The major phytocannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), affect the function of CatSper calcium channels in human sperm
Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland
Swiss Centre for Applied Human Toxicology (SCAHT), Basel, Switzerland
Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany
Service of Clinical Chemistry and Toxicology, Central Institute of Hospitals, Hospital of Valais, Sion, Switzerland
Department of Internal Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland
Correspondence address. Department of Genetic Medicine and Development, University of Geneva, Rue Michel-Servet 1, 1206 Geneva, Switzerland. E-mail: serge.nef@unige.ch (S.N.); Department of Genetic Medicine and Development, University of Geneva, Rue Michel-Servet 1, 1206 Geneva, Switzerland. E-mail: rita.rahban@unige.ch (R.R.)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.
Introduction
Infertility and problems of impaired fecundity affect an estimated 10–15% of couples worldwide, and the male factor contributes to approximately 50% of all infertility cases (Boivin et al., 2007; Agarwal et al., 2015; Barratt et al., 2017). Among the many etiological factors associated with male infertility, environmental and lifestyle factors such as diet, obesity, physical activity, and substance abuse are thought to play an important role (Cazzaniga et al., 2020; Del Giudice et al., 2020). In particular, there is evidence that cannabis consumption may impair fertility; however, its effect on semen parameters and sperm fertilizing capacities remains controversial (Belladelli et al., 2021).
In mammals, the principal sperm plasma membrane Ca2+ channel is the flagellar-specific Cation channel of Sperm (CatSper), which acts as an essential modulator of downstream mechanisms involved in fertilization (reviewed by Kaupp and Strünker, 2017; Sun et al., 2017; Rahban and Nef, 2020; Wang et al., 2021; Hwang and Chung, 2023). CatSper is a multisubunit channel that can be activated by physiological stimuli such as depolarization of the membrane potential (Vm), alkalization of the intracellular pH (pHi) as well as binding of natural ligands such as progesterone (P4) and prostaglandins released in the oviduct (Lishko et al., 2011; Strünker et al., 2011). Upon channel activation, the intracellular calcium concentration ([Ca2+]i) increases significantly. This increase is involved in key sperm functions, including capacitation (Sumigama et al., 2015), hyperactivation (Alasmari et al., 2013; Williams et al., 2015; Young et al., 2024), penetration into viscous media (Rennhack et al., 2018; Luo et al., 2019; Young et al., 2024) and, possibly, acrosome reaction (AR, Tamburrino et al., 2014; Luo et al., 2019; Prajapati et al., 2022). Several studies have shown that CatSper is a promiscuous chemosensor (Brenker et al., 2012; Rahban and Nef, 2020; Jeschke et al., 2021). In addition to endogenous ligands, its activity is modulated by a variety of exogenous compounds that might interfere with the ability of sperm to reach and fertilize the egg. These include endocrine-disrupting chemicals (EDCs) (Tavares et al., 2013; Schiffer et al., 2014; Yuan et al., 2020), odorants (Brenker et al., 2012), pharmaceutical compounds (Tavares et al., 2013; Schiffer et al., 2014; Rehfeld et al., 2016, 2022; Majzoub and Agarwal, 2018; Yuan et al., 2020; Birch et al., 2021; Rahban et al., 2021; Wang et al., 2021), and steroids (Jeschke et al., 2021; Wehrli et al., 2023).
Cannabis sativa, also known as marijuana, is the third most commonly used recreational drug worldwide after alcohol and tobacco (Capriotti and Sapp, 2021). The prevalence of cannabis use is increasing, particularly among people of reproductive age, in part due to widespread legalization and increased social acceptability and accessibility (World Drug Report, 2017). According to the 2022 annual report of the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), 15.5% of European Union (EU) residents aged 15–34 years had consumed cannabis at least once in the previous year (European Monitoring Centre for Drugs and Drug Addiction, 2022). Similarly, a survey conducted by the US National Center for Drug Abuse Statistics found that 45% of people aged 12 years and older had used marijuana at least once in their lives (US National Center for Drug Abuse Statistics, 2022). Today, over 50 countries have legalized or decriminalized the medical use of marijuana. Although the legalization of its main active component, delta-9-tetrahydrocannabinol (THC), is highly controversial, the cultivation of cannabidiol (CBD), its non-psychoactive analog, is no longer restricted in the EU or the USA if the THC concentration does not exceed 0.3% or 0.2%, respectively (Pannico et al., 2022).
Here, we investigated the effects of THC and CBD on [Ca2+]i and CatSper-mediated Ca2+ responses using kinetic [Ca2+]i fluorimetry. We show that both THC and CBD increase [Ca2+]i with CBD inducing a greater increase compared to THC. These Ca2+ signals were abolished in men with homozygous deletion of the CATSPER2 gene (CATSPER2−/−) demonstrating that they are mediated through CatSper. Moreover, both THC and CBD suppress ligand- and pH-dependent activation of CatSper, with THC inhibiting the channel more potently than CBD. Using functional assays, we show that THC, but not CBD, reduced total motility, hyperactivity, and penetration in viscous medium and induced a small increase in AR downstream of CatSper. Overall, we provide the first experimental evidence that the major phytocannabinoids THC and CBD disrupt the human sperm cell Ca2+ signaling and function, which may have implications for human fertilization.
Materials and methods
Reagents
(−)-Trans-Δ9-THC [Chemical Abstracts Service (CAS) #: 1972-08-3] and CBD (CAS#: 13956-29-1) were purchased from Sigma-Aldrich (Buchs, Switzerland) at a stock concentration of 1 mg/ml in methanol with a certified reference material from Ceriliant®. For the functional tests, THC was purchased from Lipomed (Arlesheim, Switzerland, CAS#: 1972-08-3) and dissolved in ethanol (EtOH) at a stock concentration of 2 mM. CBD was purchased from Tocris Bioscience (CAS#: 13956-29-1; Bristol, UK) and dissolved at a stock concentration of 20 mM in EtOH. The following phytocannabinoid metabolites were purchased from Sigma-Aldrich, Buchs, Switzerland, at a stock concentration of 1 mg/ml in methanol with a certified reference material from Ceriliant®: (−)-11-hydroxy-Δ8-THC (OH-THC, CAS#: 28646-40-4), (±)-11-Nor-Δ9-THC carboxylic acid (COOH-THC, CAS#: 104874-50-2), 7-hydroxy CBD (7-OH-CBD, CAS#: 1101886-10-5), and 7-carboxy CBD (COOH-CBD, CAS#: 1101886-13-8). Ionophore A23178 (CAS#: 52665-69-7), P4 (CAS#: 57-83-0), and prostaglandin E1 (PGE1) (CAS#: 745-65-3) were purchased from Sigma-Aldrich (Buchs, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 20 mM. The fluorescent Ca2+ indicator Fluo-4 AM (CAS#: 273221-67-3) was purchased from Invitrogen (California, USA) and dissolved in DMSO at a stock concentration of 2 mM. Human serum albumin (HSA, CAS#: 70024-90-7) was obtained from Polygon Diagnostics (Lucerne, Switzerland). Propidium iodide (PI) was purchased from Thermo Fisher Scientific (Massachusetts, USA) and dissolved in water at a stock concentration of 1 mg/ml. Fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was purchased from Sigma-Aldrich (Buchs, Switzerland) and was dissolved in phosphate-buffered saline (PBS) at a stock concentration of 1 mg/ml. Hoechst-33342 was purchased from Sigma-Aldrich (Buchs, Switzerland) and dissolved in water at a stock concentration of 1.5 mg/ml.
Semen sample collection and preparation
Human semen samples were collected from healthy volunteers and a man with homozygous deletion of the CATSPER2 gene (CATSPER2−/−) (Young et al., 2024) with prior written consent, under approval from the ethical committees of the Medical Association Westfalen-Lippe, the Medical Faculty of the University of Münster (4INie, 2021-402-f-S) and the Cantonal Ethics Committee of the State of Geneva (CCER #PB_2019-00119/#14-147). Semen samples were produced by ejaculation into plastic containers after a recommended 48-h period of sexual abstinence. All semen samples were liquefied at 37°C for 15–30 min. Motile spermatozoa were selected by swim-up as previously described (Strünker et al., 2011). Briefly, 1 ml of semen was overlayed with 4 ml of human tubular fluid (HTF) medium in a 45°-tilted tube and incubated at 37°C for 60 min. The supernatant was collected in 15 ml Falcon tubes and centrifuged twice at 700× g for 15 min. Sperm cells were resuspended in HTF medium supplemented with HSA (0.3% w/v) to obtain a final sperm concentration of 1 × 107 cells/ml. The HTF medium was prepared as follows (in mM): 93.8 NaCl, 4.7 KCl, 0.2 MgSO4, 0.369 KH2SO4, 2.04 CaCl2, 20.98 HEPES, 2.78 glucose, 21.4 Na-lactate, 4 NaHCO3, 0.33 Na-pyruvate, pH adjusted to 7.35 with NaOH.
Ca2+ fluorimetry measurements
Cells were loaded with the fluorescent Ca2+ indicator Fluo-4 AM at a final concentration of 2.5 µM in the presence of Pluronic F-127 (0.05% w/v) for 30 min at 37°C. After incubation, the excess dye was removed by centrifugation for 5 min at 700× g, and cells were resuspended in HTF to obtain a final concentration of 5 × 106 cells/ml. A volume of 50 µl of Fluo-4-loaded cells (corresponding to 125 000 cells) was added to each well in a 384-well plate for fluorescence measurement. Fluorescence was measured in a fluorescent plate reader (FLUOstar Omega, BMG Labtech, Germany) at 30°C with an excitation wavelength of 480 nm and an emission wavelength of 520 nm with bottom optics. Fluorescence was recorded before and after the application of 25 µl (1:3 dilution) of buffer, ligands, or phytocannabinoids with an electronic multichannel pipette yielding technical duplicates for each condition within each experiment. Changes in Fluo-4 fluorescence are depicted as ΔF/F0 (%), that is, the change in fluorescence (ΔF) relative to the mean basal fluorescence (F0) before application of buffer or stimuli (25 µl) in order to correct for intra- and inter-experimental as well as compound-induced variations in basal fluorescence among individual wells.
Patch-clamp recordings
Patch-clamp recordings from human sperm were performed in the whole-cell configuration, as previously described (Strünker et al., 2011). The standard extracellular solution (HEPES-Saline, HS) contained (in mM): 135 NaCl, 5 KCl, 1 MgSO4, 2 CaCl2, 5 glucose, 10 lactic acid, and 20 HEPES, pH adjusted to 7.4 with NaOH. The sodium-based divalent-free solution (NaDVF) contained (in mM): 140 NaCl, 40 HEPES, 1 EGTA, pH adjusted to 7.4 with NaOH; the pipette solution contained (in mM): 130 Cs-aspartate, 50 HEPES, 5 EGTA, 5 CsCl, pH adjusted to 7.3 with CsOH.
CatSper-Activity-Test
A modified CatSper-Activity-Test (Young et al., 2024) was performed in 96-well plates (Nunc Microwell, Thermo Fisher). Wells were filled with 95 µl HTF, 95 µl Ca2+-free HTF (HTF0Ca, negative control), or HTF0Ca mixed with the compounds to be analyzed. The HTF0Ca contained (in mM): 82 NaCl, 4.7 KCl, 0.369 KH2PO4, 5 EGTA, 5 EDTA, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 4 NaHCO3, and 20.98 HEPES, 0.03% HSA (w/v), pH was adjusted to 7.35 using NaOH. A 5 µl aliquot of swim-up sperm sample was added to each well, and the plate was incubated at 37°C. After 30 min, motility was analyzed with an inverted microscope (Olympus IX73) placed in a home-built housing at 37°C, equipped with 10× objective (UPlanFI, Olympus) and a 1× magnification adapter (Olympus U-TV1x-2). Brightfield videos of the bottom of the wells were recorded with CMOS camera (UI-3140CP-M-GL R2, iDS GmbH) at 60 frames per second (fps), and the contrast was enhanced in Image J (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA) using the ‘find edges’ algorithm. An open-source CASA program (Alquézar-Baeta et al., 2019) was then used to determine the number (in the field of view) of motile sperm in HTF, HTF0Ca (control), and HTF0Ca containing the test compounds. Sperm having a maximal amplitude of lateral head displacement (ALHmax) ≥1 µm were considered motile. The number of motile sperm in HTF0Ca (control) and HTF0Ca containing the test compounds relative to that in HTF (set to 100%) was calculated and depicted.
Sperm motility assessment
Swim-up prepared sperm cells were incubated for 3 h at a concentration of 12.5 × 106/ml in capacitating media (HTF+) containing (in mM): 93.8 NaCl, 4.7 KCl, 0.2 MgSO4, 0.369 KH2PO4, 2.04 CaCl2, 20.98 HEPES, 2.78 glucose, 21.4 Na-lactate, 25 NaHCO3, 0.33 Na-pyruvate, and the pH was adjusted to 7.35 with NaOH. After capacitation, sperm cells were incubated in the absence (buffer) or presence (10 µM) of selected compounds for an additional 1 h at 37°C. A 2 µl aliquot was placed in a 20 µm depth counting chamber (Leja, Nieuw-Vennep, The Netherlands). The slide was placed on a thermostatic (37°C) microscope stage and analyzed using computer-assisted sperm analysis (CASA) (Hamilton Thorne Ceros II, Beverly, MA). A minimum of 200 sperm cells in at least five randomly selected fields were captured at a rate of 65 fps using a 10× objective. Progressive motility was classified based on the average path velocity (VAP) with the following parameters: slow ≤ 1 > medium ≤ 25 > rapid. Sperm cells were considered hyperactive if they had a VAP > 50 µm/s, a curvilinear velocity (VCL) > 90 µm/s, and an ALH > 5 µm.
Sperm penetration in viscous media
Sperm penetration was assessed using the Kremer test according to a previously published study (Rahban et al., 2021). Briefly, swim-up recovered sperm cells (3 × 106/ml) were allowed to capacitate in HTF+ media for 3 h at 37°C. Cells were then incubated in the absence (buffer) or presence of compounds at 10 µM for one hour at 37°C. A glass capillary (0.2 × 4.0 × 50 mm, CM Scientific, UK), filled with 1% (w/v) methylcellulose (MC, 4000 centipoise, CAS#: 9004-67-5, Sigma-Aldrich, Buchs, Switzerland) prepared with HTF media containing 0.3% (w/v) HSA, THC, or CBD at 10 µM or buffer and sealed on one end with wax (Vitrex, UK), was prepared. After incubation, the open end of the capillary was added to the sperm cells for an additional hour. The number of sperm cells reaching the 1 cm mark inside the capillary tube was counted using a microscope with a 10× objective.
Assessment of acrosome reaction
The evaluation of AR on live sperm cells was performed as previously described (Wehrli et al., 2023). Briefly, sperm cells (5 × 106 cells/ml) were allowed to capacitate for 4 h at 37°C in capacitating media HTF+. After capacitation, sperm cells were incubated with 5 µg/ml FITC-PSA and 10 µg/ml Hoechst-33342 for 30 min. Samples were centrifuged at 700× g for 10 min to remove excess dye and resuspended in HTF+. Sperm cells were then incubated for 45 min in the absence (EtOH 0.5%) or presence of the compounds at 10 µM. The positive control, ionophore A23187, was used at 2 µM for 30 min. All the samples were then briefly incubated for <10 min with PI (0.5 µg/ml) prior to analysis. Samples were analyzed by BDFACSAria (BD Biosciences, San Jose, CA, USA). FITC-PSA fluorescence was detected by excitation at 475 nm and emission at 560/35 nm, PI was detected by excitation at 530 nm and emission at 675/75 nm and Hoechst was detected by excitation at 346 nm and emission at 460 nm. Hoechst-positive and PI-negative cells were classified as live cells. Doublet exclusion was performed by two-dimensional dot plot analysis of forward-scatter width (FSC-W) versus forward-scatter height (FSC-H) and side-scatter width (SSC-W) versus side-scatter height (SSC-H) on the pre-selected singlets. Data were collected from 40 000 FITC-PSA-negative events per sample to define the sperm population. Based on the selected population, PI-negative and FITC-PSA-positive cells were selected as live acrosome-reacted cells, whereas PI-negative and FITC-PSA-negative cells were selected as live acrosome-intact cells.
Statistical analysis
Data are shown as mean ± SD with ‘n’ referring to the number of independent experiments performed using sperm samples from ≥3 different donors. Experiments on human sperm lacking functional CatSper channels were performed with an n = 3 on swim-up sperm prepared from one individual with a homozygous deletion of the CATSPER2 gene. Statistical analysis and fitting of dose–response relation to generate sigmoidal curves were performed on raw data using GraphPad Prism, version 8.1.1 (Prism, La Jolla, CA, USA). Sigmoidal curves were generated after converting the compound concentrations to their respective log values. A four-parameter Hill equation with a variable Hill coefficient was fitted to generate both the half-maximal activating concentrations (EC50), the half-maximal inhibitory concentrations (IC50), and the 2 percentiles of maximal inhibitory effect (IC02). Functional in vitro assays were compared using one-way ANOVA corrected for multiple comparisons with the Dunnett method. A P-value of <0.05 was considered significant.
Results
Phytocannabinoids THC, CBD, and their metabolites act on CatSper in human sperm
We studied the effect of the major phytocannabinoids, THC and CBD, and their metabolites OH-THC, COOH-THC, OH-CBD, and COOH-CBD on [Ca2+]i of human sperm loaded with a fluorescent Ca2+ indicator. Fluorescence was measured before and after the addition of increasing doses of phytocannabinoids, positive control (progesterone, P4), and the buffer control (indicated as ‘0’ in gray) (Fig. 1A–C and E–G). P4 induced the prototypical pronounced Ca2+ signal previously described, while THC and its metabolites COOH-THC and OH-THC induced only a small, but significant transient Ca2+ increase (Fig. 1A–D and J; Supplementary Fig. S1A–C). The EC50 for THC, COOH-THC, and OH-THC were 1.03 ± 1.20 µM, 0.87 ± 0.53 µM and 5.33 ± 2.30 µM, respectively (Fig. 1D). Compared to THC, CBD induced a more pronounced Ca2+ increase with an EC50 of 1.17 ± 1.17 µM (Fig. 1E and H; Supplementary Fig. S1). The CBD metabolites OH-CBD and COOH-CBD also induced Ca2+ increases with an EC50 of 1.80 ± 1.40 µM and 1.82 ± 1.34 µM, respectively, that were, however, smaller in amplitude compared to that induced by CBD (Fig. 1; Supplementary Fig. S1). In sperm of men with homozygous deletion of the CATSPER2 gene, leading to a loss of CatSper function (previously described in Schiffer et al., 2020; Jeschke et al., 2021; Young et al., 2024), the Ca2+ signals induced by THC, CBD, and their metabolites were abolished, demonstrating they rest on Ca2+ influx via CatSper (Fig. 1I and J).
THC and CBD suppress the ligand-induced activation of CatSper in human sperm
We investigated the action of the phytocannabinoids and their metabolites on P4- and PGE1-induced Ca2+ influx. THC suppressed the P4- and the PGE1-induced Ca2+ increase with half-maximal inhibitory concentrations (IC50) of 1.88 ± 1.15 µM and 0.98 ± 1.10, respectively (Fig. 2A–C). Similarly, CBD suppressed the P4- and PGE1-induced Ca2+ signal with IC50 values of 2.47 ± 1.12 µM and 6.14 ± 1.08 µM, respectively (Fig. 2D–F). The P4 and PGE1 responses were also suppressed by THC and CBD metabolites, yet with greatly reduced potency and/or efficacy (Supplementary Fig. S2).
THC and CBD might affect ligand activation of CatSper at pharmacologically relevant concentrations
To determine the pharmacological relevance of the THC and CBD action on CatSper in human sperm, we calculated the concentration with 2% of the maximal inhibitory effect (IC02), an estimated lowest effective dose used in previous publications (Schiffer et al., 2014; Rehfeld et al., 2016; Frederiksen et al., 2021). The IC02 values were compared with the maximal reported plasma concentration (Cmax) of THC and CBD (Fig. 3). The IC02 of THC for inhibition of the P4-induced Ca2+ signal was 0.95 ± 0.22 µM and, thus, higher than the reported Cmax of 0.51 µM, while the IC02 for the inhibition of the PGE1 response (0.39 ± 0.02 µM) was lower than the Cmax, suggesting that THC might affect ligand activation of CatSper at pharmacologically relevant concentrations (Fig. 3A). The IC02 of CBD to inhibit the P4 response (IC02 of 0.28 ± 0.01 µM) but not that to inhibit the PGE1 response (1.03 ± 0.20 µM) was lower than the Cmax of 0.40 µM, suggesting that also CBD might affect CatSper at pharmacologically relevant concentrations (Fig. 3B).
THC and CBD also suppress pH-induced activation of CatSper
We then investigated whether THC and CBD also inhibit the ligand-independent activation of CatSper, such as the Ca2+ influx evoked by intracellular alkalization via NH4Cl. Both THC and CBD suppressed the alkaline-induced Ca2+ signal, with THC featuring a higher potency compared to CBD; the IC50 values were 0.90 ± 1.11 µM and 3.98 ± 1.08 µM, respectively (Fig. 4A-C). Of note, neither THC nor CBD alone affected the sperm intracellular pH (Supplementary Fig. S3). In conclusion, THC and CBD also inhibit the ligand-independent activation of CatSper in a dose-dependent manner. This indicates that both THC and CBD are genuine CatSper inhibitors rather than progesterone and PGE1 antagonists.
THC and CBD directly inhibit the CatSper channel
We scrutinized whether THC and CBD directly inhibit CatSper by two independent techniques: a motility-based assay and the patch-clamp electrophysiology. Lowering the extracellular Ca2+ ([Ca2+]o) concentration to nanomolar levels renders human sperm immotile (Torres-Flores et al., 2011; Schiffer et al., 2020). This cessation of motility by low [Ca2+]o is mediated by CatSper and can thus be suppressed by CatSper inhibitors (Young et al., 2024). This finding allows for a simple motility-based assay to assess the activity of CatSper in human sperm, called the CatSper-Activity-Test (Young et al., 2024). We used this test to study the action of THC and CBD on CatSper in human sperm. To this end, we incubated human sperm for 30 min in standard HTF (considered as the reference media), Ca2+-free HTF (HTF0Ca, control media), and Ca2+-free HTF media containing the CatSper inhibitor TS150 (Schierling et al., 2023), THC, CBD, or THC/CBD metabolites. Compared to standard HTF media (set to 100%), the fraction of motile sperm in control HTF0Ca media dropped to 0% (Fig. 5A), i.e. the sperm became immotile. TS150, but also THC and CBD, suppressed this motility decrease. In the presence of the compounds, the fraction of motile sperm increased to about 53%, 72%, and 63%, respectively. This demonstrates that similar to TS150, THC and CBD also directly inhibit CatSper in human sperm. Of note, at the test concentration of 10 µM, THC-OH, THC-COOH, CBD-OH, and CBD-COOH did not suppress the motility decrease (Fig. 5A). It is noteworthy to mention that the THC and CBD metabolites completely inhibit the channel only at concentrations >30 µM (Supplementary Fig. S2). Thus, taken altogether, this confirms the results of the Ca2+-fluorimetric experiments. In addition, we recorded CatSper-mediated membrane currents from human sperm using the patch-clamp technique. Perfusion of the sperm cell with THC suppressed CatSper currents (Fig. 5B and C). At +100 mV, the presence of THC (10 µM) reduced the current amplitude to 8 ± 6% (n = 4) of that recorded in its absence.
THC significantly reduced hyperactive motility and penetration into viscous media and induced AR
We analyzed the swimming behavior of sperm cells in a non-viscous medium in the presence and absence of THC and CBD at 10 µM. Incubation of sperm with THC for 1 h induced a significant reduction in total, progressive, and hyperactive motility, whereas CBD did not affect sperm motility (Fig. 6A–C). When assessing sperm penetration in a viscous medium using a modified Kremer test, the number of penetrating sperm cells in the presence of THC was significantly reduced to 0.38 ± 0.33 of the control at a 1 cm distance (Fig. 6D). CBD, however, did not induce a significant change (1.33 ± 0.60-fold) (Fig. 6D). In addition, we investigated the action of THC and CBD on AR using live-cell flow cytometry. THC alone induced an increase in AR, whereas CBD did not (Fig. 6E). Taken together, the inhibition of CatSper by THC impairs motility, including hyperactivation as well as penetration in a viscous medium. The effects of THC, but not CBD, on sperm motility and AR as well as the lack of an effect of CBD might be because THC, but not CBD, also affects the viability of human sperm (Supplementary Fig. S4).
Discussion
The widespread use of cannabis among women and men of reproductive age is of concern, particularly given the limited and often conflicting data on its potentially harmful effects on the reproductive system. The sperm-specific Ca2+ channel CatSper involved in multiple important processes leading to successful fertilization in vivo and in vitro (Williams et al., 2015; Luo et al., 2019; Young et al., 2024). It has been shown on multiple occasions that CatSper is highly promiscuous and can be activated by structurally diverse molecules (reviewed in Rahban and Nef, 2020; Hwang and Chung, 2023). We investigated the action of the major phytocannabinoids, THC and CBD, on CatSper and human sperm function in vitro. We show that THC and CBD act on CatSper and inhibit the ligand- and alkalization-induced activation of the channel. Functionally, THC, but not CBD, reduces sperm hyperactivation and penetration in viscous media and induces the AR.
An important question to first address is whether the results from our in vitro study are of pharmacological relevance in vivo. The serum concentration of THC and CBD varies depending on whether they are used for therapeutic or recreational purposes (Leung, 2011; Freeman et al., 2019). In therapeutic use, e.g. dronabinol for the treatment of nausea and vomiting caused by cancer chemotherapy, the concentration of THC can reach a plasma peak concentration of 0.025 µM (± 0.015) after 1.5 h (Food and Drug Administration, 2006). When used for recreational purposes, the concentration of these cannabinoids depends on their presence in the cigarette, the chronicity of consumption, and the route of absorption (Misner et al., 2021). When inhaled, THC is detected in the plasma within seconds of the first puff, and peak plasma concentrations are reached within 3–10 min (Huestis et al., 1992; Grotenhermen, 2003). In subjects smoking a cigarette containing cannabis with high THC or CBD content, the mean peak plasma concentrations were 0.51 µM and 0.40 µM, respectively (Lindgren et al., 1981; Perez-Reyes and Wall, 1982; Huestis et al., 1992; McGilveray, 2005; Fabritius et al., 2013; Gelmi et al., 2021). We observed that the IC02 used as an estimate of the lowest effective dose of THC for PGE1 was 0.40 ± 0.02 µM which is similar to the maximal concentration found in the blood. The IC02 of CBD for P4 was 0.28 ± 0.01 µM which is lower than the maximal concentration in the blood, suggesting that both THC and CBD might act on CatSper at physiologically relevant concentrations.
The undeniable importance of CatSper in the fertilization process lies in the fact that CatSper loss-of-function abolishes sperm flagellum hyperactivation and causes male infertility in both mice and humans (Avidan et al., 2003; Zhang et al., 2007; Avenarius et al., 2009; Hildebrand et al., 2010; Smith et al., 2013; Brown et al., 2018; Luo et al., 2019; Young et al., 2024). Evaluating the action of THC, CBD, and their metabolites on men with loss of CatSper function featuring homozygous deletions of CATSPER2 demonstrates that they directly act on CatSper to increase [Ca2+]i.
One of the landmarks of CatSper activation is inducing sperm hyperactive motility, which is characterized by a vigorous flagellar beat producing a whip-like motion that allows the sperm to overcome the viscous fluid of the upper reproductive tract to reach the egg and ultimately fertilize it (Suarez, 2008). When assessing whether the effects of THC on CatSper activation are functionally translated, we showed that THC, but not CBD, interferes with major processes downstream of CatSper, such as hyperactivation and penetration in a viscous medium, which are key steps required for fertilization. We also found that THC induced an increase in AR. This is particularly relevant as the timing of the AR is also critical for successful fertilization. Indeed, it has been shown in humans that spontaneous AR leads to poor in vitro fertilization success (Wiser et al., 2014). Although the role of CatSper-mediated Ca2+ influx in regulating the acrosomal response remains unclear (Tamburrino et al., 2014; Uñates et al., 2014; Singh and Rajender, 2015; Beltrán et al., 2016; Prajapati et al., 2022; Young et al., 2024), THC may interfere with this mechanism, thereby increasing the number of spontaneous acrosomal exocytoses. The increase in AR could be, however, explained by a decrease in sperm viability in the presence of THC after 45 min of incubation (Supplementary Fig. S4).
In order to assess whether THC could affect these sperm function and fertilization in vivo, data on its concentration in reproductive fluids such as seminal fluid and female genital tract fluids are required. To the best of our knowledge, the concentration of THC in seminal and/or oviductal fluid has never been reported. However, THC and its primary metabolites have been detected in follicular fluid (Fuchs Weizman et al., 2021). Moreover, it has been reported that women regularly using cannabis have traces of THC in their reproductive organs (El Marroun et al., 2009), including in vaginal fluids (Schuel et al., 2002). THC is rapidly metabolized to OH-THC and COOH-THC (Sharma et al., 2012; Chayasirisobhon, 2020). Since the two major metabolites of THC in serum are OH-THC and COOH-THC, we tested their ability to interfere with CatSper activation by P4 and PGE1. The THC metabolites featured a much lower potency and/or efficacy to modulation of CatSper, suggesting that THC alone is responsible for reducing the ligand-dependent and -independent activation of the channel.
The precise molecular mechanism underlying the inhibition of CatSper by THC and CBD remains to be elucidated. Both the CatSper-Activity-Test and the patch-clamp experiments demonstrate that THC and CBD act directly on the CatSper channel. This also indicates that the mechanism of inhibition is not related to antagonism of the receptor alpha/beta hydrolase domain-containing protein 2 (ABHD2), which is reportedly involved in steroid activation of CatSper. Notably, CatSper is modulated by a wide range of physiological ligands (Brown et al., 2017; Jeschke et al., 2021; Taiwo et al., 2021), as well as pharmaceuticals and synthetic compounds (Brenker et al., 2012, 2018a, 2018b; Tavares et al., 2013; Schiffer et al., 2014; Rehfeld et al., 2016; Zou et al., 2017; Rehfeld, 2020; Wang et al., 2020; Birch et al., 2021; Rahban et al., 2021; Xiang et al., 2022; Torrezan-Nitao et al., 2023; He et al., 2024). These molecules can act as full or partial CatSper agonists, inhibitors, or exhibit dual agonistic and inhibitory actions. This diversity underscores the complexity of the CatSper pharmacology, which likely involves multiple activator- and inhibitor-binding sites that might be allosterically coupled. Drugs can simultaneously bind to more than one of these sites, resulting in complex pharmacological effects on CatSper and [Ca2+]i (Rennhack et al., 2018; Wang et al., 2020; Schierling et al., 2023). Such complexity appears to apply to THC and CBD. In particular, CBD induces a more pronounced increase in [Ca2+]i compared to most previously described dual-action CatSper inhibitors, suggesting that its pharmacological action on CatSper is atypical. Another example of such an atypical dual-action CatSper modulator is SKF96365 (Torrezan-Nitao et al., 2023). This compound is a potent CatSper agonist but transitions into a CatSper inhibitor when applied over an extended time course. Further studies are required to determine whether CBD and SKF96365 share a similar molecular mechanism in modulating the CatSper channel.
Marijuana is composed of over 120 cannabinoids, of which THC and CBD are the most abundant (Peng and Shahidi, 2021). Cannabinoids activate specific endocannabinoid receptors throughout the body, including in the reproductive tract. More specifically, THC acts mainly on the cannabinoid receptors type 1 and 2 (CB1 and CB2), which have been reported to be specifically expressed in human sperm (Pertwee, 2006; Agirregoitia et al., 2010; Nielsen et al., 2019). Recent studies have investigated the effects of cannabis and phytocannabinoids on reproductive health and particularly on sperm function, with conflicting results. Some reports suggest that cannabis consumption impairs sperm motility (Hembree et al., 1979; Hong et al., 1982; Whan et al., 2006; Carroll et al., 2020), sperm morphology (Pacey, 2012; Carroll et al., 2020; Hehemann et al., 2021; Belladelli et al., 2023), and AR (Whan et al., 2006). Cannabis consumers were found to have lower total sperm count (Kolodny et al., 1974; Hembree et al., 1979; Gundersen et al., 2015) and serum testosterone levels (Kolodny et al., 1974). However, other studies have reported no changes in semen parameters or serum testosterone levels among cannabis consumers (Cushman, 1975; Thistle et al., 2017; Nassan et al., 2019; Zufferey et al., 2024). A recent systematic review and meta-analysis reported no clinically meaningful associations between cannabis use and testicular function, including semen parameters, serum testosterone, and gonadotropin concentrations (Belladelli et al., 2021). In women, cannabis use has been associated with changes in reproductive hormone levels and profiles (Mendelson and Mello, 1984; Mendelson et al., 1986), menstrual cycle disturbance (Jukic et al., 2007), and reduced oocyte retrieval and fertilization among couples undergoing in vitro fertilization. Here we show that the main psychoactive component of cannabis, THC, acts on CatSper and alters key sperm functions, which could disturb the fertilization process in vivo and reduce fertility in males, females, or both. Further studies are needed to fully understand the mechanism of action and the extent to which THC may affect CatSper function and sperm fertilizing capacities. In a research setting, THC can be used as a CatSper inhibitor. Indeed, compared to previously described CatSper inhibitors (Rennhack et al., 2018; Carlson et al., 2022; Schierling et al., 2023), THC reduced P4- and the PGE1-induced CatSper activation with the highest potency. We argue that THC can also be used as a starting point for the development of non-hormonal contraception targeting CatSper.
Supplementary Material
Acknowledgements
We thank Yves Cambet and Vincent Jaquet (R.E.A.D.S platform, University of Geneva) and Gregory Schneiter, Cécile Gameiro, and Jean-Pierre Aubry (F.A.C.S platform, University of Geneva) for technical assistance.
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
Funding
This work was supported by the Swiss Centre for Applied Human Toxicology (SCAHT) and the Département de l’Instruction Publique of the State of Geneva. T.S. and C.B. are supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation): project number 329621271 (CRU326).
Conflict of interest
The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the research reported.