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Division of Anatomy & Cell Biology,3 Department of Pathology & Anatomical Sciences
Department of Gynecology & Obstetrics,4 School of Medicine & Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCONCLUSIONSDEDICATIONREFERENCES |
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9tetrahydrocannabinol [(–)9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (–)9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (–)9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.
acrosome reaction, anandamide, cannabinoid receptors, capacitation, fertilization, hyperactivated motility, neurotransmitters, signal transduction, sperm, synaptic vesicles
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCONCLUSIONSDEDICATIONREFERENCES |
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Invertebrate and mammalian sperm express receptors for many neurotransmitters and neuromodulators, e.g., acetylcholine (nicotinic and muscarinic types) [3–4, 9], adenosine and ATP [4], serotonin and catecholamines [4, 10, 11], prostaglandins and leukotrienes [12, 13], progesterone and estrogen [4, 14], amino acids [4], peptides [2, 4–6, 15, 16], and odorants [4]. Sperm also express receptors for psychoactive drugs, e.g., nicotine [3, 4, 9], cocaine [4], opioids [17], and cannabinoids [4, 18–24]. Receptors for these neuroactive agents modulate normal sperm functions essential for fertilization, including respiration, motility, chemotaxis, capacitation, and AR.
Endocannabinoids (ECBs) are fatty-acid derivates (arachidonoylethanolamide [AEA], certain other N-acylethanolamides [NAEs], 2-arachidonoyl glycerol [2-AG], noladin ether, virodhamine, etc) that are endogenous ligands for CNRs [21, 22, 25]. AEA was the first ECB discovered [26]. This novel ecosanoid mimics many of the pharmacological properties of (–)9tetrahydrocannabinol [(–)9THC], the primary psychoactive substance in Cannabis [25]. At higher concentrations, AEA is an agonist for type-1 vanilloid receptors (now called transient receptor potential channel vanilloid receptor subunit 1 [TRPV1]), can directly inhibit voltage-operated type-L Ca2+ and K+ (types Shaker-related and TASK-1) channels, and can directly activate glutamate ionotropic receptors [27]. Both AM-356 and 2-AG directly block acetylcholine-mediated responses in Xenopus oocytes transfected with cholinergic receptor nicotinic alpha polypeptide 7 receptor (IC50 168 and 178 nM, respectively), while (–)9THC and synthetic cannabinoid agonists (CP55,940 and WIN 55,212–2) do not affect function of this nicotinic receptor [28]. AEA and its congeners are released from membrane phospholipid precursors, N-acyl-phosphatidylthanolamines (NAPEs), by action of N-acyl-phosphatidylthanolamine-hydrolyzing phospholipase D (NAPE-PLD) when neurons and other cells are stimulated by depolarizing agents, neurotransmitters, and hormones [25, 29, 30]. High levels of NAPE-PLD are found in brain and testis [30]. Released AEA is quickly eliminated by membrane-bound fatty acid amide hydrolase (FAAH), indicative of roles in cell signaling [25, 31]. Significantly, boar sperm contain both NAPE-PLD and FAAH to produce and degrade their own AEA [24].
CNRs belong to the superfamily of G-protein-coupled receptors [32]. Two subtypes have been cloned and characterized. Cannabinoid receptor 1 (CNR1; previously known as CB1) was originally cloned from rat and human brain [33, 34]. It is widely distributed in neural and nonneural cells in reproductive and other peripheral organs [21, 22, 35]. Functional CNR1 receptors are expressed in human and dog testis [34, 36], mouse Leydig cells [37], human and porcine sperm [23, 24], normal and malignant human prostatic epithelium [38], mouse oviductal muscularis [39], human uterus [36] and myometrium at term [40], mouse uterine epithelial and stromal cells and preimplantation embryos [41], human fetus and placenta [21, 22, 42]. Cannabinoid receptor 2 (CNR2; previously known as CB2) was originally cloned from human promyelocytic leukemia HL 60 cells [43] and has important roles in modulating immune responses [44]. Functional CNR2 receptors are expressed in human uterus and myometrium [36, 40], rat testis [45], mouse Sertoli cells [46], normal and malignant human prostatic epithelium [38], porcine sperm [24], preimplanation mouse embryos [41], and human placental macrophages [42]. These observations indicate multiple roles for ECB signaling in reproductive physiology, which may be potential targets for exogenous cannabinoids derived from marijuana smoke [18–24, 31, 39–42, 47, 48]. Additional CNR subtypes may exist [27, 35], e.g., ionotropic CNRs in sensory neurons that are activated by (–)9THC, cannabinol, mustard oil, and capsaicin, but not by AEA or 2-AG [49].
CNR signaling by differential activation of G-protein subtypes 1) modulates multiple signal transduction pathways (activates inwardly rectifying K+channels, inhibits opening of voltage-gated Ca2+ [types N and P/Q] and K+ [type A] channels, inhibits activation of protein kinases, and can both inhibit and stimulate cAMP accumulation, etc) [32, 44]; 2) inhibits exocytotic release of neurotransmitters by central and peripheral neurons [25, 35, 39, 50, 51], hormones by the pituitary gland [52], inflammatory mediators by mast cells [53], and cytokines by immune cells [44]; 3) inhibits fertilization in sea urchins by blocking AR in sperm [18, 54–57]; and 4) modulates capacitation and fertilizing potential of human and boar sperm in vitro [20–24].
Retrograde Endocannabinoid Signaling in Brain
A growing body of evidence indicates that AEA signaling modulates release of chemical messengers in central and peripheral neurons [25, 35, 50, 51]. This hypothesis is based on observations that 1) CNR1 receptors are localized to presynaptic axon terminals; 2) CNR1 agonists inhibit opening of Ca2+ channels, cAMP production, and exocytotic release of neurotransmitters from synaptic vesicles; 3) CNR1 antagonists block inhibitory effects of cannabinoids on neurotrasmitter release by neurons; 4) release of AEA and other ECBs by postsynaptic neurons is Ca2+ dependent; 5) FAAH is localized in neurons postsynaptic to neurons that express CNR1; and 6) increased AEA concentrations are observed in brain after administration of FAAH inhibitors. These findings suggest that retrograde AEA signals released by postsynaptic neurons inhibit release of additional neurotransmitters at synapses [50]. According to this model (Fig. 1), 1) neurotransmitter secreted by presynaptic neurons binds to its receptor on the surface of postsynaptic neurons; 2) in response to this stimulus, free intracellular Ca2+ increases within postsynaptic neurons, which activates a membrane-bound transacylation-phosphodiesterase pathway to release AEA from membrane phospholipids into the synaptic cleft; 3) released AEA then binds to CNR1 on presynaptic neurons, leading a to Gi-protein-mediated signal cascade that reduces free intracellular Ca2+ and cAMP production and elevates intracellular K+ to inhibit secretion of additional neurotransmitter by presynaptic neurons; 4) AEA then dissociates from CNR1, is taken up by postsynaptic neurons and hydrolyzed by membrane-bound FAAH to terminate AEA-signaling. Analogous processes have been implicated in preventing polyspermy during fertilization in sea urchins [18, 22, 57, 58].
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Evidence for CNRs in Sperm
[3H]CP,55940 is a potent bicyclic synthetic cannabinoid agonist used to demonstrate CNRs in mammalian brain and other somatic tissues [34, 41, 59–61]. Specific binding of [3H]CP55,940 to sea urchin (Stronglyocentrotus purpuratus) and human sperm occurs at low ligand concentrations (0.5–50 nM) and levels off when all receptors are occupied [18, 20]. These data satisfy pharmacologic requisites for affinity and saturability of CNRs [59]. Binding properties of [3H]CP55,940 to CNRs in sea urchin, human, and porcine sperm are remarkably similar to those obtained with mammalian brain and other somatic tissues (Table 1). [3H]CP55,940 binds with high affinity to human CNR1 and CNR2. Consistent with studies on rat brain and cloned human CNR1, (–)9THC is more potent than (+)9THC in displacing [3H]CP55,940 from specific binding sites on sea urchin sperm (P < 0.02) [18]. Similarly, Hill transformation of [3H]CP55,940-specific binding data obtained with sea urchin and human sperm and rat cerebral cortex membranes yields straight lines and Hill coefficients of ~1, which indicates that these cells contain a single class of CNRs and the absence of significant cooperative interactions [18, 20, 59]. Radioligand binding studies also indicate the presence of TRPV1 receptors in boar sperm [24].
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An orthologue of vertebrate CNR has been cloned in the urchordate Ciona intestinalis, commonly known as the sea squirt [62]. Sea squirt CNR shares 28% sequence identity with human CNR1 and 24% sequence identity with human CNR2, which suggests that the ancestor of vertebrate CNRs originated in a deuterostomian invertebrate. Furthermore, phylogenetic analysis indicates that the common ancestor of sea squirt CNR and vertebrate CNRs predates a gene duplication event that gave rise to mammalian CNR1 and CNR2 [63–65]. CNR mRNA is expressed in brain and peripheral organs in the sea squirt, including ovary and testis, which also contain AEA and 2-AG. Consistent with these findings, radioligand-binding studies detected high affinity binding sites in Ciona tissues for ligands that are selective for mammalian CNR1 and CNR2, as well as enzymes responsible for the formation and removal of these lipid-signal molecules [63]. As will be discussed below, properties of sea urchin sperm CNR are not identical to those of known mammalian CNRs [18, 55–57, 66].
Immunochemical studies using Western blots demonstrated the presence of CNR1 in human and boar sperm [23, 24]. A weak signal for CNR2 also was detected in porcine sperm [24]. Consistent with these observations, approximately ~85% of specific binding of [3H]CP55,940 to boar sperm was blocked by the CNR1 selective antagonist SR141716A, while ~15% was blocked by the CNR2 selective antagonist SR144528 [24]. TRPV1 and FAAH were also detected in boar sperm by Western blots. CNR1, TRPV1, and FAAH are colocalized to the head, middle piece, and tail of boar sperm by immunocytochemistry, which supports their roles in modulating acrosomal status and motility [20–24]. Boar sperm also contain NAPE-PLD and AEA [24]. Together, these findings indicate that mammalian sperm produce and degrade their own AEA to modulate their own functions via CNR and TPRV1 receptors. This situation appears to be analogous to the cholinergic autoregulatory system that modulates motility of sea urchin and mammalian sperm [4]. CNR1 mRNA was also detected in ejaculated human sperm by RT-PCR [23]. While a complex population of mRNAs is present in ejaculated human sperm, their fates and biological functions are poorly understood [67].
CNR-Signaling Modulates Sea Urchin Sperm Fertility
Psychoactive drugs, such as morphine, cocaine, nicotine, the volatile anesthetic halothane, and propranolol (a ß-adrenergic receptor blocker) promote polyspermic fertilization in pretreated sea urchin eggs that are inseminated with excess sperm [68–71]. Unexpectedly, we observed that (–)9THC-treated eggs showed a lower incidence of polyspermy than controls under these conditions, suggesting that the drug inhibits fertilization [54]. This possibility was examined using a minimal sperm density just sufficient to fertilize ~90% of eggs cultured in sea water. Eggs pretreated with (–)9THC do not show a reduction in receptivity to untreated sperm. However, pretreatment of sperm with (–)9THC results in a concentration-dependent reduction in fertilizing potential (Table 2). Sperm fertilizing capacity also is reduced by 1) AEA and ethanolamides of related unsaturated fatty acids (oleic, palmitic), but not by ethanolamides of saturated fatty acids (mysteric, stearic, and arachidic) [57, 66]; and 2) by synthetic cannabinoid ligands WIN 55,212–2 and SR141716A (Table 2). WIN55,212–2 is a potent agonist for mammalian CNR1 and CNR2, while SR141716A is a potent CNR1-selective inverse agonist/antagonist [72]. However, SR141716A does not block inhibitory effects of (–)9THC on sperm fertility [66]. It actually potentiates inhibitory effects elicited by (–)9THC. These findings suggest that (–)9THC and SR141716A act at the same molecular target in sea urchin sperm, possibly a receptor with some CNR1-like properties. Intriguingly, SR141716A mimics inhibitory effects of AEA and 2-AG on opening voltage-gated Ca2+ channels in differentiated NG108–15 cells [73]. Additional work is required to determine whether SR141716A is a "...protean..." agonist that elicits inverse responses in some systems and positive responses in others [74] or also produces biological effects via non-CNR1-mediated processes [32]. Two major nonpsychoactive cannabinoids in marijuana, cannabinol [CBN] and cannabidiol [CBD] [75], mimic inhibitory effects of (–)9THC on sea urchin sperm fertility and AR [54, 56]. CBD and CBN exhibit low affinities for rat brain CNR1 (Ki 5300 and 3200 nM, respectively) [60], while CBN has a greater affinity for human CNR2 than (–)9THC (Ki 250 and 320 nM, respectively) [43]. Differences in the properties of sea urchin and mammalian CNRs may reflect molecular divergence from a common deuterostomian invertebrate ancestor [62–65]. Sperm cultured in AEA and (–)9THC swim more vigorously than control sperm for 10–90 min [19, 54, 57]. Because sea urchin eggs are fertilized within seconds after insemination [66], these data implicated AR as a potential target for CNR signaling.
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Cannabinoids inhibit the acrosome reaction
AEA, (–)9THC, CP55,940, and WIN55,212–2 inhibit AR stimulated by solubilized egg jelly [EJ] (Table 2). Spontaneous ARs are also prevented by (–)9THC [55]. These responses mimic those observed in neurons where CNR agonists inhibit evoked and spontaneous secretion of neurotransmitters [35]. Sperm regain their ability to undergo EJ-stimulated AR and to fertilize eggs upon removal of (–)9THC and AEA [55–57]. The rank order of potency to inhibit specific binding of [3H]CP,55940 to live sea urchin sperm and to inhibit EJ-stimulated AR is CP55,940 > (–)9THC > (+)9THC (Tables 1 and 2). Observed differences in potency of (–) and (+)9THC in displacing [3H]CP55,940 from its specific binding sites on sperm, and in blocking egg jelly-stimulated ARs are statistically significant (P < 0.02 and P < 0.025, respectively) [18]. EJ-stimulated AR in sea urchin sperm is associated with elevated intracellular levels of cAMP [5, 6], similar to those associated with evoked release of neurotransmitters at synapses [25, 35, 50, 51]. The inhibitory effects of cannabinergic agonists on sea urchin sperm flunctions are comparable with those observed on inhibition of forskolin-induced cAMP accumulation in cultured rat cerebellar neurons and in cells transfected with cloned human CNR1 and CNR2 (Table 2). This provides additional support for the presence of functional CNRs in sea urchin sperm because there is a large body of evidence that activated CNR1 and CNR2 block forskolin-induced cAMP accumulation in cells that naturally express these receptors and in cells that are transfected with cloned CNR1/and CNR2 receptors [61, 76, 77].
CNR signaling modulates ion channels in neurons and other somatic cells to inhibit exocytotic release of chemical messengers [25, 35, 39, 44, 50–53]. Stimulation of AR by egg jelly is associated with opening of ligand- and voltage-gated ion channels resulting in net influx of Ca2+ and Na+, and net efflux of H+ and K+, which changes membrane potential and elevates pHi [5, 6]. However, AEA and (–)9THC do not block AR in sea urchin sperm artificially induced by ionophores that transport specific cations (Ca2+, Na+, K+) across biological membranes and by NH4OH that increases pHi [55, 57]. Similarly, AEA does not block Ca2+ influx and AR in human sperm artificially induced by ionomycin [23]. These results suggest that CNR signaling affects stimulation-secretion-coupling event(s) in sea urchin and human sperm before opening of ion channels.
AR in sea urchin sperm is a complex process involving 1) acrosomal exocytosis that exposes matrix constituents such as bindin required for sperm egg-binding and 2) polymerization of monomeric actin in the subacrosomal fossa that extends the acrosomal tubule enclosed by its binded-coated plasma membrane, which is destined to fuse with the egg's plasma membrane [5, 6, 78]. Ultrastructural studies on sea urchin sperm showed that the membrane fusion step in acrosomal exocytosis is blocked by (–)9THC, CBD, and CBN [56]. Surprisingly, these cannabinoids also cause formation of lipid deposits in the subacrosomal fossa and centriolar fossa of treated sperm. The nuclear envelope is fragmented in close proximity to lipid deposits in the subacrosomal fossa, possibly resulting from localized lipase activity. These deposits disappear from treated sperm after cannabinoids are removed by washing, and sperm regain their capacity to undergo egg jelly-stimulated AR and to fertilize eggs. Interestingly, lipid bodies in activated eosinophils contain Ca2+-dependent phospholipase A2 to release arachidonic acid (AA) from arachidonyl-phospholipids and oxidative enzymes to convert AA into potent eicosanoids (prostaglandins and leukotrienes) [79]. Biochemical studies showed that (–)9THC and CBN activate Ca2+-dependent PLA2 activity in homogenates of S. purpuratus and Lytechinus pictus sperm [80]. Furthermore, (–)9THC is significantly more potent than (+)9THC (P < 0.01, ANOVA) [81]. Cannabinoid-induced activation of Ca2+-dependent phospholipase A2 in neurons and other somatic cells is mediated via CNRs, which leads to production of eicosanoids that elicit autocrine and paracrine effects [82, 83]. Are AA-oxidation products similarly involved in the effects of AEA and other cannabinergic ligands on functions of sea urchin sperm?
Stereoselectivity is a characteristic feature of receptor-mediated reactions [75]. Differences in the potency of (–)9THC and (+)9THC in displacing [3H]CP-55,940 from specific binding sites on sea urchin sperm, in blocking egg jelly-stimulated AR, and in activating Ca2+-dependent phospholipase A2 in sperm homogenates [18, 81] are comparable with studies on CNRs in mammalian brain [60, 61, 75]. These findings support the presence of functional CNRs in sea urchin sperm [18].
Retrograde AEA-signaling blocks polyspermy The prevention of polyspermy is a critical event in normal fertilization [68]. As soon as the first sperm activates the egg, other sperm in the vicinity represent a potential hazard to normal development. Secretion of the egg's cortical granules (cortical reaction) is one of the early responses of sea urchin eggs to stimulation by the fertilizing sperm. The cortical reaction begins at the site of binding of the fertilizing sperm to the egg's vitelline coat, spreads in a wave-like manner around the egg's surface, and promotes detachment of the vitelline coat to form the fertilization envelope (FE), which acts as a mechanical barrier to further sperm penetration. The cortical reaction is completed within ~60 sec following insemination in S. purpuratus. Because most sea urchin eggs are activated within ~1 sec after insemination [68, 84], they are potentially vulnerable to penetration by additional sperm until FE elevation is completed [85]. Other processes operate to limit sperm penetration during this period: 1) rapid Na+-dependent electrical depolarization of the egg's plasma membrane that prevents fusion with additional sperm [68, 84, 86, 87], 2) release of H2O2 by the egg that reacts with a sperm peroxidase to reduce sperm fertilizing capacity and inhibits EJ-stimulated AR [88–93], and 3) processes involving prostaglandins and leukotrienes [94, 95].
AEA signaling also may help prevent polyspermy in sea urchins [18, 57]. This hypothesis is based on effects of AEA and cannabinoids on sperm fertilizing capacity and AR described above, and is supported by work from Di Marzo's group [58] showing that 1) ovarian sea urchin eggs, but not sperm, contain AEA, palmitoyl-ethanolamide, and stearoyl-ethanolamide, as well as NAPEs, the phospholipid precursors of NAEs in mammalian tissues; 2) homogenates of ovarian eggs contain NAPE-PLD-like activity capable of releasing [3H]AEA from synthetic [3H]N-arachidonoyl-phosphatidyl-ethanolamine; and 3) homogenates of ovarian eggs, but not sperm, contain FAAH-like activity, which catalyzes hydrolysis of [3H]AEA and [3H]palmitoyl-ethanolamide. These results indicate that sea urchin eggs have the capacity to release and degrade AEA during fertilization to modulate sperm fertilizing capacity. According to our model (Fig. 2), binding of egg jelly-coat-ligand to its receptor on the sperm surface stimulates opening of Ca2+ channels, resulting in Ca2+ influx that promotes AR [5, 6]. Binding of sperm to its receptor on the egg's surface activates the egg and promotes release of Ca2+ from intracellular stores [96], resulting in activation of NAPE-PLD in the egg to release AEA from membrane lipids. Binding of released AEA to CBR on other nearby sperm inhibits opening of Ca2+ channels to block AR and prevents that sperm cell from refertilizing the egg before completion of the cortical reaction [57]. Reuptake and hydrolysis of AEA by eggs is likely to be mediated by FAAH within the egg, analogous to the processes that terminate AEA signaling in neurons [25, 61, 50–58]. Additional research is required to test the validity of this model to determine whether AEA is actually released by eggs during fertilization and to determine whether removal of released AEA promotes polyspermy, as shown previously with respect to egg-derived H2O2 [88, 89]. The proposed model closely resembles retrograde AEA signaling in brain, where AEA released from depolarized postsynaptic neurons inhibits neurotransmitter release at presynaptic terminals (see Fig. 1, above). Within this context, the acrosome in sea urchin sperm is functionally analogous to synaptic vesicles in presynaptic neurons, while the egg is functionally analogous to postsynaptic neurons.
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AEA-Signaling Modulates Human Sperm Functions
Unlike sea urchin sperm, which can fertilize eggs immediately after spawning into sea water [5, 6], ejaculated sperm from humans or other mammals, bathed in male secretions comprising the seminal plasma, are not yet capable of fertilizing an egg [97]. Mammalian sperm acquire the capacity to fertilize eggs following removal from seminal plasma and exposure for several hours to female reproductive tract fluids in vivo or by incubation in appropriate culture medium in vitro [4, 5, 97–99]. Capacitated sperm exhibit vigorous hyperactivated motility (HA) required for fertilization and can undergo physiological AR at the egg's surface.
Fertilization in vivo requires that the proper number of sperm arrive at the oviductal ampulla in the appropriate physiological state coincident with a viable unfertilized egg. Sperm are sequentially exposed to seminal plasma, oviductal fluid, follicular fluid, and secretions of granulosa cells in the cumulus matrix surrounding ovulated eggs as they travel from the vagina to the site of fertilization in the oviduct. AEA is synthesized in rat testis [100], mouse uterus and oviduct [31, 39, 41, 101], and human uterus [102]. FAAH is expressed in mouse and human uterine epithelial cells [31, 47, 102]. These observations suggested that sperm may be exposed to AEA as they transit male and female reproductive tracts. Biochemical studies showed that AEA is present in human seminal plasma [12.1 ± 2.1 nM], midcycle oviductal fluid [10.7 ± 2.5 nM], and follicular fluid [2.9 ± 0.9 nM], analyzed by HPLC and quantitated by isotope dilution [20, 21]. AEA also is present in boar seminal plasma [24]. Because plasma levels of AEA during the follicular and luteal phases of the menstrual cycle in women are 1.68 ± 0.16 nM and 0.87 ± 0.19 nM, respectively [48], it is unlikely that midcycle oviductal fluid simply is an ultrafiltrate of blood. Palmitylethanolamide (PEA) and oleylethanolamide (OEA) are enzymatically released together with AEA from membrane phospholipid precursors (NAPEs) when neurons are stimulated [25, 30]. PEA and OEA are also found in human seminal plasma, midcycle oviductal fluid, and follicular fluid [22], but their biological functions remain to be determined. Because progesterone and prostaglandin E (physiological ligands that stimulate AR) are synthesized by granulosa cells in ovarian follicles and in the cumulus matrix of ovulated eggs [4, 12], it is tempting to speculate that AEA and other NAEs in follicular fluid may likewise be synthesized by granulosa cells.
Capacitation and fertilizing potential The presence of CNRs and TRVP1 receptors in mammalian sperm and of AEA in reproductive-tract fluids suggest functional roles for AEA signaling in regulating sperm capacitation and fertilizing potential [20–24]. Significantly, levels of CNR1 binding, TRPV1 binding, NAPE-PLD activity, AEA reuptake activity, and FAAH activity in boar sperm decline during capacitation [24].
Hyperactivated motility
Vigorous hyperactivated motility [HA] enables sperm to arrive at the egg surface and assists penetration of the zona pellucida [97–99, 103]. R-methanandamide (AM-356) is a potent CNR1-selective agonist and metabolically stable substitute for AEA [72]. Effects of AM-356 on HA in swim-up sperm were quantitatively evaluated using a Hamilton-Thorne IVOS computerized semen analyzer to identify motile sperm that simultaneously exhibit curvilinear velocity > 100 µm/sec; head amplitude > 7.5 µm; linearity < 65 [20]. These characteristics define a sperm with high velocity and nonlinear swimming, which correlates with fertilizing potential [98, 99, 104]. AM-356 elicits biphasic effects on the incidence of HA in human sperm between 1 and 6 h: at [2.5 nM] it inhibits HA, while at [0.25 nM] it stimulates HA (P < 0.05; ANOVA) [20]. Biphasic responses are a characteristic feature of CNR and other G-protein-coupled receptors [105, 106]. The biphasic effects of AM-356 on HA in human sperm resemble those produced by AEA and (–)9THC on 1) Ca2+ channels in neuroblastoma cells [107, 108], 2) AA metabolism in neurons and other somatic cells [82, 83], 3) testosterone secretion in vivo and in vitro [109], 4) trophoblast outgrowth in culture [110, 111], and 5) phagocytosis in neutrophils and behavior in mice [106].
Studies on demembranated mammalian sperm suggest that a rise in free intracellular Ca2+ alters flagellar beat patterns characteristic of HA [112]. Observed effects of AM-356 on HA are consistent with previous findings by Burkman and colleagues on modulation of rabbit sperm HA in the oviduct that pointed to an unknown inhibitory substance in the isthmus, which was negated upon 10-fold dilution [113, 114]. Factors in the rabbit isthmic environment provide regulatory control: initially inducing sperm quiescence and, at the appropriate time, stimulating vigorous HA motility, thus enabling sperm to escape and swim to the ampulla. Bovine isthmic and ampullary fluids produce different effects on motility, AR, zona binding, and fertility of bull sperm [115–117].
Acrosomal status
Acquisition of acrosomal competence during capacitation represents an intermediate state preparing sperm to undergo AR in response to physiological stimuli at the egg's surface [97] and appears to be correlated with alteration of acrosomal caps [118–121]. Here, localized transient fusion events expose acrosomal matrix proteins required for initial binding of sperm to the egg's zona pellucida (ZP) and acrosomal exocytosis [121]. These processes resemble kiss-and-run fusion pores observed during exocytosis of synaptic vesicles in neurons [122, 123] and secretory granules in other somatic cells [124]. Using an expanded triple-stain procedure to evaluate acrosomal status and sperm viability [125], we observed a time-dependent increase in the incidence of live human sperm with altered acrosomal caps [20]. Punctate white spots were observed within altered acrosomal caps of some sperm [20], possibly reflecting localized loss of exposed matrix proteins [121]. Initially ~20%, then ~50% (at 2 h), and finally ~70% (at 6 h) of viable sperm displayed altered caps. AM-356 (1.0 and 2.5 nM) or (–)9THC (150 and 1500 nM) did not affect alteration of acrosomal caps between 0 and 2 h. By contrast at a later time period, (–)9THC and AM-356 at these concentrations completely blocked acrosomal cap alterations in another subpopulation of sperm that respond between 2 and 6 h. Both AM-356 and (–)9THC potently inhibited acrosomal modifications in a concentration-dependent manner during the later period of capacitation: IC50 5.9 ± 0.6 pM for AM-356 and 3.5 ± 1.5 nM for (–)9THC [20]. Much higher concentrations of these ligands are required to activate putative ionotropic CNRs and TRPV1 receptors [26, 47, 126]. AM-356 also inhibits capacitation of boar sperm evaluated by chlortetracycline fluorescence staining [24]. The inhibitory effects AM-356 on capacitation of porcine sperm are blocked by the CNR1-selective antagonist SR141716A.[24]. These findings, together with the presence of CNR1 in human and procine sperm [23, 24], indicate a critical role for ECB-signaling via CNR1 receptors in modulating acquisition of acrosomal competence during capacitation.
Consistent with previous results with sea urchin sperm [55, 57], AEA and AM-356 inhibit spontaneous AR during capacitation of human and boar sperm [23, 24]. However, boar sperm capacitated in the presence of the TRPV1 selective antagonist capsazepine show a ~3-fold higher incidence of spontaneous ARs than controls (P < 0.01) [24]. Because endogenous levels of AEA in boar sperm increase during capacitation, these findings suggest a physiological role for AEA signaling via TRPV1 receptors in preventing spontaneous ARs during capacitation [24].
Sperm fertilizing potential Ethical concerns preclude using live human eggs to study sperm fertility. Nevertheless, fertilizing potential of human sperm can be evaluated on the basis of tight binding of capacitated sperm to the ZP of nonviable eggs in the Hemizona assay (HZA), which is predictive of sperm fertility for human in vitro fertilization [127–131]. One half of a bisected ZP was inseminated with sperm that had been capacitated in culture medium containing AM-356, and the matching half was inseminated with sperm incubated in medium containing vehicle, to serve as an internal control [20]. Tight binding of sperm was reduced 49.8% ± 5.1% by 1 nM AM-356 under these conditions (P < 0.001). Previous HZA studies showed that >80% of tightly bound sperm are acrosome reacted [130, 131]. Similarly, boar sperm capacitated in the presence AM-356 show a ~3-fold reduction in AR stimulated by solublized ZP compared with controls (P < 0.01) that are associated with decreased levels of intracellular cAMP [24]. Additional research is required to determine whether these results obtained with AM-356 reflect effects on sperm capacitation and/or a direct effect on ZP-stimulated AR in capacitated sperm. Similarly, future studies should examine possible effects of CNR ligands on AR stimulated by other physiological triggers, e.g., progesterone [4] and prostaglandin E [12], etc.
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CONCLUSIONS |
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9THC flood endocannabinoid-signal systems because they are slowly metabolized, accumulate in fat stores, and produce persistent effects with potentially damaging consequences on sperm production, especially in chronic marijuana smokers [19–23, 137]. Does smoking marijuana also adversely impact sperm capacitation and fertilizing potential in vivo? The answer to this particular question awaits determination of concentrations of (–)9THC and other cannabinoids in reproductive tract fluids of chronic marijuana smokers. Are any of the effects of AEA on sperm elicited by non-CNR or non-TRVP1 mediated processes? Elucidation of these phenomena will enhance our understanding of normal sperm physiology, may account for certain currently unexplained types of infertility, may lead to the development of novel drugs for use in reproductive medicine, and provide new insights into modulation of synaptic transmission. |
DEDICATION |
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TOPABSTRACTINTRODUCTIONCONCLUSIONSDEDICATIONREFERENCES |
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FOOTNOTES |
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2 Correspondence: Herbert Schuel, Division of Anatomy and Cell Biology, Department of Pathology and Anatomical Sciences, School of Medicine and Biomedical Sciences, 206 Farber Hall, University at Buffalo, SUNY, Buffalo, NY 14214. FAX: 716 829 2911; schuel{at}acsu.buffalo.edu
Received: 28 April 2005.
First decision: 11 May 2005.
Accepted: 12 August 2005.
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7-nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes. J Pharmacol Exp Ther 2004 310:1152-11609-tetrahydrocannabinol. Biochem Pharmacol 1991 42:899-904[CrossRef][Medline]
