<0,05 vs human sperm. C) Evaluation by immunofluorescence of TRPV1 protein localization (green) in whole human seminiferous tubule, testis cell populations from fine needle aspiration specimens (D) and ejaculated spermatozoa (E). Samples were counterstained with DAPI (blue). In the negative control (Neg insert in C) primary antibody was omitted. As additional control condition, immunogen peptide was added in the reaction mixture (+ Peptide).
Fig 2
Fig 2. Functional characteristics of sperm cells selected by thermotaxis.
A) Quantification of sperm cells selected by thermotaxis in non-capacitated and capacitated conditions. Migration towards a 31–37°C gradient (T gradient), towards 31°-no gradient control (Ctrl 31°C) and towards 37°-no gradient control (Ctrl 37°C) were compared. Data are reported as millions of sperm cells. Significance: * = P<0,05 vs both Ctrl 31°C and Ctrl 37°C; ǂ = P<0,05 vs corresponding non-capacitated conditions. B) Assessment of sperm acrosome reaction, intracellular calcium levels and apoptosis by flow cytometry. Data are reported respectively as percentage of the CD46-positive (Acrosome reaction), calcium orange positive cells (Intracellular Ca2+) and annexin-V-positive cells (Apoptosis) cells migrated towards the T gradient, the Ctrl 31°C or the Ctrl 37°C in either non-capacitated or capacitated conditions. Significance: * = P<0,05 vs Ctrl 31°C; ǂ = P<0,05 vs corresponding non-capacitated conditions. C) Sperm motility parameters of migrated cells assessed by automated Sperm Class Analyser. Sperm curvilinear velocity (VLC), straight-line (rectilinear) velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR as VSL/VAP), wobble (WOB as VAP/VLC), beat-cross frequency (BFC), hypermotility (Hyper) were considered in cells migrated towards the T gradient, the Ctrl 31°C or the Ctrl 37°C in either non-capacitated or capacitated conditions. Significance: * = P<0,05 vs Ctrl 31°C; ǂ = P<0,05 vs corresponding non-capacitated conditions.
Fig 3
Fig 3. TRPV1 levels in sperm cells selected by thermotaxis.
TRPV1 protein expression of the sperm cells migrated towards the 31°-no gradient control (Ctrl 31°C), towards the37°-no gradient control (Ctrl 37°C) and towards a 31–37°C gradient (T gradient). Cells were analyzed by cytometry (Panel I) and immunofluorescence (Panel II). In panel I, histogram plots of cells stained with anti-TRPV1 antibody (thick continuous line), anti-TRPV1 antibody and immunogen peptide (thin continuous line) and negative control with no primary antibody are compared. Anti-TRPV1 antibody cell staining intensity was distinguished within as low (yellow areas), high (green areas) and intermediate (orange areas). In panel II, TRPV1 staining at immunofluorescence appears as green whilst cell nuclei are counterstained with DAPI (blue). Data are representative of six independent experiments.
Fig 4
Fig 4. Sperm cells migration under pharmacological modulation of TRPV1.
A) Dose-response curve of sperm cells migration towards a capsaicin (CPS) gradient at the concentrations indicated. Significance: * = P<0,05 between the indicated condition; ** = P<0,01 vs CPS 0 μM. B) Dose response curve of acrosome reaction (Acr. Reaction), intracellular calcium levels (Intr. Ca2+) and apoptosis in sperm cells migrated towards a CPS gradient at the concentrations indicated. Data, obtained by flow cytometry, are reported respectively as percentage of CD46-positive (acr. reaction), calcium orange positive cells (Intr. Ca2+) and annexin-V-positive cells (apoptosis). Significance vs CPS 0 μM: * = P<0,05; ** = P<0,01; ǂ = P<0,001 C) Dose-response curve of sperm cells migration towards a 10 μM CPS gradient, pre-incubated with capsazepine (CPZ) for 15 minutes at the concentrations indicated. Significance: * = P<0,05 between the indicated conditions; ** = P<0,01 vs CPZ 0 μM. D) Dose response curve of acrosome reaction, intracellular calcium levels and apoptosis in sperm cells migrated towards a 10 μM CPS gradient pre-incubated with CPZ for 15 minutes at the concentrations indicated. Data, obtained by flow cytometry, are reported respectively as percentage of CD46-positive (acr. reaction), calcium orange positive cells (intr. Ca2+) and annexin-V-positive cells (apoptosis). Significance vs CPS 0 μM: * = P<0,05; ** = P<0,01
Fig 5
Fig 5. Analysis of TRPV1-dependent calcium trafficking in human spermatozoa.
A)Western blot analysis of TRPV1 expression in isolated sperm cytoplasm and sperm membrane obtained by differential centrifugation. β-Actin was used as cytoplasm marker. Images are representative of three independent experiments. D) Dynamic of intracellular calcium concentration in sperm cells stimulated with 10 μM capsaicin (CPS), CPS pre incubated with 10 μM capsazepine (CPS + CPZ), CPS pre incubated with 2,5 μM rabbit polyclonal anti-TRPV1 antibody (CPS + Ab), CPS with chelation of extracellular calcium obtained with addition of 6mM EDTA (CPS + EDTA) compared to unstimulated conditions (CTRL). In C) values of area under the curves normalized on controls (Normalized A.U.C.) are compared. Significance: * = P<0,05 vs CTRL B) Dynamic of intracellular calcium concentration in sperm cells stimulated with 10 μg/mL progesterone (P4), P4 pre incubated with 10 μM capsazepine (P4 + CPZ), P4 pre incubated with 2,5 μM rabbit polyclonal anti-TRPV1 antibody (P4 + Ab), P4 with chelation of extracellular calcium obtained with addition of 6mM EDTA (P4 + EDTA) compared to unstimulated conditions (CTRL). In E) values of area under the curves normalized on controls (Normalized A.U.C.) are compared. Significance: * = P<0,05 vs CTRL
Fig 6
Fig 6. Functional characteristics of sperm cells selected by thermotaxis under pharmacological modulation of TRPV1.
A) Quantification of sperm cells migrated towards a 31–37°C temperature gradient (T gradient) or 10 μM gradient of capsaicin (CPS) or towards 31° no gradient control (Ctrl 31°C), without (w/o) or with pre-incubation with 10 μM capsazepine (CPZ) or 2,5 μM rabbit polyclonal anti-TRPV1 antibody (Ab). Data are reported as millions of cells migrated. Significance vs Ctrl 31°C w/o CZP or Ab: * = P<0,05; ** = P<0,01. B) Acrosome reaction, intracellular calcium levels and apoptosis in the sperm migrated towards the T gradient or the CPS gradient or towards Ctrl 31°, without or with pre-incubation with CPZ or Ab. Data, obtained by flow cytometry, are reported respectively as percentage of CD46-positive (acr. reaction), calcium orange positive cells (intr. Ca2+) and annexin-V-positive cells (apoptosis). Significance: * = P<0,05 vs corresponding condition without CPZ; ǂ = P<0,05 vs Ctrl 31°C. C) Sperm motility parameters assessed by automated Sperm Class Analyser (C). Sperm curvilinear velocity (VLC), straight-line (rectilinear) velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR as VSL/VAP), wobble (WOB as VAP/VLC), beat-cross frequency (BFC), hypermotility (Hyper) were considered in cells migrated towards the T gradient or the CPS gradient or towards Ctrl 31°, without or with pre-incubation with CPZ or Ab. Significance: * = P<0,05 vs 31°C.
Fig 7
Fig 7. TRPV1 levels in of sperm cells selected by pharmacological modulation of TRPV1.
A) TRPV1 protein expression of sperm cells migrated towards a 10 μM CPS gradient (CPS) or towards a 10 μM CPS gradient with pre-incubation with 10 μM CPZ (CPS+CPZ) or towards a 31–37°C temperature gradient with pre-incubation with 10 μM CPZ (T gradient + CPZ). Analysis was performed by flow cytometry (Panel I) and immunofluorescence (Panel II). In panel I, histogram plots of cells stained with anti-TRPV1 antibody (thick continuous line), anti-TRPV1 antibody and immunogen peptide (thin continuous line) and negative control with no primary antibody are compared. Anti-TRPV1 antibody cell staining intensity was distinguished within as low (yellow areas), high (green areas) and intermediate (orange areas). In panel II, TRPV1 staining an immunofluorescence appears as green whilst cell nuclei are counterstained with DAPI (blue). Data are representative of six independent experiments.B-C) RT-PCR analysis of the expression of TRPV1 mRNA sperm cells migrated migrated towards the T gradient or the CPS gradient or towards the Ctrl 31°, without (w/o) or with pre-incubation with CPZ (panel B) or 2,5 μM rabbit ployclonal anti-TRPV1 antibody (Anti TRPV1 IgG, panel C). Data are reported as normalized relative quantification (nRQ). Relative expression was also reported as gel electrophoresis analysis of the target amplification products at 186 base pairs (TRPV1) and GAPDH as housekeeping gene (108 base pairs). Significance: * = P<0,05 vs Ctrl 31°C

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