The reduction in sperm motility by THC was initially reported in sea urchin by Schuel’s group [14–16]. Series of studies later showed that cannabinoid receptors activation by anandamide (an endocannabinoid agonist) or THC inhibits sperm functions like motility, velocities, capacitation and acrosome reaction in frog, rat [6, 17], boar [26], and human [19, 27–30]. Furthermore, the spermatotoxic potential and the consequent anti-fertility effect of marijuana [7, 9, 10] and its primary psychoactive cannabinoid (THC) [17] are well-known.
Ejaculated sperm capacitation process in the female reproductive tract involves membrane and metabolic modifications such as generation of reactive oxygen species (ROS), increase in intracellular ions and protein tyrosine phosphoryation, and changes in motility and plasma membrane fluidity [31, 32]. Events like sperm binding to the zona pellucida (ZP), acrosome reaction, and oocyte fertilisation all depend on successful capacitation process [33], prevention of which will lead to fertilisation failure [34]. During this process, sperms acquire a motility pattern called hyper-activated motility (HAM) [35] that is characterised by asymmetrical flagellar beating [36], which is needed for fast swimming and generation of enough force necessary to penetrate cumulus cells and ZP during fertilisation [37]. Because the HAM patterns of mammalian sperms capacitated in-vivo and in-vitro have been reported to be similar [38, 39], it has been very easy for researchers to study different aspects of sperm physiology by in-vitro capacitation. Our data in this study showed that THC reduced the progressive sperm motility of sperm incubated in capacitation medium, which corroborates its spermatotoxic effect.
Vitamin C has been previously reported to improve rat’s semen parameters in-vivo [9] and ameliorate the spermatotoxicity induced by cisplatin in rats [40, 41] and p-dimethylaminoazobenzene in mice [42]. In a randomised controlled clinical trial that included cases after varicocele surgery, vitamin C supplementation significantly increased sperm motility and morphology but not count in infertile young adult males with low quality sperm, suggesting that it positively affects qualitative but not quantitative characteristics of semen analysis in this condition [43]. Apart from the reported improvement in sperm quality in smokers by increasing vitamin C dose from 200 to 1000 mg [44], another observational study on healthy non-smoking Americans showed that vitamin C-containing diet or supplement increased sperm count and motility [45]. When combined with vitamin E, vitamin C has also been reported to enhance rabbit male fertility [46] by increasing sperm concentration and total motile sperm, and decreasing abnormal and dead sperm [46, 47]. Combination of vitamin C and E has also been shown to ameliorate the oxidative stress and sperm toxicity induced by endosulfan in rats [48].
Does vitamin C have spermatoprotective effect in-vitro? Previous reports have shown that vitamin C is the most important seminal anti-oxidant, accounting for about 65% of the seminal anti-oxidant capacity, and is currently being used in-vitro to enhance sperm quality in infertility clinics [49, 50]. An Egyptian study has linked smoking to decrease in seminal plasma vitamin C, which was shown to consequently reduce semen parameters and fertilisation potential [51]. Our dose-response and time-course of modulation study showed that vitamin C increased the percentage motility of sperms in a dose-dependent but not time-dependent manner. For instance, 100 µM, 1 mM, 5 mM, and 10 mM, but not 10 µM, significantly increased the sperm motility throughout the observation period when compared to the baseline. Furthermore, incubation of sperms in 5 mM of vitamin C solution also increased the sperm motility by 22% when compared to control. The vitamin C-induced increase in sperm motility was associated with increase in VAP, VCL, VSL, ALH and BCF but decrease in straightness and linearity. These data provide evidences that vitamin C increases sperm motility by enhancement of sperm kinematics.
Can vitamin C ameliorate the THC-induced spermatotoxicity? In our previous in-vivo studies, we reported that the Cannabis sativa-induced spermatotoxicities (and the associated endocrine disruption and oxidative stress) were exacerbated by either vitamin C or melatonin when administered separately, but ameliorated when vitamin C was co-administered with melatonin [7, 9, 10]. However, our more recent in-vitro study showed that melatonin attenuated THC-induced reduction in rat sperm motility and kinematics. Based on our observation that the modulatory effects of melatonin on spermatotoxicity induced by Cannabis sativa or THC are different in-vivo and in-vitro, we were interested to know the effect of vitamin C on THC-induced spermatotoxicity in-vitro. In the present in-vitro study where sperms were incubated with THC and vitamin C in the capacitation medium, we observed for the first time that vitamin C completely abolished the THC-induced reduction in sperm progressive motility by maintaining it at 94% of the control (which is not significantly different from the control level). We also observed that vitamin C attenuated the THC-induced reduction of VAP, VCL, ALH, and BCF but abolished the THC-induced reduction in VSL. These data provide evidences that vitamin C ameliorates the THC-induced spermatotoxicity, especially as regards sperm motility and kinematics. Taken together with our previous in-vivo study, it also suggests that vitamin C ameliorates cannabinoid-induced spermatotoxicity in-vitro but exacerbates it in-vivo. It is thus similar to our recent study where we reported that melatonin ameliorates cannabinoid-induced spermatotoxicity in-vitro but exacerbates it in-vivo [17].
Is there a cross-talk between vitamin C and cannabinoid signalling? The existence of cannabinoid receptors (CbRs) and endocannabinoid system in mammalian sperms has been well-reported [30, 52, 53]. We have reported that THC-induced reduction in the hyperactivated motility of sperm was abolished when both CbRs 1 and 2 were blocked, but was only attenuated when either CbR 1 or CbR 2 was selectively blocked. We also showed that CbR 1 contributed more than CbR 2 in THC-induced spermatotoxicity [17]. Since the involvement of both CbRs in the regulation of sperm motility has been established, the current study further investigated if the lack of complete abolishment of some sperm kinematics by vitamin C are associated with CbRs activation by THC in sperms incubated with combination of THC and vitamin C. We blocked both CbRs (SR141716 as CB− 1 and AM-630 as CB− 2), and then added THC and vitamin C to the capacitation medium. We observed that prior blockade of CbRs cancelled the spermatotoxic effect of THC as vitamin C abolished the effect of THC on VSL, ALH and BCF, and even raised the progressive motility, VAP and VCL above the control value. This shows that vitamin C caused more inhibition of THC-induced spermatotixicity when CbRs were blocked, and is a pointer to the fact that there is a cross-talk between vitamin C and cannabinoid signalling.
Is the ameliorative effect of vitamin C on sperm motility related to sperm kinematics? Sperm competition to fertilise ovum favours traits that enhance male fertilising ability across a wide range of animals like birds [54], fishes [55] and mammals [56]. The sperm requires high flagellar BCF to penetrate different barriers along its tract [57]. Hyper-activated motility (HAM) of sperm and the hih-energy state required for successful fertilisation are signified by high VCL and ALH [58]. Moreover, sperm motility and velocity parameters reflect their mitochondrial function and energy status while specific motion parameters have been correlated with fertility of sperm [59]. The VAP, VCL, VSL, ALH, BCF, straightness and linearity had positive correlation with progressive motility while wobble negatively correlates with it in bulls [56, 60, 61]. In our recent study, sperm velocities (VAP, VCL and VSL), ALH and BCF showed strong power to predict HAM, suggesting that these kinematics determine the response of sperm capacitation-induced HAM to cannabinoids and melatonin [17]. In the present study, the probabilities that there were differences in the velocity ratios between progressively motile and non-motile sperms were very low, while there were very high probabilities for differences in the velocities (VAP, VCL, and VSL), ALH and BCF of progressively motile and non-motile sperms. This similarly suggests that velocities, ALH and BCF determine the modulation of sperm capacitation-induced HAM by cannabinoids and vitamin C.