The major phytocannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), affect the function of CatSper calcium channels in human sperm

Abstract

Study question: Do the main psychoactive phytocannabinoid delta-9-tetrahydrocannabinol (THC) and its non-psychoactive analog cannabidiol (CBD) affect human sperm function?

Summary answer: THC and CBD affect the sperm-specific Ca2+ channel CatSper, suppress activation of the channel by progesterone (P4) and prostaglandin E1 (PGE1), and THC also alters human sperm function in vitro.

What is known already: Marijuana (Cannabis sativa) is one of the most commonly used recreational drugs worldwide. Although the effects of phytocannabinoids on semen parameters have been studied, there is no evidence of a direct impact of THC and CBD on human sperm.

Study design, size, duration: We investigated the effects of the major psychoactive phytocannabinoid, THC, its non-psychoactive analog, CBD, and their major metabolites on Ca2+ influx via CatSper in human spermatozoa. THC and CBD were selected to further evaluate their action on P4-, PGE1-, and pH-induced activation of CatSper. The effects of THC and CBD on sperm motility, penetration into viscous media, and acrosome reaction (AR) were also assessed.

Participants/materials, setting, methods: The effects of phytocannabinoids on CatSper activity were investigated on semen samples from healthy volunteers and men with homozygous deletion of the CATSPER2 gene using kinetic Ca2+ fluorimetry and patch-clamp recordings. Motility was assessed by computer-assisted sperm analysis (CASA). Sperm penetration into viscous media was assessed using a modified Kremer test. The AR was evaluated by flow cytometry using Pisum sativum agglutinin-stained spermatozoa.

Main results and the role of chance: Both THC and CBD increased the intracellular calcium concentration with CBD inducing a greater increase compared to THC. These Ca2+ signals were abolished in men with homozygous deletion of the CATSPER2 gene demonstrating that they are mediated through CatSper. THC suppressed the P4- and the PGE1-induced Ca2+ increase with a half-maximal inhibitory concentration (IC50) of 1.88 ± 1.15 µM and 0.98 ± 1.10, respectively. CBD also suppressed the P4- and PGE1-induced Ca2+ signal with an IC50 of 2.47 ± 1.12 µM and 6.14 ± 1.08 µM, respectively. The P4 and PGE1 responses were also suppressed by THC and CBD metabolites, yet with greatly reduced potency and/or efficacy. THC and CBD were found to inhibit the Ca2+ influx evoked by intracellular alkalization via NH4Cl, with THC featuring a higher potency compared to CBD. In conclusion, THC and CBD inhibit both the ligand-dependent and -independent activation of CatSper in a dose-dependent manner. This indicates that these phytocannabinoids are genuine CatSper inhibitors rather than P4 and PGE1 antagonists. Finally, THC, but not CBD, impaired sperm hyperactivation and penetration into viscous media and induced a small increase in AR.

Limitations, reasons for caution: Future studies are needed to assess whether cannabis consumption can affect fertility since this study was in vitro.

Wider implications of the findings: The action of THC and CBD on CatSper in human sperm may interfere with the fertilization process, but the impact on fertility remains to be elucidated. THC inhibits the P4 and the PGE1 response more potently than CBD and most previously described CatSper inhibitors. THC can be used as a starting point for the development of non-hormonal contraceptives targeting CatSper.

Study funding/competing interest(s): This work was supported by the Swiss Center for Applied Human Toxicology (SCAHT), the Département de l'Instruction Publique (DIP) of the State of Geneva and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). The authors declare that no conflicts of interest have been identified that might affect the impartiality of the research reported.

Trial registration number: N/A.

Keywords: CBD; CatSper; THC; acrosome reaction; calcium signaling; cannabis; human sperm; phytocannabinoids; sperm motility.

Figure 1.

The action of delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and their main metabolites on the intracellular Ca²⁺ concentration in human sperm. Representative Ca²⁺ signals in human sperm loaded with a fluorescent Ca²⁺ indicator evoked by progesterone (P4) and increasing concentrations of (A–C) THC, 11-hydroxy-Δ⁸-THC (OH-THC), 11-Nor-Δ⁹-carboxy-THC (COOH-THC), and (E–G) CBD, 7-hydroxy cannabidiol (OH-CBD), and 7-carboxy cannabidiol (COOH-CBD). ΔF/F₀ (%) indicates the percentage change in fluorescence (ΔF) with respect to the mean basal fluorescence (F₀) before application (at t = 54 s) and continuous presence of the compounds. Dose–response curves were fitted to the maximal signal amplitudes (mean ± SD), and EC₅₀ (± SE of the fit) were generated for (D) THC (n = 11), OH-THC (n = 7), COOH-THC (n = 7) as well as (H) CBD (n = 10), OH-CBD (n = 8), and COOH-CBD (n = 4). (I) Representative Ca²⁺ signals evoked by progesterone (P4), THC, CBD, and metabolites before application (at t = 210 s) and continuous presence of the compounds (at 10 µM) with a subsequent application of the Ca²⁺-ionophore ionomycin (2 µM) in sperm of a man lacking the CATSPER2 gene (CATSPER2^−/−), leading to a loss of CatSper function. (J) Scatter dot plot showing mean ± SD of maximal signal amplitudes evoked by P4 (5 µM) as well as THC, CBD, and metabolites (all at 10 µM) in control (same data as shown in D and H) and CATSPER2^−/− sperm.

Figure 2.

The action of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on progesterone (P4)- and prostaglandin E1 (PGE1)-evoked Ca ²⁺  influx in human sperm. Representative Ca²⁺ signals in human sperm evoked by P4 (5 µM) and PGE1 (5 µM) in the absence (0 µM) and presence of increasing concentrations of (A, B) THC or (D, E) CBD. Sperm were incubated for ≥5 min with phytocannabinoids prior to the addition of P4 or PGE1. Dose–response curves for signal amplitudes normalized to buffer control (set to 100%) are generated for (C) THC showing the P4 inhibition (IC₅₀ = 1.88 ± 1.15 µM; n = 6) and the PGE1 inhibition (IC₅₀ = 0.98 ± 1.10 µM, n = 5). Similar dose–response curves were generated for (F) CBD showing the inhibition of P4 (IC₅₀ 2.47 ± 1.12 µM, n = 5) and PGE1 responses (IC₅₀ = 6.14 ± 1.08 µM, n = 6). Dots in panels (C) and (F) represent mean values ± SD.

Figure 3.

Delta-9-tetrahydrocannabinol (THC) acts on the progesterone-evoked Ca ²⁺  influx at pharmacologically relevant concentrations. Toxicological metrics are shown for (A) THC and (B) cannabidiol (CBD) based on the results from the progesterone (P4)- and prostaglandin E1 (PGE1)-induced Ca²⁺ influx (n ≥ 10). The reported maximal plasma concentration (C_max) based on the literature search is represented by a red triangle. The concentration at which there is a 2% inhibitory effect (IC₀₂) is represented by a blue dot (mean ± SD) and is used as an estimate of the minimum inhibitory concentration. The half-maximal inhibiting concentration (IC₅₀) is shown as black bar plot showing the mean values ± SD.

Figure 4.

The action of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on alkaline-evoked Ca ²⁺  signals in human sperm. Ca²⁺ signals evoked by NH₄Cl (3 mM) in the absence (0 µM) and the presence of increasing concentrations of (A) THC and (B) CBD are shown. (C) Dose–response curves for signal amplitudes normalized to vehicle control (set to 100%) were generated for THC and CBD with a half-maximal inhibitory concentration (IC₅₀) of 0.90 µM ± 1.11 µM (n = 4) and 3.98 µM ± 1.08 µM (n = 4), respectively. Dots in panel (C) represent mean values ± SD.

Figure 5.

Delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) inhibit CatSper in human sperm. (A) Assessment of the action of THC, CBD, and their metabolites on CatSper in human sperm using the CatSper-Activity-Test. Fraction of motile sperm (mean ± SD) 30 min after dilution in Ca²⁺-free human tubular fluid (HTF) media (HTF0Ca, control) (n = 7) or Ca²⁺-free HTF containing CatSper inhibitor TS150 (60 µM), THC, CBD, 7-hydroxy CBD (CBD-OH), 7-carboxy CBD (CBD-COOH), 11-hydroxy-Δ⁸-THC (OH-THC), or 11-Nor-Δ⁹-carboxy-THC (COOH-THC) (all at 10 µM) (n ≥ 3), relative to that upon dilution in standard HTF (set to 100%). All conditions were compared to Ca²⁺-free HTF (HTF0Ca, control), ****P < 0.0001. (B) Representative current–voltage relationship of currents recorded from a human sperm cell superfused with Ca²⁺- and Mg²⁺-containing solution (HEPES-saline, HS) (control), followed by divalent-free extracellular solution (NaDVF) and, finally, NaDVF-containing THC (10 µM). The monovalent currents in NaDVF are mediated by CatSper. (C) Relative current amplitude (mean ± SD) at +100 mV in the presence of THC (10 µM) relative to that in its absence (set to 1) (n = 4). Gray dots indicate individual values.

Figure 6.

The action of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on sperm motility, progesterone-induced acrosomal exocytosis, and sperm penetration into viscous media. (A) Sperm motility was assessed by a computer-assisted sperm analyzer (CASA) (n > 9). THC, but not CBD, significantly reduced (A) overall motility, (B) progressive motility, and (C) hyperactive motility. (D) After incubation of sperm cells with progesterone (P4) (5 µM), prostaglandin E1 (PGE1) (5 µM), phytocannabinoids (10 µM), or buffer for 1 h, cells were allowed to migrate in glass capillaries filled with methylcellulose 1% for a further hour. Cells were counted at 1 cm after incubation and normalized to vehicle control (ethanol, EtOH 0.5%) shown in a dotted gray line (n = 11). (E) Assessment of acrosomal exocytosis in live human sperm was performed using loaded sperm with fluorescent dyes to stain the acrosomal inner membrane (FITC-conjugated Pisum sativum agglutinin), viability (propidium iodide), and the nucleus (Hoechst). Sperm cells were incubated with P4 (5 µM), PGE1 (5 µM), THC, or CBD (10 µM). EtOH 0.5% was used as a vehicle control and ionophore A23187 (2 µM) as a positive control (n = 7). All analyses were compared with buffer; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bar plots with individual data points represent mean values ± SD.