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, 165 (8), 2575-83

Δ9-Tetrahydrocannabinol (Δ9-THC) Attenuates Mouse Sperm Motility and Male Fecundity

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Δ9-Tetrahydrocannabinol (Δ9-THC) Attenuates Mouse Sperm Motility and Male Fecundity

Daniel J Morgan et al. Br J Pharmacol.

Abstract

Background and purpose: Numerous studies have shown that N-arachidonoylethanolamine (AEA) can inhibit sperm motility and function but the ability of cannabinoids to inhibit sperm motility is not well understood. We investigated the effects of WIN 55,212-2, a CB(1) cannabinoid receptor agonist, and Δ(9) -tetrahydracannabinol (Δ(9) -THC) on the ATP levels and motility of murine sperm in vitro. In addition, the effects of acute administration of Δ(9) -THC on male fecundity were determined.

Experimental approach: Effects of Δ(9) -THC on basal sperm kinematics were determined using computer-assisted sperm analysis (CASA). Stop-motion imaging was performed to measure sperm beat frequency. The effect of Δ(9) -THC on sperm ATP was determined using a luciferase assay. Male fertility was determined by evaluating the size of litters sired by Δ(9) -THC-treated males. KEY RESULTS Pretreatment of sperm for 15 min with 1 µM Δ(9) -THC reduced their basal motility and attenuated the ability of bicarbonate to stimulate flagellar beat frequency. Treatment with 5 µM WIN 55,212-2 or 10 µM Δ(9) -THC for 30 min reduced sperm ATP levels. In sperm lacking CB(1) receptors this inhibitory effect of WIN 55,212-2 on ATP was attenuated whereas that of Δ(9) -THC persisted. Administration of 50 mg·kg(-1) Δ(9) -THC to male mice just before mating caused a 20% decrease in embryonic litter size.

Conclusions and implications: Δ(9) -THC inhibits both basal and bicarbonate-stimulated sperm motility in vitro and reduces male fertility in vivo. High concentrations of WIN 55,212-2 or Δ(9) -THC inhibit ATP production in sperm; this effect of WIN 55,212-2 is CB(1) receptor-dependent whereas that of Δ(9) -THC is not. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Figures

Figure 1
Figure 1
Basal and bicarbonate-stimulated motility is inhibited by Δ9-THC. (A) Treatment with 1 µM Δ9-THC or 10 µM Δ9-THC for 15 min progressively reduced the percentage of motile sperm. (n= 111–138 cells). At least eight cells were examined from each animal in two to three independent experiments. #P < 0.05 (untreated vs. 1 µM THC), *P < 0.05 (untreated vs. 10 µM THC). (B) Averaged flagellar beat frequency was determined for wild-type sperm that were bathed in HS medium containing 1 µM Δ9-THC (THC) for 15 min. (n= 22–47 cells). At least eight cells were examined from each animal in two to three independent experiments. #P < 0.05 (untreated vs. 1 µM THC). (C) Sperm were bathed in HS medium alone or HS medium containing 1 µM or 10 µM Δ9-THC (THC) for 15 min and subsequently perfused with HS medium containing 15 mM HCO3- (BC) for 1 min. Bicarbonate-stimulated beat frequency was reduced in sperm treated with 1 µM Δ9-THC relative to sperm treated with HS medium containing bicarbonate. #P < 0.05 (BC vs. BC+1 µM THC). Treatment of sperm with 10 µM Δ9-THC completely blocked the stimulating effect of bicarbonate on beat frequency. *P < 0.001 (BC vs. BC +10 µM THC). The number of sperm used for each condition is designated in parentheses. Unpaired t-tests were used to calculate P values. Error bars represent SEM.
Figure 2
Figure 2
Sperm kinematics are rapidly inhibited by 10 µM Δ9-THC. Dose–response curves over a range of Δ9-THC concentrations were constructed for CASA kinematic analysis (A, C, E). Treatment of sperm with 10 µM Δ9-THC for 30 min significantly inhibited VSL (A) and average path velocity (C), while VCL (E) was inhibited by both 1 and 10 µM Δ9-THC. (n= 2113–4498 cells for A, C, and E.) *, P < 0.05 (untreated vs. 10 µM THC and 1 µM THC). Onset of the Δ9-THC effect was rapid, with treatment of wild-type sperm with 10 µM Δ9-THC decreasing the VSL (B), VAP (D) and VCL (F) within 15 min. (n= 791–1603 cells for HS in B, D, and F). (n= 1240, 886, 252, 113 and 620 cells at 0, 15, 30, 60 and 90 min for HS +THC in B, D and F) *P < 0.05 (HS vs. THC). Unpaired t-tests were used to calculate P-values. Error bars represent SEM.
Figure 3
Figure 3
Δ9-THC and WIN 55,212-2 reduce sperm ATP levels. Treatment of sperm for 60 min with 10, 30 and 100 µM Δ9-THC reduced ATP levels (A). 10 µM Δ9-THC reduced ATP levels in sperm from wild-type mice relative to untreated controls (HS) in a time-dependent fashion (B). Significantly, the effect of Δ9-THC on ATP levels was present in sperm lacking CB1 receptors, suggesting that the inhibitory effect of Δ9-THC on ATP levels was not CB1 mediated (C). Treatment with 5 µM WIN 55,212-2 (WIN2) causes a 35% reduction in ATP levels in wild-type sperm. WIN 55,212-3 (WIN3), which does not bind with high affinity to CB1 receptors, had no effect on sperm ATP levels (D). The effect of WIN 55,212-2 on sperm ATP was absent in sperm lacking the CB1 cannabinoid receptor (E). Student's unpaired t-test was used to calculate significance (*P < 0.05). Error bars represent SEM.
Figure 4
Figure 4
Acute administration of Δ9-THC reduces male fertility. The acute effect of Δ9-THC on male fertility was determined by measuring embryonic (e12.5) litter sizes sired by vehicle treated CD1 wild-type (WT) males (n= 7 males with 20 litters) or CD1 wild-type males treated with 50 mg·kg−1Δ9-THC (n= 9 males with 17 litters). The effect of vehicle (n= 5 males with 6 litters) and 50 mg·kg−1Δ9-THC (n= 5 males with 6 litters) on litter size was also examined in mice lacking CB1 receptors (KO). Error bars indicate SEM and P-values were calculated using two-way repeated measures anova with Bonferroni's post test (*P < 0.01).

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