TRPM8, a versatile channel in human sperm

Abstract

Background: The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca(2+) dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+) ([Ca(2+)]i). Ca(2+) triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored.

Principal findings: Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM) and 80% by BCTC (1.6 microM). Activation of TRPM8 either by temperature or menthol induced [Ca(2+)]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM) and BCTC (1.6 microM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway.

Conclusions: This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

Figure 1. TRPM8 channels are present in human sperm.

A) RT-PCR showing a TRPM8 fragment amplified from human semen. Total RNA was extracted from human semen and cDNA was prepared. Specific oligonucleotides were used for TRPM8. Identity of the expected size (in white numbers) fragment was confirmed by DNA sequencing. B) Immunocytochemistry showing the presence of TRPM8 in heads and flagella of human sperm. Left panels show the confocal fluorescent images with pseudocolor and right panels show their corresponding phase contrast images. C) Western blot experiments showing TRPM8 reactivity (∼130 kDa) in sperm lysates (anti TRPM8 from Gene Tex). n≥3.

Figure 2

A) Menthol induces the AR in human sperm in a dose dependent manner independently of capacitation. Human sperm were separated by swim-up and capacitated (black bars) or not (white bars) during 5 hours. The % Acrosomal Reaction Index (see methods) mean values±SD from control sperm (spontaneous) and treated with various menthol concentrations, ionomycin (iono) and DMSO (the solvent) are shown. B) TRPM8 antagonists block the AR induced by menthol. After swim-up separation, human sperm were capacitated for at least 5 hours. The AR was induced with 500 µM menthol in the presence or absence of BCTC (1.6 µM) or capsazepine (Cz, 20 µM). Both inhibitors blocked the menthol induced AR [** (P< = 0.01), n≥5]. Controls using the inhibitors alone or solvent alone (DMSO) are also shown (black bars).

Figure 3. Menthol causes a capsazepine sensitive [Ca²⁺]i increase in human sperm.

Human sperm were loaded with the fluorescent Ca²⁺ indicator Fluo3-AM (2 µM) and the fluorescence intensity visualized before and after menthol addition as described in methods. Representative single cell (A and C) and group of cells (B and D) spatio-temporal [Ca²⁺]i changes after adding menthol (500 µM, indicated with the light gray bar) in the absence (A and B) and presence of 20 µM Cz (C and D, indicated with the dark gray bar). The panels to the right illustrate representative traces showing the fluorescence change after addition of menthol (light gray bar), in the absence or presence of capsazepine (dark gray bar). The time frame is indicated in each panel. Scales indicate (F/F0) -1 vs time (sec). Note: ∼50% of cells responded to menthol. Color coding: black (−) to red (+) indicates low to high [Ca²⁺]i. n≥3.

Figure 4. The menthol induced [Ca²⁺]i increase in human sperm populations is sensitive to capsazepine and BCTC and depends on external Ca²⁺.

Human sperm were loaded with Fluo3-AM (2 µM) and the fluorescence intensity measured in cell population before and after menthol addition as described in methods. (A) Representative traces showing the fluorescence change after addition of 500 µM menthol (gray circles), this increase was partially blocked by capsazepine (Cz, 20 µM) (black circles), the duration of the stimulus is indicated by the bars above the graph. (B) Summary of the menthol response inhibition caused by 20 µM Cz or 1.6 µM BCTC (% relative fluorescence normalized to the fluorescence obtained after the addition menthol). (C) Representative menthol responses to 500 µM menthol in media containing 100 nM (open circles) or 2 mM (closed circles) external [Ca²⁺] ([Ca²⁺]_e), the duration of menthol application is indicated by the black bar (n = 6, *** p< = 0.001).

Figure 5. Lowering temperature from 25 to 13°C increases [Ca²⁺]i in human sperm.

[Ca²⁺]i was monitored after loading cells with Fluo3-AM (2 µM) (see methods). Single cell (A and C) and group of cells (B and D) spatio-temporal [Ca²⁺]i changes induced by cooling (25–13°C) (red to blue bar) in the absence (A and B) or presence (C and D) of 20 µM Cz (gray bar). The panels to the right illustrate representative [Ca²⁺]i traces of the corresponding field during the temperature ramp in the absence (A and B) or presence (C and D) of 20 µM Cz. The time frame is indicated in each panel. Scales indicate (F/F0) -1 vs time (sec). Note: about 28% of cells responded to menthol. Color coding: black (−) to red (+) indicates low to high [Ca²⁺]. n≥3.

Figure 6. TRPM8 antagonists do not inhibit the human sperm AR induced by ZP3 or progesterone (Pg).

Sperm were capacitated for ≥5 hours (see Methods) and the AR induced with 4 µM Pg (A) or recombinant human ZP3 (B) in the presence or absence of BCTC (1.6 µM) or Cz (20 µM). The ZP3 or Pg induced AR was insensitive to both inhibitors. Controls using the inhibitors alone are also shown (black bars). (C) Representative [Ca²⁺]i changes monitored after loading cells with Fluo3-AM in cell population experiments. The presence of capsazepine (black circles) did not significantly inhibit the progesterone (gray circles) induced [Ca²⁺]i increase. Scales indicate (F/F0) -1 vs time (sec). (D) Summary of the progesterone response in the absence (gray bar) or presence of 20 µM Cz or 1.6 µM BCTC (black bars), (% relative fluorescence normalized to the fluorescence obtained after the addition progesterone; n≥5).