MOLECULAR REPRODUCTION AND DEVELOPMENT 2960-71 (1991)
Reduction of the Fertilizing Capacity of Sea Urchin Sperm by Cannabinoids Derived From Marihuana. If’. Ultraitructural Changes Associated With Inhibition of the Acrosome Reaction MICHAEL C. CHANG AND HERBERT SCHUEL Department of Anatomical Sciences, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York
Pretreatment of SfrongykxentroABSTRACT tus purpuratus sperm with A’-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electrondense deposits are present in the subacrosomal fossa and in the centriolar fossa.The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with ch1oroform:methanol (2:l) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 (IM THC. The electrondense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization. Key Words: Sea urchin, Sperm, Fertilization,Acrosome reaction, Egg jelly coat, Marihuana, Membrane perturbation, Cannabinoids, A’-tetrahydrocannabinol, Cannabidiol, Canna binol
INTRODUCTION Sea urchin sperm are stimulated to undergo the acrosome reaction during gamete interaction at fertilization by a specific ligand (fucose sulfate glycoconjugate) derived from the jelly coat of the egg (Dan et al., 1964; SeGall and Lennarz, 1979; Kopf and Garbers,
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1980; Garbers, 1989). The acrosome reaction involves the fusion of the sperm’s acrosomal membrane with its plasma membrane, exocytosis of the acrosomal granule, and the formation of the acrosomal filament (Tilney, 1985). This process is an essential prerequisite for fertilization since it exposes the sperm membrane that will attach to, and fuse with, the egg. Ag-Tetrahydrocannabinol (THC) inhibits fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of the sperm (Schuel et al., 1987). THC reduces the fertilizing of S. purpuratus sperm by blocking the acrosome reaction (Schuel et al., 1991).Pretreatment of sperm with THC prevents the triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 pM)and time (0-5 min)dependent manner (Schuel et al., 1991). Induction of the acrosome reaction is inhibited in approximately 90% of sperm pretreated with 100 FM THC for 5 min, while motility is not reduced compared with vehicle and SW treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 ~J.MTHC for 5 min and with 100 pM THC after 20.8 sec. The adverse effectsof THC (pretreatment for 5 min with 100 pM) on the acrosome reaction and sperm-fertilizing capacity are reversible. Two other cannabinoids-cannabidiol (CBD) and cannabinol (CBN)-inhibit fertilization by affecting sperm function in a manner similar to that exhibited by THC (Schuel et al., 1987, 1991). Cannabinoids have a very high lipid-aqueous partition coefficient (Seeman et al., 1972; Roth and Williams, 1979). Therefore, they may affect cellular processes by altering membrane structure (Hillard and Bloom, 1983; Martin, 1986; Burstein, 1987). Cannabinoids are known to induce morphological abnormalities in a variety of somatic cells (Hout, 1976). The present study was conducted to determine the effects of cannabinoids on the ultrastructure of S.purReceived October 29, 1990; accepted December 12, 1990. Address reprint requests to Dr. Herbert Schuel, Department of Anatomical Sciences, University at Buffalo, SUNY, Buffalo, NY 14214.
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM purutus sperm. We now report that THC, CBD, and CBN prevent the fusion reaction between the sperm plasma membrane and acrosomal membrane that normally is stimulated by egg jelly to initiate the acrosome reaction. Blockade of the acrosome reaction by cannabinoids is associated with localized disruption of the nuclear envelope and the formation of lipid deposits within the sperm. Preliminary accounts of this study have been presented previously (Chang and Schuel, 1989, 1990).
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OsO, and processed for electron microscopy (EM). The method of Wigglesworth (1988) was used to stain for lipid electron cytochemically. After sperm cells were postfixed in 1%OsO,, they were incubated in thymol (0.1% in 10% sucrose) at 22"C, then partitioned in 10% farnesol (in 60% ethanol for 45 min), washed in 50% ethanol, and followed by renewal in 1%OsO, (1 hr). Sperm cells were dehydrated with a graded series of alcohol and embedded in Spurr's medium. Cannabinoids were graciously provided by the National Institute of Drug Abuse (Bethesda, MD) or were MATERIALS AND METHODS purchased from Sigma Chemical Co. (St. Louis, MO). Strongylocentrotus purpuratus were purchased from Glutaraldehyde and OSO, (EM grade) and embedding Pacific Biomarine Laboratories (Venice, CA) and main- media were purchased from Ladd Research Industries tained in a marine aquarium at 12°C. Gametes were (Burlington, VT). Thymol and farnesol were purchased obtained by the injection of 0.5 M KC1 into the coelomic from Sigma. cavity. Eggs were shed into artificial seawater (SW), while semen were collected dry. Male and female RESULTS gametes from several individuals were pooled and Ultrastructure of Cannabinoid-Treated Sperm examined under the light microscope to assess quality The ultrastructure of sperm pretreated with 100 pM and fertility (Schuel et al., 1987, 1991). Sperm density was determined turbidometrically at 340 nm (Vacquier THC is compared with that of control sperm cultured in and Payne, 1973). Experiments were conducted in MBL SW and vehicle (Fig. 1).Vehicle controls (Fig. l C , D) are ultrastructurally identical to sperm incubated in formula artificial SW (Cavanaugh, 1956). The cannabinoids were dissolved in 95% ethanol SW (Fig. lA, B). In unstimulated sperm (Fig. lA, C) the (EtOH) to a concentration of 100-200 mg/ml. A work- plasma membrane, acrosomal membrane, and the nuing suspension (1 mg/ml) was prepared immediately clear envelope are intact. The diffuse electron-dense before each experiment (20-40 p1 of cannabinoid, plus material within the subacrosomal fossa of these sperm 0.4 ml propylene glycol, plus 3.6 ml of SW). Aliquots of (profilactin) represents a reservoir of monomeric actin these preparations were added to SW for all subsequent (Tilney, 1985). Stimulation of the sperm with solubidilutions. Vehicle (solvent) controls contained equiva- lized egg jelly results in induction of the acrosome lent amounts of EtOH and propylene glycol dissolved in reaction (Dan et al., 1964). Morphological events that comprise the acrosome reaction include fusion of the SW (Schuel et al., 1987). Solubilized jelly coats were obtained from suspen- plasma membrane with the acrosomal granule, and sions of unfertilized eggs according to the method of polymerization of actin in the subacrosomal fossa to Schackman et al. (1978). The fucose content of the jelly promote elongation of the acrosomal process (Dan et al., preparations was determined spectrophotometrically 1964; Tilney, 1985). The acrosome reaction has been completed in sperm cultured in SW (Fig. 1B) and (Dische and Shettles, 1948). Sperm suspensions (2.7 x 108-1.35 X l o 9 cellsiml) vehicle (Fig. 1D) for 5 min, stimulated with jelly, and were pretreated in SW, vehicle (solvent control), or fixed 3 min later. The acrosomal process is evident at 5-100 pM cannabinoid for 1-10 min at 17°C (Schuel the apex of the sperm head. The outer surface of the et al., 1991). Solubilized egg jelly (1.2 pg fucose acrosomal process is coated with electron-dense mateequivalentsiml, final) or SW was added to the sperm rial derived from the matrix of the unreacted acrosomal cultures. After 3-min incubation, the sperm cultures granule. Actin fibers extend within the acrosomal were fixed by addition of an equal volume of 2.5% process (Tilney, 1985) from the subacrosomal fossa to glutaraldehyde in 0.4 M cacodylate buffer at pH 7.7. the tip of the acrosomal process. In most THC-preCells were postfixed (3 hr) in 1% Os04 (in 0.1 M treated sperm, electron-dense deposits are present in phosphate buffer, pH 7.3), dehydrated with a graded the subacrosomal and centriolar fossae (Fig. lE, F). series of ethanol to propylene oxide and then embedded Intact acrosomal granules are present in THC-prein Araldite. Ultrathin sections were stained with ura- treated sperm that are unstimulated (Fig. 1E) and after nyl acetate followed by lead citrate and then examined addition of egg jelly (Fig. 1F). The nuclear matrix, with a JOEL 100 CXII transmission electron micro- mitochondrion, and flagellum appear to be morphologically identical in both control and THC-treated sperm. scope. Lipid deposits within the sperm were characterized There is no evidence of polymerization of profilactin to by means of extraction with organic solvents and by an promote elongation of the acrosomal process in THCelectron-positive staining reaction. Lipids were ex- pretreated sperm exposed to egg jelly (Fig. 1F). The acrosomal and centriolar regions of sperm are tracted from glutaraldehyde-fixed sperm using a 2: 1 mixture of ch1oroform:methanol (Folch et al., 1957). shown at higher magnification in Figures 2 and 3. Fixation of delipidated sperm was renewed in 1% Examination of the acrosomal region of THC (100glutaraldehyde (3 hr). The cells were postfixed in 1% pM)-pretreated sperm cells shows that the membrane
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Fig. 1. Ultrastructure of sea urchin (S. purpurutus) sperm (1.35 x lo9 celldml) incubated in (A) seawater; (B)seawater plus jelly; ( C ) vehicle; (D) vehicle plus jelly; (E) 100 pM THC; (F) 100 pM THC plus jelly. n, nucleus; m, mitochondrion; f, flagellum; a, acrosome; ap, acrosomal process; p, profilactin (monomeric actin) in subacrosomal fossa; d, electron-dense deposit. Sperm were pretreated with THC for 5 min a t 17°C. Solubilized egg jelly, SW, or vehicle was added to sperm culture, allowed to incubate for 3 min, and then immediately fixed with glutaraldehyde.
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
Fig. 2. High magnification of acrosomal region of control and THC-treated sperm (1.35 x lo9 cellsiml). Unstimulated sperm in seawater (A) and vehicle (B) showing intact plasma membrane (arrow), acrosomal membrane (arrowhead), and nuclear envelope (ne). THC-treated sperm (C) and THC-treated sperm following jelly activation (D) showing intact plasma and acrosomal membranes. The nuclear envelope is eroded within the subacrosomal fossa of THC-treated sperm cells, and electron-dense deposits (d) are found in this area. a , acrosome; n, nucleus; p, profilactin in subacrosomal fossa.
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Fig. 3. High magnification of the centriolar fossa showing electron-dense deposits (d) in THC-treated sperm (A) and jelly-stimulated THC-treated sperm (B). f, flagellum; n, nucleus; m, mitochondrion.
fusion reaction between the plasma membrane and the acrosomal membrane is completely blocked (Fig. 2). In all THC-treated cells, whether unstimulated (Fig. 2C) or exposed to egg jelly (Fig. 2D), these membranes are well preserved and morphologically similar to those seen in unstimulated and unreacted control sperm (Fig. ZA, B). The electron-dense deposits observed within the subacrosomal fossa (Fig. 2C, D) and the centriolar fossa (Fig. 3) are not bound by a limiting membrane. They might be lipid in nature and possibly represent the hydrolytic products of membrane. Such is the case for the nuclear membrane bordering the subacrosomal fossa, where it is fragmented and lies in close proximity to, or becomes associated with, electron-dense deposits after THC treatment (Fig. ZC,D). Since the deposit within the centriolar fossa adheres to the nuclear or the mitochondria1 membrane, and sometimes both, it is difficult to ascertain the source of probable hydrolysis. Since the deposits are seen in both unstimulated and egg jelly-treated sperm pretreated with THC, they must have formed during the THC pretreatment step and are not a consequence of the subsequent stimulation by egg jelly. CBD and CBN (100 pM) also block the acrosome reaction and produce similar electron-dense deposits (data not shown). THC produces similar ultrastructural changes in Lytechinuspictus sperm (data not shown). The effects of incubation time and THC concentration on the ultrastructure of sperm were examined.
Sperm were fixed after 1-, 5 - , and 10-min treatment with 100 pM THC to determine how quickly the ultrastructural changes take place (Fig. 4).Electrondense deposits are seen as early as after l-min treatment with THC (Fig. 4A). The presence and location of the deposits are similar for each incubation period (Fig. 4A-E). Sperm also were fixed after 8-min treatment with 5 , 25, 50, and 100 pM THC. At all these concentrations, THC produced a localized disruption of the nuclear envelope and the accumulation of electrondense deposits within the subacrosomal and centriolar fossae of the sperm. Figure 5 shows electron-dense deposits in sperm treated with 5 pM THC. The IDs0for inhibition of the egg jelly induced acrosome reaction was found to be 6.6 pM THC (Schuel et al., 1991). In the preceding study (Schuel et al., 19911, we showed that pretreatment of sperm for 5 min with 100 pM THC greatly reduced their ability to undergo the acrosome reaction upon stimulation by egg jelly and also produced a corresponding reduction in spermfertilizing capacity. These sperm functions were restored to normal levels after the removal of THC. We conducted an ultrastructural study of sperm treated under comparable conditions (Fig. 6). Sperm were pretreated with 100 pM THC, vehicle, or SW for 5 min. Batches of these cultures were fixed immediately. The remaining cultures were centrifuged at 3,000 rpm for 5 min, the supernatant was removed, and the sperm were resuspended in 100 pM THC, vehicle, or SW. After
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
Fig. 4. Ultrastructure of sperm (1.35 x lo9 cellsiml) fixed after 1-min (A), 5-min (B, C), and 10-min (D, E) treatment with 100 pM THC. Electron-dense deposits (d)are present in the subacrosomal fossa and the centriolar fossa. a, acrosome; m, mitochondrion; n, nucleus.
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M.C. CHANG AND H. SCHUEL acrosome reaction when stimulated (Fig. 6D). Pretreated sperm resuspended in 100 pM THC show severe membrane damage within the subacrosomal fossa with enlarged electron-dense deposits (Fig. 6A). Electrondense deposits also were present in the centriolar fossa. Control sperm incubated with vehicle or SW and washed under the same conditions showed the same normal ultrastructure as untreated and unstimulated cells in SW (see Figs. 1-3).
Fig. 5. Ultrastructure of sperm (2.7 x 10' cellsiml) fixed after treatment with 5 pM THC for 8 min. Electron-dense deposits (d) are present in the subacrosomal fossa (A, B) and in the centriolar fossa (A, C). m, mitochondrion; n, nucleus.
Characterization of Electron-Dense Deposits in Cannabinoid Treated Sperm The electron-dense deposits within cannabinoid treated sperm were characterized ultrastructurally in terms of extraction with organic solvents (Fig. 7) and staining for lipids using thymol and farnesol (Fig. 8). The combination of ch1oroform:methanol (2:1) is commonly used to extract lipids from cells and tissues (Folch et al., 1957). Extraction of glutaraldehyde-fixed sperm pretreated with THC completely removed the electron-dense deposits from the subacrosomal and centriolar fossae (Fig. 7). Similar results were obtained with sperm pretreated with CBN and CBD (data not shown). Cannabinoid-treated sperm not extracted with chloroform-methanol (2:l) showed electron-dense deposits in both fossae (data not shown). These findings suggest that the electron-dense deposits are lipid in nature. Cytochemical staining of lipids for electron microscopy was performed on cannabinoid-treated and -untreated sperm cells (Fig. 8). The visualization of lipid, using thymol and farnesol, is a method for distinguishing osmium bound to lipid from osmium bound to protein (Wigglesworth, 1988). Lipid staining of control cells resulted in an overall reduction of contrast with no electron-positive lipid deposits (Fig. 8A). However, observations of THC-treated sperm after thymol-farnesol staining reveal strong osmiophilic electron dense deposits in both the acrosomal (Fig. 8B, D) and the centriolar (Fig. 8C, E) fossae. The deposits are distinguished by the increase in osmium uptake after the farnesol partition with a loss of local contrast to the rest of the cell. Stained deposits were absent from THCtreated sperm extracted with chloroform-methanol (2:l) prior to staining with thymol and farnesol (Fig. 8F, G). Taken together, the results of the cytochemical staining with thymol and farnesol, and the extraction with chloroform-methanol (2:l) show that the electrondense deposits in cannabinoid-treated sperm are lipid. High magnification of the lipid-stained electron-dense deposits (Fig. 8E) reveal an infrastructure that resembles a mosaic of concentric lamellar inclusions. Such a lamellar-like periodicity is analogous to that found in lipid storage diseases, e.g., Tay-Sachs (Terry, 1971).
5-min incubation, the washed sperm were fixed and processed for electron microscopy. Sperm that were pretreated with 100 pM THC for 5 min showed electron-dense deposits in both the subacrosomal and centriolar fossae, as described previously (see Fig. 4B, C). When THC was removed from pretreated sperm by washing with SW, the electron-dense deposits were DISCUSSION absent from both fossae (Fig. 6B). The nuclear envelope within the subacrosomal fossa was fragmented. The The electron microscopic observations described plasma membrane at the apex of the sperm head and above reveal that THC inhibits the triggering of the the acrosomal membrane were intact (Fig. 6B, C). acrosome reaction stimulated by egg jelly by blocking Sperm washed with SW were able to undergo the the fusion of the acrosomal membrane with the plasma
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
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Fig. 6. Ultrastructure of THC pretreated sperm following removal of THC by washing. Sperm (1.35 x lo9 cells/ml) were pretreated with 100 pM THC for 5 min, sedimented by centrifugation, and then resuspended in 100 pM THC (A)or in seawater (B-D) and fixed 5 min later. m, mitochondrion; n, nucleus. A. Electron-dense deposits (d)are present in the subacrosomal fossa (note fragmentation of the nuclear envelope in this region) and the centriolar fossa cultured continuously in 100 FM THC. B. Electron-dense deposits are no longer present in the sperm after removal of THC by washing. Fragmentation of the
nuclear envelope in the subacrosomal fossa persists. C. High magnification of acrosomal region of sperm shown in B, fixed after removal of THC by washing. The nuclear envelope is fragmented within the subacrosomal fossa. The plasma membrane and the acrosomal membrane are intact. D. Sperm stimulated to undergo acrosome reaction after removal of THC by washing. The acrosomal process (ap) is present at the apex of the sperm head. Note fragmentation of the nuclear envelope within subacrosomal fossa.
membrane in sea urchin sperm. There are ultrastructural changes in the THC-treated sperm associated with the inhibition of the acrosome reaction. THC causes the localized breakdown of the nuclear envelope within the subacrosomal fossa of the sperm and also promotes the formation of electron-dense deposits
within the subacrosomal and centriolar fossae. These deposits are lipid because they lack a limiting membrane; are extractable with chloroform-methanol(2:1), which is known to remove lipids from cells (Folch et al., 1957); and stain positively for lipid with thymol and farnesol (Wigglesworth, 1988). CBD and CBN produce
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Fig. 7. Ultrastructure of sperm delipidated by extraction with chloroform-methanol. The sperm cells (1.35 x lo9 cellsiml) were fixed following pretreatment with 100 pM THC and exposure to egg jelly. Note the absence of electron-dense deposits in the subacrosomal fossa and the centriolar fossa. Electron-dense deposits were likewise absent in extracted sperm pretreated with THC but not stimulated by egg jelly (data not shown). a, acrosome; n, nucleus; m, mitochondrion.
similar morphological effects on the sperm. Since cannabinoids have a high lipid solubility (Martin, 1986), it is possible that these compounds might partition with lipids or membrane vesicular material within the electron-dense deposits. These ultrastructural alterations in the sperm are evident within 1 min of treatment with 100 p,M THC and after treatment with 5-100 pM THC for 8 min. The lipid deposits disappear from THC-treated sperm after removal of THC by washing with SW under conditions that have previously been shown (Schuel et al., 1991) to restore normal sperm function i.e., the capacity to undergo the acrosome reaction upon stimulation by egg jelly and to fertilize eggs. The formation of lipid deposits within sea urchin sperm appears to be a morphological correlate of the adverse effects of cannabinoids on the acrosome reaction and sperm-fertilizing capacity. Studies with humans and other mammals have shown that THC can affect sperm production in the male. This is manifested by a reduction in sperm concentration and motility (Hembree et al., 1978) and by an increased number of sperm showing abnormalities in the development of the sperm head (Zimmerman et al., 1979) and the acrosome (Issidorides, 1978). Treatment of ejaculated bull sperm with THC in vitro suppresses motility and produces ultrastructural damage to the membranes of the middle piece region and the mitochondria (Shahar et al., 1975). THC (10-100
p,M) does not appear to affect the motility of sea urchin sperm (Schuel et al., 1987 and 1991) and does not alter the ultrastructure of the mitochondria1 matrix and cristae (this study). The localized breakdown of the nuclear envelope associated with the formation of lipid deposits in cannabinoid-treated sperm may be the result of an increased hydrolysis of membrane lipids stimulated by THC, CBD, and CBN. An accumulation of free fatty acids andlor diglycerides, as a consequence of elevated phospholipase activity, might lead to the formation of the electron-positive lipid deposits observed in cannabinoid-treated sperm. Lipid bodies that are commonly observed in somatic cells may represent intracellular stores of free arachidonic acid and other fatty acids (Dvorak et al., 1983). Cannabinoids are known to stimulate phospholipase A2 activity in somatic cells, which results in the release of free arachidonic acid from phospholipids (Burstein and Hunter, 1978, 1981; Chaudhry et al., 1988; Laychock et al., 1986;Reichman et al., 1988; White and Tansik, 1980). We recently obtained evidence that THC activates phospholipase A2 activity in homogenates of S.purpuratus sperm (Chang et al., 1990). Free arachidonic acid liberated from membrane phospholipids by the action of phospholipase A2 can be oxidized within cells to produce extremely potent bioregulatory products, such as prostaglandins and leu-
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
Fig. 8. Electron cytochemical staining for lipid in sperm cells (1.35 x lo9 cellsiml) pretreated with seawater or 100 pM THC. Unreacted sperm in seawater (A) show absence of lipid-positive deposits in the subacrosomal fossa and the centriolar fossa. THCpretreated sperm show lipid-positive deposits (d) in the acrosomal fossa (B) and the centriolar fossa (C). THC-pretreated sperm stimu-
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lated by egg jelly also show lipid-positive deposits in the acrosomal fossa (D) and the centriolar fossa (E).THC-pretreated sperm stimulated by egg jelly and delipidated by chloroform-methanol extraction do not show lipid-positive deposits in the subacrosomal and the centriolar fossae (F, G ) .a, acrosome; n , nucleus; ne, nuclear envelope, m, mitochondrion; f, flagellum.
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and its metabolites as second messengers. Trends Neurosci 11:117123. Burstein S, Hunter SA (1978): Prostaglandins and cannabis-VI. Release of arachidonic acid from HeLa cells by Al-tetrahydrocannabinol and other cannabinoids. Biochem Pharmacol27:1275-1280. Burstein S, Hunter SA (1981): Prostaglandins and cannabis-VII. Elevation of phospholipase A, activity by cannabinoids in whole cells and subcellular preparations. J Clin Pharmacol21:240%2488. Burstein S, Hunter SA, Ozman K, Renzuli L (1984):Prostaglandins and cannabis-XIII. Cannabinoid-induced elevation of lipoxygenase products in mouse peritoneal macrophages. Biochem Pharmacol 33~2653-2656. Burstein SH (1987):The inhibitory and stimulatory effects of cannabinoids on eicosanoid synthesis. Natl Inst Drug Abuse Res Monogr Ser 79:158-172. Cavanaugh GM (1956): “Formulae and Methods V. of the Marine Biological Laboratory.” Woods Hole: Marine Biological Laboratory, pp 54-55. Chang MC, Schuel H (1989): The effect of delta-9-tetrahydrocannabinol (THC) on sea urchin (Strongylocentrotuspurpurutus)sperm cell ultrastructure. J Cell Biol 109:251a. Chang MC, Schuel H (1990): Ultrastructural changes in delta-9tetrahydrocannabinol treated sea urchin sperm associated with the inhibition of the acrosome reaction. J Cell Biol 111:114a. Chang MC, Berkery D, Laychock S, Schuel H (1990): Delta-9-tetrahydrocannabinol activates phospholipase A, in sea urchin sperm homogenate. J Cell Biol, 111:114a. Chaudhry A, Thompson RH, Rubin RP, Laychock SG (1988): Relationship between A-9-tetrahydrocannabinol-induced arachidonic acid release and secretagogue-evoked phosphoinositide breakdown and Ca2+mobilization of exocrine pancreas. Mol Pharmacol34:543548. Dan J, Ohori Y, Kushida H (1964): Studies on the acrosome. VII. Formation of the acrosomal process in sea urchin spermatozoa. J Ultrastruct Res 11508-524. Dische Z, Shettles LB (1948): A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J Biol Chem 175:595-603. Dvorak AM, Dvorak HF, Peters SP, Schulman ES, MacGlashan DW, Pyne K, Harvey VS, Galli SJ, Lichtenstein LM (1983):Lipid bodies: cytoplasmic organelles important to arachidonate metabolism in macrophages and mast cells. J Immunol 131:2965-2976. Folch J , Lees M, Sloane-Stanley GH (1957): A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509. Garbers G (1989):Molecular basis of fertilization. Annu Rev Biochem 58:719-742. Gerrard J M (1985): “Prostaglandins and Leukotrienes: Blood and Vascular Cell Function.” New York: Dekker, pp 3-73. Hembree WC 111, Nahas GG, Zeidenberg P, Huang HFS (1978): Changes in human spermatozoa associated with high dose marihuana smoking. In GG Nahas, WDM Paton (eds): “Marihuana: Biological Effects.” New York: Pergamon Press, pp 429439. Hillard CJ, Bloom AS (1983): Possible role of prostaglandins in the effects of the cannabinoids on adenylate cyclase activity. Eur J Pharmacol91:21-27. Huot J (1976): Cellular and biochemical alterations induced in vitro by A’-tetrahydrocannabinol: Effects on cell proliferation, nucleic acids, plasma cell membrane ATPase, and adenylate cyclase. In GG Nahas (ed): “Marihuana: Chemistry, Biochemistry, and Cellular Effects.” New York: Springer-Verlag, pp 313427. ACKNOWLEDGMENTS Issidorides MR (1979):Observations in chronic hashish users: Nuclear We thank Dr. T. Szczesny for his advice on electron aberrations in blood and sperm and abnormal acrosomes in spermatozoa. In GG Nahas and WDM Paton (eds): “Marihuana: Biologmicroscopy, Mrs. D. Berkery for her technical assisical Effects.” New York: Pergamon Press, pp 377-388. tance, and Ms. T. D’Angelo for typing this manuscript. Kopf GS, Garbers DL (1980): Calcium and a fucose-sulfate-rich This work was supported by National Institute of Drug polymer regulate sperm cyclic nucleotide metabolism and the Abuse grant DA-05000 to H.S. acrosome reaction. Biol Reprod 22:1118-1126. Laychock SG, Putney JW (1982): Roles of phospholipid metabolism in secretory cells. In Conn PM (ed): “Cellular Regulation of Secretion REFERENCES and Release.” New York: Academic Press, pp 53-105. Axelrod J, Burch RM, Jelsema CL (1988): Receptor-mediated activa- Laychock SG, Hoffman JM, Meisel E, Bilgin S (1986):Pancreatic islet tion of phospholipase A, via GTP-binding proteins: Arachidonic acid arachidonic turnover and metabolism and insulin release in re-
kotrienes (Laychock and Putney, 1982; Sammuelsson, 1983). This process is known as the arachidonic acid cascade. Cannabinoids are known to activate the arachidonic cascade in other cell systems. This may be a common mechanism for the diverse biological effects of cannabinoids (Burstein and Hunter 1978, 1981; Burstein et al., 1984). We postulate that activation of the arachidonic acid cascade may be responsible for the reduction in the fertilizing capacity of sea urchin sperm and the blockade of the acrosome reaction caused by cannabinoids. Since arachidonic acid metabolites are produced very rapidly by cells and are extremely potent, they can affect cellular function within a short period of time (Gerrard, 1985). This is consistent with the morphological observations of the time course study in which inhibition of the acrosome reaction and appearance of the lipid deposits occur as early as after 1-min pretreatment with 100 pM THC. The acrosome reaction is inhibited by 50% with 100 pM THC after 20.8 sec (Schuel et al., 1990). Our proposal is consistent with previous studies implicating arachidonic acid derived metabolites in modulating gamete interactions during fertilization (Schuel et al., 1984, 1985). Cannabinoids inhibit the ligand (egg jelly)-stimulated induction of the acrosome reaction in sea urchin sperm by blocking the fusion of the acrosomal membrane with the sperm’s plasma membrane. The ultrastructural changes observed in cannabinoid treated sperm suggest that they may act by perturbing phospholipid metabolism. This may explain the reduction in the fertilizing capacity of the sperm promoted by cannabinoids. However, THC does not inhibit the acrosome reaction induced by ionophores such as ionomycin and nigericin (Schuel et al., 1991). These findings suggest that THC blocks the stimulation-secretion-coupling mechanism in the sperm. We believe that THC may activate phospholipase A2 in the sperm to produce the ultrastructural changes observed in this study and to release free arachidonic acid from membrane phospholipids. Arachidonic acid and/or a related metabolite may act as a second messenger to block signal transduction (Axelrod et al., 1988). Elucidation of the molecular mechanisms responsible may lead to an understanding of endogenous factors that normally modulate sperm-fertilizing capacity and of signal transduction involved in triggering acrosome reaction, as well as provide the basis for understanding how cannabinoids affect other cellular systems.
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM sponse to delta-9-tetrahydrocannabinol. Biochem Pharmacol 35: 2003-2008. Martin BR (1986): Cellular effects of cannabinoids. Pharmacol Rev 38:45-74. Reichman M, Nen W, Hokin LE (1988): Ag-Tetrahydrocannabinol increases arachidonic acid levels in guinea pig cerebral cortex slices. Mol Pharmacol 34:823-828. Roth SH, Williams P J (1979): The non-specific membrane binding properties of A’-tetrahydrocannabinol and the effects of various solubilizers. J Pharm Pharmacol 31:224-230. Sammuelsson B (1983): Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation. Science 220:568-575. Schackman RW, Eddy EM, Shapiro BM (1978):The acrosome reaction of Strongylocentrotus purpurutus sperm. Ion requirements and movements. Dev Biol 65:483-495. Schuel H, Traeger E, Schuel R, Boldt J (1984): Anti-inflammatory drugs promote polyspermic fertilization in sea urchins. Gamete Res 10:9-19. Schuel H, Moss R, Schuel R (1985): Induction of polyspermic fertilization in sea urchins by the leukotriene antagonist FPL-557 12 and the 5-lipoxygenase inhibitor BW755C. Gamete Res 11:41-50. Schuel H, Schuel R, Zimmerman AM, Zimmerman S (1987): Cannabinoids reduce fertility of sea urchin sperm. Biochem Cell Biol 65:130-136. Schuel H, Berkery D, Schuel R, Chang MC, Zimmerman AM, Zimmerman S (1991): Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly. Mol Reprod Dev 2951-59.
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Seeman P, Chau-Wong M, Moyen S (1972):The membrane binding of morphine, diphenylhydantoin and tetrahydrocannabinol. Can J Physiol Pharmacol 50:1193-1200. SeGall GK, Lennarz WJ (1979): Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Dev Biol 71:33-48. Shahar A, Bin0 T, Kalay D, Homonnai TZ (1975): Effect of A’tetrahydrocannabinol (THC) on the kinetic morphology of spermatozoa. In BA Afzelius (ed): “The Functional Anatomy of the Spermatozoa.” New York: Pergamon Press, Vol 23, pp 189-193. Terry RD (1971): Some morphological aspects of the lipidoses. In J Bernsohn and H J Grossman (eds): “Lipid Storage Diseases. Enzymatic Defects and Clinical Implications.” New York: Academic Press, pp 3-25. Tihey LG (1985):The acrosomal reaction. In CB Metz and A Monroy (eds): “Biology of Fertilization.” New York: Academic Press, Vol 2, pp 158-213. Vacquier VD, Payne J E (1973): Methods for quantitating sea urchin sperm-egg binding. Exp Cell Res 82:227-235. White HL, Tansik RL (1980): Effects of A’-tetrahydrocannabinol and cannabidiol on phospholipase and other enzymes regulating arachidonate metabolism. Prostaglandins Med 4:409411. Wigglesworth VB (1988): Histological staining of lipids for the light and electron microscope. Biol Rev 63:417-431. Zimmerman AM, Bruce WR, Zimmerman S (1979): Effects of cannabinoids on sperm morphology. Pharmacology 18:143-148.
Reduction of the Fertilizing Capacity of Sea Urchin Sperm by Cannabinoids Derived From Marihuana. If’. Ultraitructural Changes Associated With Inhibition of the Acrosome Reaction MICHAEL C. CHANG AND HERBERT SCHUEL Department of Anatomical Sciences, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York
Pretreatment of SfrongykxentroABSTRACT tus purpuratus sperm with A’-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electrondense deposits are present in the subacrosomal fossa and in the centriolar fossa.The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with ch1oroform:methanol (2:l) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 (IM THC. The electrondense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization. Key Words: Sea urchin, Sperm, Fertilization,Acrosome reaction, Egg jelly coat, Marihuana, Membrane perturbation, Cannabinoids, A’-tetrahydrocannabinol, Cannabidiol, Canna binol
INTRODUCTION Sea urchin sperm are stimulated to undergo the acrosome reaction during gamete interaction at fertilization by a specific ligand (fucose sulfate glycoconjugate) derived from the jelly coat of the egg (Dan et al., 1964; SeGall and Lennarz, 1979; Kopf and Garbers,
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1980; Garbers, 1989). The acrosome reaction involves the fusion of the sperm’s acrosomal membrane with its plasma membrane, exocytosis of the acrosomal granule, and the formation of the acrosomal filament (Tilney, 1985). This process is an essential prerequisite for fertilization since it exposes the sperm membrane that will attach to, and fuse with, the egg. Ag-Tetrahydrocannabinol (THC) inhibits fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of the sperm (Schuel et al., 1987). THC reduces the fertilizing of S. purpuratus sperm by blocking the acrosome reaction (Schuel et al., 1991).Pretreatment of sperm with THC prevents the triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 pM)and time (0-5 min)dependent manner (Schuel et al., 1991). Induction of the acrosome reaction is inhibited in approximately 90% of sperm pretreated with 100 FM THC for 5 min, while motility is not reduced compared with vehicle and SW treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 ~J.MTHC for 5 min and with 100 pM THC after 20.8 sec. The adverse effectsof THC (pretreatment for 5 min with 100 pM) on the acrosome reaction and sperm-fertilizing capacity are reversible. Two other cannabinoids-cannabidiol (CBD) and cannabinol (CBN)-inhibit fertilization by affecting sperm function in a manner similar to that exhibited by THC (Schuel et al., 1987, 1991). Cannabinoids have a very high lipid-aqueous partition coefficient (Seeman et al., 1972; Roth and Williams, 1979). Therefore, they may affect cellular processes by altering membrane structure (Hillard and Bloom, 1983; Martin, 1986; Burstein, 1987). Cannabinoids are known to induce morphological abnormalities in a variety of somatic cells (Hout, 1976). The present study was conducted to determine the effects of cannabinoids on the ultrastructure of S.purReceived October 29, 1990; accepted December 12, 1990. Address reprint requests to Dr. Herbert Schuel, Department of Anatomical Sciences, University at Buffalo, SUNY, Buffalo, NY 14214.
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM purutus sperm. We now report that THC, CBD, and CBN prevent the fusion reaction between the sperm plasma membrane and acrosomal membrane that normally is stimulated by egg jelly to initiate the acrosome reaction. Blockade of the acrosome reaction by cannabinoids is associated with localized disruption of the nuclear envelope and the formation of lipid deposits within the sperm. Preliminary accounts of this study have been presented previously (Chang and Schuel, 1989, 1990).
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OsO, and processed for electron microscopy (EM). The method of Wigglesworth (1988) was used to stain for lipid electron cytochemically. After sperm cells were postfixed in 1%OsO,, they were incubated in thymol (0.1% in 10% sucrose) at 22"C, then partitioned in 10% farnesol (in 60% ethanol for 45 min), washed in 50% ethanol, and followed by renewal in 1%OsO, (1 hr). Sperm cells were dehydrated with a graded series of alcohol and embedded in Spurr's medium. Cannabinoids were graciously provided by the National Institute of Drug Abuse (Bethesda, MD) or were MATERIALS AND METHODS purchased from Sigma Chemical Co. (St. Louis, MO). Strongylocentrotus purpuratus were purchased from Glutaraldehyde and OSO, (EM grade) and embedding Pacific Biomarine Laboratories (Venice, CA) and main- media were purchased from Ladd Research Industries tained in a marine aquarium at 12°C. Gametes were (Burlington, VT). Thymol and farnesol were purchased obtained by the injection of 0.5 M KC1 into the coelomic from Sigma. cavity. Eggs were shed into artificial seawater (SW), while semen were collected dry. Male and female RESULTS gametes from several individuals were pooled and Ultrastructure of Cannabinoid-Treated Sperm examined under the light microscope to assess quality The ultrastructure of sperm pretreated with 100 pM and fertility (Schuel et al., 1987, 1991). Sperm density was determined turbidometrically at 340 nm (Vacquier THC is compared with that of control sperm cultured in and Payne, 1973). Experiments were conducted in MBL SW and vehicle (Fig. 1).Vehicle controls (Fig. l C , D) are ultrastructurally identical to sperm incubated in formula artificial SW (Cavanaugh, 1956). The cannabinoids were dissolved in 95% ethanol SW (Fig. lA, B). In unstimulated sperm (Fig. lA, C) the (EtOH) to a concentration of 100-200 mg/ml. A work- plasma membrane, acrosomal membrane, and the nuing suspension (1 mg/ml) was prepared immediately clear envelope are intact. The diffuse electron-dense before each experiment (20-40 p1 of cannabinoid, plus material within the subacrosomal fossa of these sperm 0.4 ml propylene glycol, plus 3.6 ml of SW). Aliquots of (profilactin) represents a reservoir of monomeric actin these preparations were added to SW for all subsequent (Tilney, 1985). Stimulation of the sperm with solubidilutions. Vehicle (solvent) controls contained equiva- lized egg jelly results in induction of the acrosome lent amounts of EtOH and propylene glycol dissolved in reaction (Dan et al., 1964). Morphological events that comprise the acrosome reaction include fusion of the SW (Schuel et al., 1987). Solubilized jelly coats were obtained from suspen- plasma membrane with the acrosomal granule, and sions of unfertilized eggs according to the method of polymerization of actin in the subacrosomal fossa to Schackman et al. (1978). The fucose content of the jelly promote elongation of the acrosomal process (Dan et al., preparations was determined spectrophotometrically 1964; Tilney, 1985). The acrosome reaction has been completed in sperm cultured in SW (Fig. 1B) and (Dische and Shettles, 1948). Sperm suspensions (2.7 x 108-1.35 X l o 9 cellsiml) vehicle (Fig. 1D) for 5 min, stimulated with jelly, and were pretreated in SW, vehicle (solvent control), or fixed 3 min later. The acrosomal process is evident at 5-100 pM cannabinoid for 1-10 min at 17°C (Schuel the apex of the sperm head. The outer surface of the et al., 1991). Solubilized egg jelly (1.2 pg fucose acrosomal process is coated with electron-dense mateequivalentsiml, final) or SW was added to the sperm rial derived from the matrix of the unreacted acrosomal cultures. After 3-min incubation, the sperm cultures granule. Actin fibers extend within the acrosomal were fixed by addition of an equal volume of 2.5% process (Tilney, 1985) from the subacrosomal fossa to glutaraldehyde in 0.4 M cacodylate buffer at pH 7.7. the tip of the acrosomal process. In most THC-preCells were postfixed (3 hr) in 1% Os04 (in 0.1 M treated sperm, electron-dense deposits are present in phosphate buffer, pH 7.3), dehydrated with a graded the subacrosomal and centriolar fossae (Fig. lE, F). series of ethanol to propylene oxide and then embedded Intact acrosomal granules are present in THC-prein Araldite. Ultrathin sections were stained with ura- treated sperm that are unstimulated (Fig. 1E) and after nyl acetate followed by lead citrate and then examined addition of egg jelly (Fig. 1F). The nuclear matrix, with a JOEL 100 CXII transmission electron micro- mitochondrion, and flagellum appear to be morphologically identical in both control and THC-treated sperm. scope. Lipid deposits within the sperm were characterized There is no evidence of polymerization of profilactin to by means of extraction with organic solvents and by an promote elongation of the acrosomal process in THCelectron-positive staining reaction. Lipids were ex- pretreated sperm exposed to egg jelly (Fig. 1F). The acrosomal and centriolar regions of sperm are tracted from glutaraldehyde-fixed sperm using a 2: 1 mixture of ch1oroform:methanol (Folch et al., 1957). shown at higher magnification in Figures 2 and 3. Fixation of delipidated sperm was renewed in 1% Examination of the acrosomal region of THC (100glutaraldehyde (3 hr). The cells were postfixed in 1% pM)-pretreated sperm cells shows that the membrane
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Fig. 1. Ultrastructure of sea urchin (S. purpurutus) sperm (1.35 x lo9 celldml) incubated in (A) seawater; (B)seawater plus jelly; ( C ) vehicle; (D) vehicle plus jelly; (E) 100 pM THC; (F) 100 pM THC plus jelly. n, nucleus; m, mitochondrion; f, flagellum; a, acrosome; ap, acrosomal process; p, profilactin (monomeric actin) in subacrosomal fossa; d, electron-dense deposit. Sperm were pretreated with THC for 5 min a t 17°C. Solubilized egg jelly, SW, or vehicle was added to sperm culture, allowed to incubate for 3 min, and then immediately fixed with glutaraldehyde.
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
Fig. 2. High magnification of acrosomal region of control and THC-treated sperm (1.35 x lo9 cellsiml). Unstimulated sperm in seawater (A) and vehicle (B) showing intact plasma membrane (arrow), acrosomal membrane (arrowhead), and nuclear envelope (ne). THC-treated sperm (C) and THC-treated sperm following jelly activation (D) showing intact plasma and acrosomal membranes. The nuclear envelope is eroded within the subacrosomal fossa of THC-treated sperm cells, and electron-dense deposits (d) are found in this area. a , acrosome; n, nucleus; p, profilactin in subacrosomal fossa.
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Fig. 3. High magnification of the centriolar fossa showing electron-dense deposits (d) in THC-treated sperm (A) and jelly-stimulated THC-treated sperm (B). f, flagellum; n, nucleus; m, mitochondrion.
fusion reaction between the plasma membrane and the acrosomal membrane is completely blocked (Fig. 2). In all THC-treated cells, whether unstimulated (Fig. 2C) or exposed to egg jelly (Fig. 2D), these membranes are well preserved and morphologically similar to those seen in unstimulated and unreacted control sperm (Fig. ZA, B). The electron-dense deposits observed within the subacrosomal fossa (Fig. 2C, D) and the centriolar fossa (Fig. 3) are not bound by a limiting membrane. They might be lipid in nature and possibly represent the hydrolytic products of membrane. Such is the case for the nuclear membrane bordering the subacrosomal fossa, where it is fragmented and lies in close proximity to, or becomes associated with, electron-dense deposits after THC treatment (Fig. ZC,D). Since the deposit within the centriolar fossa adheres to the nuclear or the mitochondria1 membrane, and sometimes both, it is difficult to ascertain the source of probable hydrolysis. Since the deposits are seen in both unstimulated and egg jelly-treated sperm pretreated with THC, they must have formed during the THC pretreatment step and are not a consequence of the subsequent stimulation by egg jelly. CBD and CBN (100 pM) also block the acrosome reaction and produce similar electron-dense deposits (data not shown). THC produces similar ultrastructural changes in Lytechinuspictus sperm (data not shown). The effects of incubation time and THC concentration on the ultrastructure of sperm were examined.
Sperm were fixed after 1-, 5 - , and 10-min treatment with 100 pM THC to determine how quickly the ultrastructural changes take place (Fig. 4).Electrondense deposits are seen as early as after l-min treatment with THC (Fig. 4A). The presence and location of the deposits are similar for each incubation period (Fig. 4A-E). Sperm also were fixed after 8-min treatment with 5 , 25, 50, and 100 pM THC. At all these concentrations, THC produced a localized disruption of the nuclear envelope and the accumulation of electrondense deposits within the subacrosomal and centriolar fossae of the sperm. Figure 5 shows electron-dense deposits in sperm treated with 5 pM THC. The IDs0for inhibition of the egg jelly induced acrosome reaction was found to be 6.6 pM THC (Schuel et al., 1991). In the preceding study (Schuel et al., 19911, we showed that pretreatment of sperm for 5 min with 100 pM THC greatly reduced their ability to undergo the acrosome reaction upon stimulation by egg jelly and also produced a corresponding reduction in spermfertilizing capacity. These sperm functions were restored to normal levels after the removal of THC. We conducted an ultrastructural study of sperm treated under comparable conditions (Fig. 6). Sperm were pretreated with 100 pM THC, vehicle, or SW for 5 min. Batches of these cultures were fixed immediately. The remaining cultures were centrifuged at 3,000 rpm for 5 min, the supernatant was removed, and the sperm were resuspended in 100 pM THC, vehicle, or SW. After
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
Fig. 4. Ultrastructure of sperm (1.35 x lo9 cellsiml) fixed after 1-min (A), 5-min (B, C), and 10-min (D, E) treatment with 100 pM THC. Electron-dense deposits (d)are present in the subacrosomal fossa and the centriolar fossa. a, acrosome; m, mitochondrion; n, nucleus.
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M.C. CHANG AND H. SCHUEL acrosome reaction when stimulated (Fig. 6D). Pretreated sperm resuspended in 100 pM THC show severe membrane damage within the subacrosomal fossa with enlarged electron-dense deposits (Fig. 6A). Electrondense deposits also were present in the centriolar fossa. Control sperm incubated with vehicle or SW and washed under the same conditions showed the same normal ultrastructure as untreated and unstimulated cells in SW (see Figs. 1-3).
Fig. 5. Ultrastructure of sperm (2.7 x 10' cellsiml) fixed after treatment with 5 pM THC for 8 min. Electron-dense deposits (d) are present in the subacrosomal fossa (A, B) and in the centriolar fossa (A, C). m, mitochondrion; n, nucleus.
Characterization of Electron-Dense Deposits in Cannabinoid Treated Sperm The electron-dense deposits within cannabinoid treated sperm were characterized ultrastructurally in terms of extraction with organic solvents (Fig. 7) and staining for lipids using thymol and farnesol (Fig. 8). The combination of ch1oroform:methanol (2:1) is commonly used to extract lipids from cells and tissues (Folch et al., 1957). Extraction of glutaraldehyde-fixed sperm pretreated with THC completely removed the electron-dense deposits from the subacrosomal and centriolar fossae (Fig. 7). Similar results were obtained with sperm pretreated with CBN and CBD (data not shown). Cannabinoid-treated sperm not extracted with chloroform-methanol (2:l) showed electron-dense deposits in both fossae (data not shown). These findings suggest that the electron-dense deposits are lipid in nature. Cytochemical staining of lipids for electron microscopy was performed on cannabinoid-treated and -untreated sperm cells (Fig. 8). The visualization of lipid, using thymol and farnesol, is a method for distinguishing osmium bound to lipid from osmium bound to protein (Wigglesworth, 1988). Lipid staining of control cells resulted in an overall reduction of contrast with no electron-positive lipid deposits (Fig. 8A). However, observations of THC-treated sperm after thymol-farnesol staining reveal strong osmiophilic electron dense deposits in both the acrosomal (Fig. 8B, D) and the centriolar (Fig. 8C, E) fossae. The deposits are distinguished by the increase in osmium uptake after the farnesol partition with a loss of local contrast to the rest of the cell. Stained deposits were absent from THCtreated sperm extracted with chloroform-methanol (2:l) prior to staining with thymol and farnesol (Fig. 8F, G). Taken together, the results of the cytochemical staining with thymol and farnesol, and the extraction with chloroform-methanol (2:l) show that the electrondense deposits in cannabinoid-treated sperm are lipid. High magnification of the lipid-stained electron-dense deposits (Fig. 8E) reveal an infrastructure that resembles a mosaic of concentric lamellar inclusions. Such a lamellar-like periodicity is analogous to that found in lipid storage diseases, e.g., Tay-Sachs (Terry, 1971).
5-min incubation, the washed sperm were fixed and processed for electron microscopy. Sperm that were pretreated with 100 pM THC for 5 min showed electron-dense deposits in both the subacrosomal and centriolar fossae, as described previously (see Fig. 4B, C). When THC was removed from pretreated sperm by washing with SW, the electron-dense deposits were DISCUSSION absent from both fossae (Fig. 6B). The nuclear envelope within the subacrosomal fossa was fragmented. The The electron microscopic observations described plasma membrane at the apex of the sperm head and above reveal that THC inhibits the triggering of the the acrosomal membrane were intact (Fig. 6B, C). acrosome reaction stimulated by egg jelly by blocking Sperm washed with SW were able to undergo the the fusion of the acrosomal membrane with the plasma
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
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Fig. 6. Ultrastructure of THC pretreated sperm following removal of THC by washing. Sperm (1.35 x lo9 cells/ml) were pretreated with 100 pM THC for 5 min, sedimented by centrifugation, and then resuspended in 100 pM THC (A)or in seawater (B-D) and fixed 5 min later. m, mitochondrion; n, nucleus. A. Electron-dense deposits (d)are present in the subacrosomal fossa (note fragmentation of the nuclear envelope in this region) and the centriolar fossa cultured continuously in 100 FM THC. B. Electron-dense deposits are no longer present in the sperm after removal of THC by washing. Fragmentation of the
nuclear envelope in the subacrosomal fossa persists. C. High magnification of acrosomal region of sperm shown in B, fixed after removal of THC by washing. The nuclear envelope is fragmented within the subacrosomal fossa. The plasma membrane and the acrosomal membrane are intact. D. Sperm stimulated to undergo acrosome reaction after removal of THC by washing. The acrosomal process (ap) is present at the apex of the sperm head. Note fragmentation of the nuclear envelope within subacrosomal fossa.
membrane in sea urchin sperm. There are ultrastructural changes in the THC-treated sperm associated with the inhibition of the acrosome reaction. THC causes the localized breakdown of the nuclear envelope within the subacrosomal fossa of the sperm and also promotes the formation of electron-dense deposits
within the subacrosomal and centriolar fossae. These deposits are lipid because they lack a limiting membrane; are extractable with chloroform-methanol(2:1), which is known to remove lipids from cells (Folch et al., 1957); and stain positively for lipid with thymol and farnesol (Wigglesworth, 1988). CBD and CBN produce
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Fig. 7. Ultrastructure of sperm delipidated by extraction with chloroform-methanol. The sperm cells (1.35 x lo9 cellsiml) were fixed following pretreatment with 100 pM THC and exposure to egg jelly. Note the absence of electron-dense deposits in the subacrosomal fossa and the centriolar fossa. Electron-dense deposits were likewise absent in extracted sperm pretreated with THC but not stimulated by egg jelly (data not shown). a, acrosome; n, nucleus; m, mitochondrion.
similar morphological effects on the sperm. Since cannabinoids have a high lipid solubility (Martin, 1986), it is possible that these compounds might partition with lipids or membrane vesicular material within the electron-dense deposits. These ultrastructural alterations in the sperm are evident within 1 min of treatment with 100 p,M THC and after treatment with 5-100 pM THC for 8 min. The lipid deposits disappear from THC-treated sperm after removal of THC by washing with SW under conditions that have previously been shown (Schuel et al., 1991) to restore normal sperm function i.e., the capacity to undergo the acrosome reaction upon stimulation by egg jelly and to fertilize eggs. The formation of lipid deposits within sea urchin sperm appears to be a morphological correlate of the adverse effects of cannabinoids on the acrosome reaction and sperm-fertilizing capacity. Studies with humans and other mammals have shown that THC can affect sperm production in the male. This is manifested by a reduction in sperm concentration and motility (Hembree et al., 1978) and by an increased number of sperm showing abnormalities in the development of the sperm head (Zimmerman et al., 1979) and the acrosome (Issidorides, 1978). Treatment of ejaculated bull sperm with THC in vitro suppresses motility and produces ultrastructural damage to the membranes of the middle piece region and the mitochondria (Shahar et al., 1975). THC (10-100
p,M) does not appear to affect the motility of sea urchin sperm (Schuel et al., 1987 and 1991) and does not alter the ultrastructure of the mitochondria1 matrix and cristae (this study). The localized breakdown of the nuclear envelope associated with the formation of lipid deposits in cannabinoid-treated sperm may be the result of an increased hydrolysis of membrane lipids stimulated by THC, CBD, and CBN. An accumulation of free fatty acids andlor diglycerides, as a consequence of elevated phospholipase activity, might lead to the formation of the electron-positive lipid deposits observed in cannabinoid-treated sperm. Lipid bodies that are commonly observed in somatic cells may represent intracellular stores of free arachidonic acid and other fatty acids (Dvorak et al., 1983). Cannabinoids are known to stimulate phospholipase A2 activity in somatic cells, which results in the release of free arachidonic acid from phospholipids (Burstein and Hunter, 1978, 1981; Chaudhry et al., 1988; Laychock et al., 1986;Reichman et al., 1988; White and Tansik, 1980). We recently obtained evidence that THC activates phospholipase A2 activity in homogenates of S.purpuratus sperm (Chang et al., 1990). Free arachidonic acid liberated from membrane phospholipids by the action of phospholipase A2 can be oxidized within cells to produce extremely potent bioregulatory products, such as prostaglandins and leu-
ULTRASTRUCTURAL CHANGES IN CANNABINOID-TREATED SPERM
Fig. 8. Electron cytochemical staining for lipid in sperm cells (1.35 x lo9 cellsiml) pretreated with seawater or 100 pM THC. Unreacted sperm in seawater (A) show absence of lipid-positive deposits in the subacrosomal fossa and the centriolar fossa. THCpretreated sperm show lipid-positive deposits (d) in the acrosomal fossa (B) and the centriolar fossa (C). THC-pretreated sperm stimu-
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lated by egg jelly also show lipid-positive deposits in the acrosomal fossa (D) and the centriolar fossa (E).THC-pretreated sperm stimulated by egg jelly and delipidated by chloroform-methanol extraction do not show lipid-positive deposits in the subacrosomal and the centriolar fossae (F, G ) .a, acrosome; n , nucleus; ne, nuclear envelope, m, mitochondrion; f, flagellum.
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and its metabolites as second messengers. Trends Neurosci 11:117123. Burstein S, Hunter SA (1978): Prostaglandins and cannabis-VI. Release of arachidonic acid from HeLa cells by Al-tetrahydrocannabinol and other cannabinoids. Biochem Pharmacol27:1275-1280. Burstein S, Hunter SA (1981): Prostaglandins and cannabis-VII. Elevation of phospholipase A, activity by cannabinoids in whole cells and subcellular preparations. J Clin Pharmacol21:240%2488. Burstein S, Hunter SA, Ozman K, Renzuli L (1984):Prostaglandins and cannabis-XIII. Cannabinoid-induced elevation of lipoxygenase products in mouse peritoneal macrophages. Biochem Pharmacol 33~2653-2656. Burstein SH (1987):The inhibitory and stimulatory effects of cannabinoids on eicosanoid synthesis. Natl Inst Drug Abuse Res Monogr Ser 79:158-172. Cavanaugh GM (1956): “Formulae and Methods V. of the Marine Biological Laboratory.” Woods Hole: Marine Biological Laboratory, pp 54-55. Chang MC, Schuel H (1989): The effect of delta-9-tetrahydrocannabinol (THC) on sea urchin (Strongylocentrotuspurpurutus)sperm cell ultrastructure. J Cell Biol 109:251a. Chang MC, Schuel H (1990): Ultrastructural changes in delta-9tetrahydrocannabinol treated sea urchin sperm associated with the inhibition of the acrosome reaction. J Cell Biol 111:114a. Chang MC, Berkery D, Laychock S, Schuel H (1990): Delta-9-tetrahydrocannabinol activates phospholipase A, in sea urchin sperm homogenate. J Cell Biol, 111:114a. Chaudhry A, Thompson RH, Rubin RP, Laychock SG (1988): Relationship between A-9-tetrahydrocannabinol-induced arachidonic acid release and secretagogue-evoked phosphoinositide breakdown and Ca2+mobilization of exocrine pancreas. Mol Pharmacol34:543548. Dan J, Ohori Y, Kushida H (1964): Studies on the acrosome. VII. Formation of the acrosomal process in sea urchin spermatozoa. J Ultrastruct Res 11508-524. Dische Z, Shettles LB (1948): A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J Biol Chem 175:595-603. Dvorak AM, Dvorak HF, Peters SP, Schulman ES, MacGlashan DW, Pyne K, Harvey VS, Galli SJ, Lichtenstein LM (1983):Lipid bodies: cytoplasmic organelles important to arachidonate metabolism in macrophages and mast cells. J Immunol 131:2965-2976. Folch J , Lees M, Sloane-Stanley GH (1957): A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509. Garbers G (1989):Molecular basis of fertilization. Annu Rev Biochem 58:719-742. Gerrard J M (1985): “Prostaglandins and Leukotrienes: Blood and Vascular Cell Function.” New York: Dekker, pp 3-73. Hembree WC 111, Nahas GG, Zeidenberg P, Huang HFS (1978): Changes in human spermatozoa associated with high dose marihuana smoking. In GG Nahas, WDM Paton (eds): “Marihuana: Biological Effects.” New York: Pergamon Press, pp 429439. Hillard CJ, Bloom AS (1983): Possible role of prostaglandins in the effects of the cannabinoids on adenylate cyclase activity. Eur J Pharmacol91:21-27. Huot J (1976): Cellular and biochemical alterations induced in vitro by A’-tetrahydrocannabinol: Effects on cell proliferation, nucleic acids, plasma cell membrane ATPase, and adenylate cyclase. In GG Nahas (ed): “Marihuana: Chemistry, Biochemistry, and Cellular Effects.” New York: Springer-Verlag, pp 313427. ACKNOWLEDGMENTS Issidorides MR (1979):Observations in chronic hashish users: Nuclear We thank Dr. T. Szczesny for his advice on electron aberrations in blood and sperm and abnormal acrosomes in spermatozoa. In GG Nahas and WDM Paton (eds): “Marihuana: Biologmicroscopy, Mrs. D. Berkery for her technical assisical Effects.” New York: Pergamon Press, pp 377-388. tance, and Ms. T. D’Angelo for typing this manuscript. Kopf GS, Garbers DL (1980): Calcium and a fucose-sulfate-rich This work was supported by National Institute of Drug polymer regulate sperm cyclic nucleotide metabolism and the Abuse grant DA-05000 to H.S. acrosome reaction. Biol Reprod 22:1118-1126. Laychock SG, Putney JW (1982): Roles of phospholipid metabolism in secretory cells. In Conn PM (ed): “Cellular Regulation of Secretion REFERENCES and Release.” New York: Academic Press, pp 53-105. Axelrod J, Burch RM, Jelsema CL (1988): Receptor-mediated activa- Laychock SG, Hoffman JM, Meisel E, Bilgin S (1986):Pancreatic islet tion of phospholipase A, via GTP-binding proteins: Arachidonic acid arachidonic turnover and metabolism and insulin release in re-
kotrienes (Laychock and Putney, 1982; Sammuelsson, 1983). This process is known as the arachidonic acid cascade. Cannabinoids are known to activate the arachidonic cascade in other cell systems. This may be a common mechanism for the diverse biological effects of cannabinoids (Burstein and Hunter 1978, 1981; Burstein et al., 1984). We postulate that activation of the arachidonic acid cascade may be responsible for the reduction in the fertilizing capacity of sea urchin sperm and the blockade of the acrosome reaction caused by cannabinoids. Since arachidonic acid metabolites are produced very rapidly by cells and are extremely potent, they can affect cellular function within a short period of time (Gerrard, 1985). This is consistent with the morphological observations of the time course study in which inhibition of the acrosome reaction and appearance of the lipid deposits occur as early as after 1-min pretreatment with 100 pM THC. The acrosome reaction is inhibited by 50% with 100 pM THC after 20.8 sec (Schuel et al., 1990). Our proposal is consistent with previous studies implicating arachidonic acid derived metabolites in modulating gamete interactions during fertilization (Schuel et al., 1984, 1985). Cannabinoids inhibit the ligand (egg jelly)-stimulated induction of the acrosome reaction in sea urchin sperm by blocking the fusion of the acrosomal membrane with the sperm’s plasma membrane. The ultrastructural changes observed in cannabinoid treated sperm suggest that they may act by perturbing phospholipid metabolism. This may explain the reduction in the fertilizing capacity of the sperm promoted by cannabinoids. However, THC does not inhibit the acrosome reaction induced by ionophores such as ionomycin and nigericin (Schuel et al., 1991). These findings suggest that THC blocks the stimulation-secretion-coupling mechanism in the sperm. We believe that THC may activate phospholipase A2 in the sperm to produce the ultrastructural changes observed in this study and to release free arachidonic acid from membrane phospholipids. Arachidonic acid and/or a related metabolite may act as a second messenger to block signal transduction (Axelrod et al., 1988). Elucidation of the molecular mechanisms responsible may lead to an understanding of endogenous factors that normally modulate sperm-fertilizing capacity and of signal transduction involved in triggering acrosome reaction, as well as provide the basis for understanding how cannabinoids affect other cellular systems.
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