MOLECULAR REPRODUCTION AND DEVELOPMENT 33:303-306 (1992)
Biosynthesis of Prostaglandins by Human Spermatozoa In Vitro and Their Role in Acrosome Reaction and Fertilization ASHIM C. ROY AND SHAN S. RATNAM Department of Obstetrics and Gynaecology, National University of Singapore, National University Hospital, Singapore ABSTRACT Five homogenates of human sperm cells were separately incubated with [14C]arachidonic acid in the presence of reduced glutathione, L-tryptophan, and haematin as cofactors. The cyclooxygenase products of arachidonic acid metabolism were extracted, separated, and measured for their radioactivity. The rate of formation of prostaglandin (PG)D,, PGE,, PGF, 6-keto PGF,, and thromboxane (TX)B, were 18.0 k 1.11, 10.9 -C 0.68, 5.8 ? 0.21, 3.9 0.13 and 6.6 2 0.52 pmol/106 cells/min, respectively. These results are discussed in relation t o the hypothesis that cyclooxygenase metabolites of certain polyunsaturated fatty acids play an important part in the sperm acrosome reaction and fertilization.
*
0 1992 Wtley-Liss, Inc.
Key Words: Arachidonic acid, Calcium ionophores, Cyclooxygenase, Cyclooxygenase inhibitors, Lipoxygenase products, Phospholipase A, Thromboxanes
Nugteren, 1971) indicated the presence of cyclooxygenase activity in rat spermatozoa (Johnson and Ellis, 19771, such activity remains to be recognized with a specific and sensitive assay method. In this study, we investigated the possible existence of cyclooxygenase activity in the human spermatozoa using [14C]AA as the substrate and assessing the formation of labelled products.
MATERIALS AND METHODS [l-14C]arachidonic acid (specific activity 55.0 mCi/ mmol) was obtained from Amersham International (Amersham, U.K.), LK6D TLC plates (20 x 20 cm) from Whatman Inc. (Clifton, N J ) and Percoll from Pharmacia (Uppsala, Sweden). Solvents and other chemicals were of analytical grade, purchased from Sigma Chemical Co. (St. Louis, MO) and Merck (Darmstadt,Federal Republic of Germany).
Preparation of Spermatozoa1 Homogenates Fresh ejaculates were collected by masturbation afThe prostaglandins (PGs) are a family of biologically ter 3 days or more of abstinence from healthy subjects active acid lipids derived from the metabolism of cer- aged between 20 and 30 years (mean 28 years) attendtain polyunsaturated fatty acids such a s arachidonic ing the male fertility clinic at the National University acid (AA) by a microsomal enzyme called fatty acid Hospital. After seminal clot liquefaction, the ejaculates cyclooxygenase (EC 1.14.99.1) (Hall and Behrman, were analyzed for volume, pH, and sperm concentra1982; Needleman e t al., 1986). These autacoids and tion, motility, and morphology. They were pooled a t their metabolites occur in abundant quantities in the random and centrifuged at 950g for 15 min. The pellet seminal plasma of man and some animals (Kelly, 1981; was uniformly suspended in Tris HC1 buffer (50 mM, Bygdeman and Gillespie, 1982). They may play a func- pH 8.0) to a concentration of 2-10 x lo6 cellsiml. The tional role in the maturation and transport of sperma- suspension was carefully layered on discontinuous 45% tozoa through the male reproductive system. and 90% Percoll in 0.9% NaCl and then centrifuged at The autacoids have also been implicated in the 10,OOOg for 10 min. The sedimented sperm pellet was events leading to sperm acrosome reaction and fertili- resuspended in 50 mM Tris HC1 buffer (pH 8.0) containzation (Lau et al., 1973; Lau and Saksena, 1979; Meizel ing 0.1% Triton X-100 to a concentration of 2-4 x lo8 and Turner, 1983, 1984; Aitken and Kelly, 1985; Ait- cellsiml and then sonicated for 30 sec at 4°C using a n ken et al., 1986; Joyce et al., 1987; Yanagimachi, 1988). Artek Sonic Dismembrator 150 (Artek System Corp., They and/or their receptors have also been located in Farmingdale, NY) at a n amplitude setting of 5. The the plasmalemma of rabbit (Bartoszewicz et al., 1975) sonicate was centrifuged for 15 min at 1,OOOg (4"C),and and human (Mercado et al., 1978) spermatozoa, respec- the resulting supernatant was immediately used for tively. Therefore, it is possible that generation of the arachidonate cyclooxygenase products by spermatozoa is important in the induction of the acrosome reaction Received February 21,1992; accepted May 18,1992. and fertilization. Although a nonspecific histochemical Address reprint requests to Dr. Ashim C. Roy, Department of Obstetstudy employing 3,3'-diaminobenzidine a s staining re- rics and Gynaecology, National University of Singapore, National agent (Graham and Karnovski, 1966; Janszen and University Hospital, Lower Kent Ridge Road, Singapore 0511.
INTRODUCTION
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A.C. ROY AND S.S. RATNAM
the assay of cyclooxygenase activity. The Percoll gradient method yielded adequate sperm concentration, with high motility and improved morphology, while eliminating other cellular elements such as leukocytes, germinal-line cells, and anucleate residual cytoplasmic masses.
Assay of Cyclooxygenase Activity The standard incubation mixture (1.0 ml) contained spermatozoal homogenate (0.8 ml), haematin (5 pM), L-tryptophan (2.5 mM), reduced glutathione (2.5 mM), and [1-14C]AA(1.0 pCi after conversion to Na salt with 0.1 M Na,C03). Haematin and L-tryptophan facilitate the formation of PGH, from AA, and reduced glutathione of PGE, from PGH, (Miyamoto et al., 1976; Yoshimoto et al., 1977; Hemler and Lands, 1980). The mixture was incubated for 120 min a t 37°C in a shaking water bath. Blanks contain a n identical mixture, but the reaction was terminated a t 0 time. The reaction was initiated by the addition of [ 14C]AA. The reaction was stopped by adding 1.0 M HC1 to pH 3-3.5, and the lipids were extracted twice with 6 ml of ethyl acetate. The combined organic phase was evaporated to dryness under a vacuum, and the prostanoids and AA were separated on a silica gel G-coated TLC plate, visualized, identified, and counted for radioactivity a s described previously (Roy et al., 1984a,b). The activity of cyclooxygenase was assessed from the conversion of exogenous [14C]AA into [14C]prostanoids. Quantitation of the prostanoid synthesis was based on the specific activity of [l4C1AAcorrected for nonenzymatic synthesis and procedural losses. Since no protease inhibitors were used in the experiments, the rate of formation of PGs and thromboxanes obtained may be somewhat less than the actual value. This difference may be due to proteolysis by endogenous proteases of the enzymes involved in the cyclooxygenase pathway of arachidonic acid metabolism.
RESULTS The results of the incubation of five homogenates of separately pooled human spermatozoa with exogenous [14C]AAare shown in Figure 1.Under the assay conditions (see Materials and Methods), the spermatozoal homogenates produced, in decreasing order, PGD,, PGE,, TXB, = PGF,,, and 6-keto PGF,, in vitro. Paired Student’s t test (two-tailed) shows the difference between each pair of formation to be significantly (P < 0.005) different except between TXB, and PGF,, (P > 0.05). TXB, and 6-keto PGF,, (6-KF1,) are the stable nonenzymatically formed products of TXA, and prostacyclin (PGI,), respectively. Therefore, these two metabolites represent the synthesis of their respective parent substances. The concentration of all cyclooxygenase products of AA metabolism increased linearly with time a t least up to 120 min. However, these products were not formed significantly during incubation with boiled homogenates. Indomethacin (50 pM), a nonsteroidal antiinflammatory drug and a potent inhibitor of cyclooxygen-
2o
r
PGb
PGE2
PGF,,
TXB2
6KFIa
Fig. 1. Biosynthesis of prostanoids by human spermatozoa from [14C]arachidonic acid (AA) in vitro. Five spermatozoal homogenates (0.8 ml) were separately incubated with I1-l4C1AA (1.0 WCi) in the presence of haematin (5 wM), L-tryptophan (2.5 mM), and reduced glutathione (2.5 mM) in a total volume of 1.0 ml for 120 min a t 37°C. The reaction was terminated with HC1 (1.0 M) to pH 3-3.5 and the lipids were extracted twice with 6.0 ml ethyl acetate. The combined organic phase was evaporated to dryness under vacuum, and the prostanoids were separated on a silica gel G-coated TLC plate, identified, and counted for radioactivity as described previously (Roy et al., 1984a,b).
ase activity (Flower, 1974; Roy et al., 1984a,b), inhibited 60-70% of the generation of prostanoids. This is in agreement with histochemical findings (Johnson and Ellis, 1977) indicating that indomethacin significantly diminished but did not totally eliminate PG synthesis in the reproductive tract of male rats.
DISCUSSION The results presented in this study constitute the first direct evidence for the occurrence of fatty acid cyclooxygenase activity in spermatozoa. From this study, however, it was not possible to determine the site(s) of the enzyme activity in the spermatozoa. Subcellularly, in the cultured fibroblasts, cyclooxygenase activity is contained in endoplasmic reticulum and nuclear membrane but not in plasma membrane or mitochondrial membrane (Needleman et al., 1986). Cyclooxygenase activity is known to insert two molecules of oxygen into arachidonate to yield a 15-hydroperoxy-9,ll-endoperoxidewith a substituted cyclopentane ring (PGG,). A hydroperoxidase activity reduces PGG, to its 15-hydroxy analogue (PGH,), the immediate precursor of bisenoic prostaglandins (e.g., PGE,, PGF,,, PGI,, TXA,). The cyclooxygenase and peroxi-
PROSTAGLANDIN SYNTHESIS BY HUMAN SPERMATOZOA dase activities reside in a single protein, called prostaglandin endoperoxide synthase (PES) (Needleman et al., 1986). Similarly, PES catalyzes the formation of monoenoic prostaglandins such as PGE, from dihomo-y -1inolenic acid. Nonsteroidal antiinflammatory agents such a s indomethacin inhibit the cyclooxygenase but not the hydroperoxidase activity of PES (Needleman e t al., 1986). It is well documented that polyunsaturated fatty acids, predominantly AA, are incorporated as a structural component of phospholipids into cell membranes and other subcellular structures of all tissues of the body. They can be released by the action of phospholipase A, or by the sequential actions of phospholipase C and 1,2-diacylglycerol lipase (Irvine, 1982). Recently, phospholipase A, activity has been detected in human sperm cells (Antaki et al., 1989). Therefore, human spermatozoa, which also possess cyclooxygenase activity (this study),should be capable ofproducing PGs and thromboxanes in vivo. Several studies have suggested the involvement of cyclooxygenase metabolites of certain polyunsaturated fatty acids in the sperm acrosome reaction and fertilization. It has been reported that PGE,, PGE,, PGF,,, and AA (the precursor of bisenoic PGs) stimulate the acrosome reaction of hamster and guinea pig spermatozoa (Meizel and Turner, 1983, 1984; Joyce e t al., 1987). PGE, increased the acrosome reaction of guinea pig spermatozoa initiated by Ca2’ (Joyce et al., 1987). Inhibitors of cyclooxygenase activity have been reported to decrease significantly the acrosome reaction of hamster and guinea pig spermatozoa induced by AA and Ca2+,respectively; the inhibition did not occur in the presence of PGE, and PGF,, (Meizel and Turner, 1984; Joyce et al., 1987). Administration of cyclooxygenase inhibitors to female mice (Lau e t al., 1973) and rabbits (Lau and Saksena, 1979) affected the fertilizing capacity of spermatozoa in vivo, and this was reversed by PGs. Cyclooxygenase inhibitors also reduced the fertilizing capacity of mouse spermatozoa in vitro (Joyce e t al., 1987). Moreover, it has been found that PGs of the E series have the ability to enhance the capacity of human spermatozoa €or penetration of zona-free hamster oocytes (Aitken and Kelly, 1985; Aitken et al., 1986). It is believed that calcium ion influx is a n essential step in the mammalian sperm acrosome reaction (Yanagimachi, 1988) and that calcium ionophores enhance the reaction (Joyce et al., 1987). PGs may act primarily a s ionophores to facilitate the movement of Ca2+ through lipoprotein membranes and thus have a role in the exocytotic event (Meizel and Turner, 1983; Aitken e t al., 1986; Joyce et al., 1987). The present study in which significant amounts of cyclooxygenase activity have been found in human sperm cells provides further support for the essential role of PGs in the sperm acrosome reaction and fertilization. Finally, we have found (unpublished data) a similar formation of various lipoxygenase products (Hall and Behrman, 1982; Needleman et al., 1986) by human spermatozoa in vitro. Some of these AA metabolites
305
have already been shown to be important for the mammalian acrosome reaction (Meizel and Turner, 1984; Basuray et al., 1990).
ACKNOWLEDGMENTS We thank Mdm. Tan Suan Mian and Mdm. Ho Lai Meng for their excellent technical assistance.
REFERENCES Aitken RJ, Irvine S, Kelly RW (1986): Significance of intracellular calcium and cyclic adenosine 3’,5’-monophosphate in the mechanisms by which prostaglandins influence human sperm function. J Reprod Fertil77:451462. Aitken FLJ, Kelly RW (1985): Analysis of the direct effects of prostaglandins on human sperm function. J Reprod Fertil 73:139-146. Antaki P, Guerette P, Chapdelaine A, Roberts KD (1989): Detection of phospholipase A, in human spermatozoa. Biol Reprod 41:241-246. Bartoszewicz W, Dandekar WP, Glass RH, Gordon M (1975):Localization of prostaglandin on the plasmalemma of rabbit sperm. J Exp Zoo1 191:151-160. Basuray R, De Jonge C, Zaneveld L J (1990): Evidence for a role of cysteinyl leukotrienes in mouse and human sperm function. J Androl 11:47-51. Bygdeman M, Gillespie A (1982):Prostaglandins in human reproduction. In Lee J B (ed):“Prostaglandins. Current Endocrinology: Basic and Clinical Aspects.” New York: Elsevier North Holland Inc., pp. 215-249. Flower FLJ (1974): Drugs which inhibit prostaglandin biosynthesis. Pharmacol Rev 26:33-67. Graham RC, Karnovski MJ (1966): The early stages of adsorption of injected horse-radish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J Histochem 14:291-302. Hall AK, Behrman HR (1982): Prostaglandins: biosynthesis, metabolism and mechanism of cellular action. In Lee J B (ed):“Prostaglandins.” Amsterdam: Elsevier North Holland, pp 1-38. Hemler ME, Lands WEM (1980): Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J Biol Chem 255:6253-6261. Irvine RF (1982): Review article. How is the level of free arachidonic acid controlled in mammalian cells? Biochem J 204:3-16. Janszen FHA, Nugteren DH (1971): Histochemical localisation of prostaglandin synthetase. Histochemistry 27: 159-164. Johnson JM, Ellis LC (1977): The histochemical localization of prostaglandin synthetase activity in reproductive tract of the male rat. J Reprod Fertil51:17-22. Joyce CL, Nuzzo NA, Wilson L J r , Zaneveld LJD (1987):Evidence for a role of cyclooxygenase (prostaglandin synthetase) and prostaglandins in the sperm acrosome reaction and fertilization. J Androl 8:7&82. Kelly RW (1981): Prostaglandin synthesis in the male and female reproductive tract. J Reprod Fertil62:293-304. Lau IF, Saksena SK (1979): Inhibition of ovulation and fertilization by indomethacin and effect of prostagIandin-F,*, on early pregnancy in the rabbit. Prostagland Med 2:425-432. Lau IF, Saksena SK, Chang MS (1973): Pregnancy blockade by indomethacin, an inhibitor of prostaglandin synthesis: Its reversal by prostaglandins and progesterone in mice. Prostaglandins 4: 195203. Meizel S, Turner KO (1983): Stimulation of an exocytotic event, the hamster acrosome reaction by cis-unsaturated fatty acids. FEBS Lett 161:315-318. Meizel S, Turner KO (1984): The effects of products and inhibitors of arachidonic acid metabolism on the hamster sperm acrosome reaction. J Exp Zoo1 231:283-288. Mercado E, Villalobos M, Dominguez R, Rosado A (1978):Differential binding of PGE-1 and PGF-2a to the human spermatozoa membrane. Life Sci 22:429436. Miyamoto T, Ogino N, Yamamoto S, Hayaishi 0 (1976): Purification
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of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 251:2629-2636. Needleman P, Turk J, Jakschik BA, Morrison AR, Lefkowith J B (1986): Arachidonic acid metabolism. Annu Rev Biochem 55:69102. Roy AC, Karim SMM, Kottegoda SR, Ratnam SS (1984a):Thromboxane A, and prostacyclin levels in molar pregnancy. Br J Obstet Gynaecol91:90%912. Roy AC, Tan SM, Kottegoda SR, Ratnam SS (1984b3: Ability ofhuman
corpora cavernosa muscle to generate prostaglandins and thromboxanes in vitro. IRCS Med Sci 12:608-609. Yanagimachi R (1988):Mammalian fertilization. In Knobil E, Neil1 J, Ewing L, Greenwald GS, Markert CL, Pfaff DW (eds):“The Physiology of Reproduction.” New York: Raven Press, pp 135-185. Yoshimoto T, Yamamoto S, Okuma M, Hayaishi 0 (1977):Solubilization and resolution of thromboxane synthesizing system from microsomes of bovine blood platelets. J Biol Chem 2525871-5874.
Biosynthesis of Prostaglandins by Human Spermatozoa In Vitro and Their Role in Acrosome Reaction and Fertilization ASHIM C. ROY AND SHAN S. RATNAM Department of Obstetrics and Gynaecology, National University of Singapore, National University Hospital, Singapore ABSTRACT Five homogenates of human sperm cells were separately incubated with [14C]arachidonic acid in the presence of reduced glutathione, L-tryptophan, and haematin as cofactors. The cyclooxygenase products of arachidonic acid metabolism were extracted, separated, and measured for their radioactivity. The rate of formation of prostaglandin (PG)D,, PGE,, PGF, 6-keto PGF,, and thromboxane (TX)B, were 18.0 k 1.11, 10.9 -C 0.68, 5.8 ? 0.21, 3.9 0.13 and 6.6 2 0.52 pmol/106 cells/min, respectively. These results are discussed in relation t o the hypothesis that cyclooxygenase metabolites of certain polyunsaturated fatty acids play an important part in the sperm acrosome reaction and fertilization.
*
0 1992 Wtley-Liss, Inc.
Key Words: Arachidonic acid, Calcium ionophores, Cyclooxygenase, Cyclooxygenase inhibitors, Lipoxygenase products, Phospholipase A, Thromboxanes
Nugteren, 1971) indicated the presence of cyclooxygenase activity in rat spermatozoa (Johnson and Ellis, 19771, such activity remains to be recognized with a specific and sensitive assay method. In this study, we investigated the possible existence of cyclooxygenase activity in the human spermatozoa using [14C]AA as the substrate and assessing the formation of labelled products.
MATERIALS AND METHODS [l-14C]arachidonic acid (specific activity 55.0 mCi/ mmol) was obtained from Amersham International (Amersham, U.K.), LK6D TLC plates (20 x 20 cm) from Whatman Inc. (Clifton, N J ) and Percoll from Pharmacia (Uppsala, Sweden). Solvents and other chemicals were of analytical grade, purchased from Sigma Chemical Co. (St. Louis, MO) and Merck (Darmstadt,Federal Republic of Germany).
Preparation of Spermatozoa1 Homogenates Fresh ejaculates were collected by masturbation afThe prostaglandins (PGs) are a family of biologically ter 3 days or more of abstinence from healthy subjects active acid lipids derived from the metabolism of cer- aged between 20 and 30 years (mean 28 years) attendtain polyunsaturated fatty acids such a s arachidonic ing the male fertility clinic at the National University acid (AA) by a microsomal enzyme called fatty acid Hospital. After seminal clot liquefaction, the ejaculates cyclooxygenase (EC 1.14.99.1) (Hall and Behrman, were analyzed for volume, pH, and sperm concentra1982; Needleman e t al., 1986). These autacoids and tion, motility, and morphology. They were pooled a t their metabolites occur in abundant quantities in the random and centrifuged at 950g for 15 min. The pellet seminal plasma of man and some animals (Kelly, 1981; was uniformly suspended in Tris HC1 buffer (50 mM, Bygdeman and Gillespie, 1982). They may play a func- pH 8.0) to a concentration of 2-10 x lo6 cellsiml. The tional role in the maturation and transport of sperma- suspension was carefully layered on discontinuous 45% tozoa through the male reproductive system. and 90% Percoll in 0.9% NaCl and then centrifuged at The autacoids have also been implicated in the 10,OOOg for 10 min. The sedimented sperm pellet was events leading to sperm acrosome reaction and fertili- resuspended in 50 mM Tris HC1 buffer (pH 8.0) containzation (Lau et al., 1973; Lau and Saksena, 1979; Meizel ing 0.1% Triton X-100 to a concentration of 2-4 x lo8 and Turner, 1983, 1984; Aitken and Kelly, 1985; Ait- cellsiml and then sonicated for 30 sec at 4°C using a n ken et al., 1986; Joyce et al., 1987; Yanagimachi, 1988). Artek Sonic Dismembrator 150 (Artek System Corp., They and/or their receptors have also been located in Farmingdale, NY) at a n amplitude setting of 5. The the plasmalemma of rabbit (Bartoszewicz et al., 1975) sonicate was centrifuged for 15 min at 1,OOOg (4"C),and and human (Mercado et al., 1978) spermatozoa, respec- the resulting supernatant was immediately used for tively. Therefore, it is possible that generation of the arachidonate cyclooxygenase products by spermatozoa is important in the induction of the acrosome reaction Received February 21,1992; accepted May 18,1992. and fertilization. Although a nonspecific histochemical Address reprint requests to Dr. Ashim C. Roy, Department of Obstetstudy employing 3,3'-diaminobenzidine a s staining re- rics and Gynaecology, National University of Singapore, National agent (Graham and Karnovski, 1966; Janszen and University Hospital, Lower Kent Ridge Road, Singapore 0511.
INTRODUCTION
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A.C. ROY AND S.S. RATNAM
the assay of cyclooxygenase activity. The Percoll gradient method yielded adequate sperm concentration, with high motility and improved morphology, while eliminating other cellular elements such as leukocytes, germinal-line cells, and anucleate residual cytoplasmic masses.
Assay of Cyclooxygenase Activity The standard incubation mixture (1.0 ml) contained spermatozoal homogenate (0.8 ml), haematin (5 pM), L-tryptophan (2.5 mM), reduced glutathione (2.5 mM), and [1-14C]AA(1.0 pCi after conversion to Na salt with 0.1 M Na,C03). Haematin and L-tryptophan facilitate the formation of PGH, from AA, and reduced glutathione of PGE, from PGH, (Miyamoto et al., 1976; Yoshimoto et al., 1977; Hemler and Lands, 1980). The mixture was incubated for 120 min a t 37°C in a shaking water bath. Blanks contain a n identical mixture, but the reaction was terminated a t 0 time. The reaction was initiated by the addition of [ 14C]AA. The reaction was stopped by adding 1.0 M HC1 to pH 3-3.5, and the lipids were extracted twice with 6 ml of ethyl acetate. The combined organic phase was evaporated to dryness under a vacuum, and the prostanoids and AA were separated on a silica gel G-coated TLC plate, visualized, identified, and counted for radioactivity a s described previously (Roy et al., 1984a,b). The activity of cyclooxygenase was assessed from the conversion of exogenous [14C]AA into [14C]prostanoids. Quantitation of the prostanoid synthesis was based on the specific activity of [l4C1AAcorrected for nonenzymatic synthesis and procedural losses. Since no protease inhibitors were used in the experiments, the rate of formation of PGs and thromboxanes obtained may be somewhat less than the actual value. This difference may be due to proteolysis by endogenous proteases of the enzymes involved in the cyclooxygenase pathway of arachidonic acid metabolism.
RESULTS The results of the incubation of five homogenates of separately pooled human spermatozoa with exogenous [14C]AAare shown in Figure 1.Under the assay conditions (see Materials and Methods), the spermatozoal homogenates produced, in decreasing order, PGD,, PGE,, TXB, = PGF,,, and 6-keto PGF,, in vitro. Paired Student’s t test (two-tailed) shows the difference between each pair of formation to be significantly (P < 0.005) different except between TXB, and PGF,, (P > 0.05). TXB, and 6-keto PGF,, (6-KF1,) are the stable nonenzymatically formed products of TXA, and prostacyclin (PGI,), respectively. Therefore, these two metabolites represent the synthesis of their respective parent substances. The concentration of all cyclooxygenase products of AA metabolism increased linearly with time a t least up to 120 min. However, these products were not formed significantly during incubation with boiled homogenates. Indomethacin (50 pM), a nonsteroidal antiinflammatory drug and a potent inhibitor of cyclooxygen-
2o
r
PGb
PGE2
PGF,,
TXB2
6KFIa
Fig. 1. Biosynthesis of prostanoids by human spermatozoa from [14C]arachidonic acid (AA) in vitro. Five spermatozoal homogenates (0.8 ml) were separately incubated with I1-l4C1AA (1.0 WCi) in the presence of haematin (5 wM), L-tryptophan (2.5 mM), and reduced glutathione (2.5 mM) in a total volume of 1.0 ml for 120 min a t 37°C. The reaction was terminated with HC1 (1.0 M) to pH 3-3.5 and the lipids were extracted twice with 6.0 ml ethyl acetate. The combined organic phase was evaporated to dryness under vacuum, and the prostanoids were separated on a silica gel G-coated TLC plate, identified, and counted for radioactivity as described previously (Roy et al., 1984a,b).
ase activity (Flower, 1974; Roy et al., 1984a,b), inhibited 60-70% of the generation of prostanoids. This is in agreement with histochemical findings (Johnson and Ellis, 1977) indicating that indomethacin significantly diminished but did not totally eliminate PG synthesis in the reproductive tract of male rats.
DISCUSSION The results presented in this study constitute the first direct evidence for the occurrence of fatty acid cyclooxygenase activity in spermatozoa. From this study, however, it was not possible to determine the site(s) of the enzyme activity in the spermatozoa. Subcellularly, in the cultured fibroblasts, cyclooxygenase activity is contained in endoplasmic reticulum and nuclear membrane but not in plasma membrane or mitochondrial membrane (Needleman et al., 1986). Cyclooxygenase activity is known to insert two molecules of oxygen into arachidonate to yield a 15-hydroperoxy-9,ll-endoperoxidewith a substituted cyclopentane ring (PGG,). A hydroperoxidase activity reduces PGG, to its 15-hydroxy analogue (PGH,), the immediate precursor of bisenoic prostaglandins (e.g., PGE,, PGF,,, PGI,, TXA,). The cyclooxygenase and peroxi-
PROSTAGLANDIN SYNTHESIS BY HUMAN SPERMATOZOA dase activities reside in a single protein, called prostaglandin endoperoxide synthase (PES) (Needleman et al., 1986). Similarly, PES catalyzes the formation of monoenoic prostaglandins such as PGE, from dihomo-y -1inolenic acid. Nonsteroidal antiinflammatory agents such a s indomethacin inhibit the cyclooxygenase but not the hydroperoxidase activity of PES (Needleman e t al., 1986). It is well documented that polyunsaturated fatty acids, predominantly AA, are incorporated as a structural component of phospholipids into cell membranes and other subcellular structures of all tissues of the body. They can be released by the action of phospholipase A, or by the sequential actions of phospholipase C and 1,2-diacylglycerol lipase (Irvine, 1982). Recently, phospholipase A, activity has been detected in human sperm cells (Antaki et al., 1989). Therefore, human spermatozoa, which also possess cyclooxygenase activity (this study),should be capable ofproducing PGs and thromboxanes in vivo. Several studies have suggested the involvement of cyclooxygenase metabolites of certain polyunsaturated fatty acids in the sperm acrosome reaction and fertilization. It has been reported that PGE,, PGE,, PGF,,, and AA (the precursor of bisenoic PGs) stimulate the acrosome reaction of hamster and guinea pig spermatozoa (Meizel and Turner, 1983, 1984; Joyce e t al., 1987). PGE, increased the acrosome reaction of guinea pig spermatozoa initiated by Ca2’ (Joyce et al., 1987). Inhibitors of cyclooxygenase activity have been reported to decrease significantly the acrosome reaction of hamster and guinea pig spermatozoa induced by AA and Ca2+,respectively; the inhibition did not occur in the presence of PGE, and PGF,, (Meizel and Turner, 1984; Joyce et al., 1987). Administration of cyclooxygenase inhibitors to female mice (Lau e t al., 1973) and rabbits (Lau and Saksena, 1979) affected the fertilizing capacity of spermatozoa in vivo, and this was reversed by PGs. Cyclooxygenase inhibitors also reduced the fertilizing capacity of mouse spermatozoa in vitro (Joyce e t al., 1987). Moreover, it has been found that PGs of the E series have the ability to enhance the capacity of human spermatozoa €or penetration of zona-free hamster oocytes (Aitken and Kelly, 1985; Aitken et al., 1986). It is believed that calcium ion influx is a n essential step in the mammalian sperm acrosome reaction (Yanagimachi, 1988) and that calcium ionophores enhance the reaction (Joyce et al., 1987). PGs may act primarily a s ionophores to facilitate the movement of Ca2+ through lipoprotein membranes and thus have a role in the exocytotic event (Meizel and Turner, 1983; Aitken e t al., 1986; Joyce et al., 1987). The present study in which significant amounts of cyclooxygenase activity have been found in human sperm cells provides further support for the essential role of PGs in the sperm acrosome reaction and fertilization. Finally, we have found (unpublished data) a similar formation of various lipoxygenase products (Hall and Behrman, 1982; Needleman et al., 1986) by human spermatozoa in vitro. Some of these AA metabolites
305
have already been shown to be important for the mammalian acrosome reaction (Meizel and Turner, 1984; Basuray et al., 1990).
ACKNOWLEDGMENTS We thank Mdm. Tan Suan Mian and Mdm. Ho Lai Meng for their excellent technical assistance.
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