The reduction in spermatozoa 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 spermatozoa functions like motility, velocities, capacitation and acrosome reaction in the frog, rat (6,17), boar (27), and human (19,28–31). 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 spermatozoa capacitation process in the female reproductive tract involves membrane and metabolic modifications such as the generation of reactive oxygen species (ROS), increase in intracellular ions and protein tyrosine phosphorylation, and changes in motility and plasma membrane fluidity (32,33). Events like spermatozoa binding to the zona pellucida (ZP), acrosome reaction, and oocyte fertilisation all depend on successful capacitation process (34), prevention of which will lead to fertilisation failure (35). During this process, spermatozoa acquire a motility pattern called hyper-activated motility (HAM) (36) that is characterised by asymmetrical flagellar beating (37), which is needed for fast swimming and generation of enough force necessary to penetrate cumulus cells and ZP during fertilisation (38). Because the HAM patterns of mammalian spermatozoa capacitated in-vivo and in-vitro have been reported to be similar (39,40), it has been very easy for researchers to study different aspects of spermatozoa physiology by in-vitro capacitation. Our data in this study showed that THC reduced the progressive motility of spermatozoa 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 (41,42) and p-dimethylaminoazobenzene in mice (43). In a randomised controlled clinical trial that included cases after varicocele surgery, vitamin C supplementation significantly increased motility and morphology but not count in infertile young adult males with low-quality spermatozoa, suggesting that it positively affects qualitative but not quantitative characteristics of semen analysis in this condition (44). Apart from the reported improvement in spermatozoa quality in smokers by increasing vitamin C dose from 200 to 1000 mg (45), another observational study on healthy non-smoking Americans showed that vitamin C-containing diet or supplement increased spermatozoa count and motility (46). When combined with vitamin E, vitamin C has also been reported to enhance rabbit male fertility (47) by increasing concentration and total motile but decreasing abnormal and dead spermatozoa (47,48). Combination of vitamins C and E reportedly ameliorate the oxidative stress and spermatotoxicity induced by endosulfan in rats (49).
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 spermatozoa quality in infertility clinics (50,51). An Egyptian study has linked smoking to decrease in seminal plasma vitamin C, which was shown to consequently reduce semen parameters and fertilisation potential (52). Our dose-response and time-course of modulation study showed that vitamin C increased the percentage motility of spermatozoa 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 spermatozoa motility throughout the observation period when compared to the baseline. Furthermore, incubation of spermatozoa in 5 mM of vitamin C solution also increased their motility by 22% when compared to control. The vitamin C-induced increase in spermatozoa motility was associated with an increase in VAP, VCL, VSL, ALH and BCF but decrease in straightness and linearity. These data provide pieces of evidence that vitamin C increases motility by enhancing the kinematics of spermatozoa. These data are consistent with the previous report of Babaie and Sivandi (24) that also reported an improvement in the motility and kinematics of caprine spermatozoa treated with vitamin C in-vitro. Similar to our study, Babaie and Sivandi (24) obtained epididymal spermatozoa from adult goat testes and incubated them in the capacitation medium (Ca2+-free Tyrode’s medium, spermatozoa TALP) supplemented with 50 µg/mL vitamin C, after which the motion parameters were assessed using the CASA at various intervals. They also observed that vitamin C increased total and progressive spermatozoa motility, VCL, VSL, linearity, ALH and BCF.
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 spermatozoa motility and kinematics of rat. 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 spermatozoa 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 progressive motility of spermatozoa 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 pieces of evidence that vitamin C ameliorates the THC-induced spermatotoxicity, especially as regards motility and kinematics of spermatozoa. 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 the endocannabinoid system in mammalian spermatozoa has been well-reported (31,53,54). We have reported that THC-induced reduction in the HAM of spermatozoa 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 spermatozoa motility has been established, the current study further investigated if the lack of complete abolishment of some kinematics by vitamin C is associated with CbRs activation by THC in spermatozoa incubated with a 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 spermatotoxicity 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 motility related to the kinematics of spermatozoa? Across a wide range of animals like birds (55), fishes (56) and mammals (57), the competition of spermatozoa to fertilise the ovum is favoured by traits (e.g. kinematics) that promote the fertilising ability of male. To penetrate different barriers along its track, the spermatozoon requires a high flagellar BCF (58). The VCL and ALH signify the hyper-activated motility (HAM) of spermatozoa and the high-energy state required for successful fertilisation (59) while motility and velocities of spermatozoa reflect their mitochondrial function and energy status that have been correlated with their fertility (60). Moreover, the progressive motility of spermatozoa has been positively correlated with the VAP, VCL, VSL, ALH, BCF, straightness and linearity but negatively correlated with wobble in bulls (57,61,62). In our recent study, velocities (VAP, VCL and VSL), ALH and BCF showed strong predictive power on HAM, suggesting that these kinematics determine the response of spermatozoa capacitation-induced HAM to cannabinoids and melatonin (17). In the present study, there was a very low probability that the progressively motile and non-motile spermatozoa differ in their velocity ratios (wobble, straightness and linearity), while there was a very high probability that they differ in their velocities (VAP, VCL, and VSL), ALH and BCF. These suggest that velocities, ALH and BCF determine the modulation of spermatozoa capacitation-induced HAM by cannabinoids and vitamin C.
Even though hydrogen peroxide is the major ROS produced in spermatozoa, a previous study has shown that 87% of infertile patients have superoxide anion (63). Spermatozoa are sensitive to assault from free radicals due to the existence of high membrane polyunsaturated fatty acids and lack of capacity for DNA repair (64). Increased ROS level has also been shown to correlate with decreased motility (65) and many structural and functional abnormalities of spermatozoa. Meanwhile, non-enzymatic antioxidants like vitamin C, pyruvate, vitamin E, taurine, vitamin A, hypotaurine, albumin, ubiquitol, urate, and enzymatic antioxidants like catalase, superoxide dismutase, and glutathione peroxidase/reductase have been reported to be contained in the seminal fluid (66), even though vitamin C is considered as the major antioxidant in the testis (67). Furthermore, the ROS-induced damage in spermatozoa has been shown to reduce with supplementation of the latter’s clinical media with antioxidant (64). Despite the physiological significance of vitamin C in the body, especially on spermatozoa, humans (unlike most animals) are unable to synthesise vitamin C, making its inclusion in diet or supplement a necessity (68). Vitamin C transporters have been reported to be expressed in the Sertoli cells, spermatocytes, spermatids, spermatozoa and the testis (69,70). However, our present study is limited due to our inability to establish the relationship between vitamin C transporters and cannabinoid receptors as regards HAM of capacitated spermatozoa, which is worthy of consideration in a future study. Another limitation of our study is our inability to estimate the seminal ROS and antioxidant levels in all the groups, which would have enabled us to understand the relationship between spermatozoa kinematics and redox system and their modulation by vitamin C and/or cannabinoid system. Notwithstanding the limitations, our study is the first to provide information on the in-vitro effect of vitamin C on hyperactivated motility and kinematics of capacitated spermatozoa treated with cannabinoids.