12
BBALIP
Biochrmica
et Biaphywu
Acta, 1043 (1990) 12- 18 Elsevier
53329
Role of lipoxygenase in the mechanism of acrosome reaction in mammalian spermatozoa Y. Lax, S. Grossman,
S. Rubinstein,
Health Sciences Research Center, Department
ofLife Science, Bar-Ilan Uniuersity, Ramut-Gun (Israel)
(Received
Key words:
Acrosome
reaction;
Acrosin;
N. Magid and H. Breitbart
5 October
Calcium:
1989)
Lipoxygenase;
Calcium
ionophore;
(Spermatozoa)
acrosome reaction (AR) in bull spermatozoa was induced by the Ca*+ -ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca*+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 PM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20,40 or 80 pg/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 pg/ml, respectively. Under identical conditions, 100 PM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 19HETE, the classical product of 15lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa. The
Introduction The acrosome reaction in mammalian spermatozoa essential to fertilization and occurs in sperm that have first undergone a series of events known as capacitation [1.2]. The Ca’+-dependent breakdown and fusion of the outer acrosomal membrane and the overlying plasma membrane are major events in the acrosome reaction and result in exocytosis of soluble acrosome content [1,3]. The acrosome reaction seems to be required for both the penetration of the zona pellucida by sperm and
Abbreviations: AR, acrosome reaction; BHT, butylated hydroxytoluene: I5-HETE, 15-hydroxy-5,8,11,13-eicosatetraenoic acid: NDGA. nordihydroguaiaretic acid; PC12, dilauroylphosphatidylcholine: 15-HPETE, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid: BAEE. benzoylarginine ethyl ester; ETYA, eicosatetraynoic acid. Correspondence: H. Breitbart, Health Sciences Research Center, Department of Life Science, Bar-Ban University, Ramat-Gran 52100.
I wael 0005.2760/90/$03.50
“, 1990 Elsevier Science Publishers
B.V. (Biomedical
the fusion with the oocyte plasma membrane. In somatic cells, metabolism of arachidonic acid via the cyclooxygenase pathway leads to prostaglandins, prostacyclins and thromboxanes [4] and metabolism involving the lipoxygenase pathway leads to hydroperoxyand hydroxyeicosatetraenoic acids and to leukotrienes [5]. The products of the oxidative metabolism of arachidonic acid appear to be important for exocytosis in somatic cells [6]. Studies by some investigators [7-131 suggest that phospholipase A, and the products from its hydrolysis of membrane phospholipids (lysophospholipids and arachidonic acid) are involved in the hamster and guinea-pig sperm acrosome reaction. Inhibitors of cycle-oxygenase and lipoxygenase pathways inhibit the acrosome reaction of hamster and guinea-pig sperm and products of these enzymes stimulate the acrosome reaction when added to capacitated sperm [14,15]. It was shown that 15-lipoxygenase is involved in arachidonic acid metabolism in ram tests [16]. The data in the present study indicate, to our knowledge for the Division)
13 first time, the involvement of 15lipoxygenase in the mechanism of acrosome reaction in bovine spermatozoa. This work was presented in preliminary form at the 21st Meeting of the American Society for the Study of Reproduction (1988). Materials and Methods Sperm preparation. Frozen bull sperm cells were thawed at 37” C by taking one frozen capsule (5 +10’ cells) into 0.875 ml of medium comprising 150 mM NaCl, 10 mM histidine (pH 7.4). The cells were washed by three centrifugations at 780 X g, at 25 o C for 10 min. The washed cells were resuspended in TALP medium [17] to reach a final concentration of 1 . lo* cells/ml. Acrosin release and calcium uptake. Incubation of cells, acrosin activity and 45Ca accumulation in the cells were essentially performed as described by us earlier
“0
1.0
2.0
30
40
5.0
mM Co’2 Fig. 1. Effect of extracellular Ca2+ concentration on acrosin release. Sperm cells (lO’/ml) were incubated in TALP medium which contained increased concentrations of Ca2+. Acrosin release, induced by 2 pM A23187, was determined after 1 h (0) or 3 h (0) of incubation, Each point represents the mean f S.E. of duplicates from three experiments (P < 0.005).
W31. Determination of lipoxygenase products. Sperm cells (5 . 108/ml) were incubated for 3 h at 37” C in TALP medium containing 5.0 PCi [l-‘4C]arachidonic acid spec. act. (56 mCi/mmol). The reaction was stopped by adding 10% formic acid to get pH 4.0, then extracted with twice the sample volume of diethyl ether. The organic phases were combined, dried over anhydrous Na,SO, and evaporated under reduced pressure. The residue was subsequently taken in a small volume of ether and dried under N,. The residue was dissolved in 0.1 ml of diethyl ether and separated by thin-layer chromatography using petroleum ether/diethyl ether/ acetic acid (100 : 50 : 1, v/v) as solvents [16]. The plates were dried and autoradiographed with Curix RP-2 Agfa film with an exposure time of lo-14 days. The film was developed and the spots in each lane were scraped, dissolved in scintillation fluid and counted in Contron liquid scintillation spectrometer. Materials. A23187 (Calbiochem) was dissolved in dimethylformamide/ethanol (3 : 1, v/v). PC12 liposomes (Sigma) were prepared as described earlier [17]. Arachidonic acid (Sigma) was dissolved in chloroform/ methanol (2 : 1, v/v) and kept at - 80” C. Before use, the solvent was removed by N, and the residue was dissolved in TALP to get 0.5 mg/ml. NDGA, BHT, 15-HETE and indomethacin were purchased from Sigma, 15HPETE and ETYA from Cayman, flufenamic acid from Aldrich and radiochemicals from Amersham. Control tubes always contained equivalent volume of solvent.
ency of acrosin release on extracellular Ca*+ concentrations. It can be seen that the optimal Ca*+ concentration for acrosin release is between 2 and 5 mM. It has been shown before that phospholipids like phosphatidylcholinedilauroyl (PC12) can stimulate the occurrence of the acrosome reaction in bull spermatozoa as detected by staining and by penetration into zona-free hamster eggs [17]. In the present study, the acrosome reaction was determined by following acrosin release from the cells; it was thus essential to see whether this phospholipid can stimulate the release of acrosin. It can be seen in Fig. 2 that acrosin release is enhanced by increased concentrations of PC12. Acrosome reaction in hamster sperm is stimulated by arachidonic acid [14]. In Fig. 3 it is shown that
500 ln
z
I
0
400-
I
,E =
300-
/;p---_
The acrosome reaction in washed bull spermatozoa was induced by Ca2+ and the Ca2+-ionophore A23187. The acrosome reaction is defined in the literature as a Ca2+-dependent process. In Fig. 1 we show the depend-
,a,
--._ 1
p
ti $ 5 E c
IQ zoo-
j/ /‘/
looI/ 0
Results
7
*@.c
/-
//
. . .0 ~,,,,.,,....,..~.....~.~~ ,.,.........'i' I I I I
2
3
4
TIME (h) Fig. 2. Effect of PC12 on acrosin release. Sperm cells (lO*/ml) were incubated in TALP medium in the presence of three concentrations of PC12 liposomes and 2 mM Ca2+. Acrosin released from the cells was determined during 4 h of incubation. The symbols are: control without PC12, 0; 90 nM PC12, 0; 180 pM PC12, 0 and 360 nM PC12, H. n = 3.
14 1000
I
I
I 1
=” s
I
750-
w$ ,;
500-
:
I I 2
I 3
L
I
1
IO*
0
TIME (h) Fig. 3. Effect of arachidonic acid on acrosin release. Sperm cells (lox/ml) were incubated in TALP medium containing 20 pg/ml arachidonic acid. Acrosin released from the cells was determined during 3 h of incubation in the presence of 2 mM Ca*+, (m), or in the absence of Ca2+ plus 0.5 mM EGTA, (0). n = 4.
arachidonic acid stimulate acrosin release from bull sperm. No indication of AR could be found with 20 pgg/ml linoleic acid (data not shown). The effects of PC12 and arachidonic acid on acrosin release, are both Ca’+-dependent (data not shown). PC12 causes small stimulation in acrosin release in absence of extracellular Ca’+. In order to find out whether arachidonic acid metabolism is involved in the mechanism of acrosome reaction, we studied the effect of lipoxygenase and cycle-oxygenase inhibitors. Nordihydroguaiaretic acid (NDGA), which inhibits the two pathways, but is more specific to the lipoxygenase pathway causes inhibition of acrosin release that was induced under various conditions (Fig. 4). NDGA (10 PM) causes 32%, 43%, 45% and 28% inhibition in the presence of Ca2+ alone, Ca2+ plus A23187, PC12 or arachidonic acid, respectively. Eicosatetraynoic acid (ETYA), which is an analogue of arachidonic acid, inhibits acrosin release that was induced by A23187 or arachidonic acid (Table I). When AR is induced by arachidonic acid, the system is more sensitive to inhibition by ETYA in comparison to the A23187 induced cells. In the presence of 2 pg/ml ETYA there is 0% or 36% inhibition of AR induced by A23187 or arachidonic acid, respectively. When 20 pg/ml ETYA was used, there is 17% or 58% inhibition of AR induced by A23187 or arachidonic acid, respectively. ETYA itself could not induce acrosin release under conditions in which arachidonic acid does. Indomethacin, an inhibitor of the cycle-oxygenase activity, showed no inhibition of the acrosome reaction induced by A23187, PC12 or by arachidonic acid. In fact, with IOOpM indomethacin, we can see 30-35% stimulation of acrosin release in the presence of A23187 or pC12. The effect of propyl gallate and butylated hydroxy-
I
10-S
10-4
NDGA (Ml Fig. 4. Effect of NDGA concentrations on acrosin release. Sperm cells (lOx/ml) were incubated in TALP containing 2 mM Ca*+ for 3 h, with increased concentrations of NDGA. Acrosin release was induced by: Ca*+ only, (0); 2 pM A23187, (0); 360 PM PC12, (0) and 20 pg/ml arachidonic acid, (m). Each point represents the mean + SE. of duplicates from three to five experiments (P i 0.005 for 10e4. 10e5 M NDGA and P i 0.025 for 10Uh M MDGA).
toluene, which inhibit the lipoxygenase and cyclooxygenase pathways, and flufenamic acid, which inhibits the cycle-oxygenase, on acrosin release is demonstrated in Fig. 5. Very small inhibition, if any, was found with butylated hydroxytoluene. Propyl-gallate and flufenamic acid at 100 PM cause 40% and 30% inhibition, respectively. In our recent paper, we had shown an increase in calcium accumulation in ram spermatozoa under capacitation conditions [18]. The data in the present paper (Fig. 6) show an increase in Ca2+ uptake with time. In the presence of the Ca ’ ‘-ionophore, A231 87, Ca2+ accumulation is about twice as high in comparison to the control without ionophore. These results are in good
TABLE
I
Effeci of indomethocin und ETYA
on acrosin release
Sperm cells (lOa/ml) were incubated in TALP containing 2 mM Ca*+ for 3 h with increased concentrations of indomethin. Acrosin release was induced by 2 PM A23187,0.36 mM PC12 or 20 pg/ml arachidonic acid. Each point represents the mean * SE. of duplicates from three experiments. The 100% acrosin activity is 395, 500 and 750 nmol BAEE/min per 10’ cells in the presence of A23187, PC12 or arachidonic acid, respectively. Inhibitor
added
None Indomethacin Indomethacin Indomethacin ETYA ETYA ETYA ETYA
Acrosin
(1 PM) (10 pM) (100 PM) (2 (20 (40 (80
pg/ml) pg/ml) pg/ml) pg/ml)
activity
(8)
A23187
PC12
arachidonic
100 89+ 4 88i 5 135514 107+ 5 83*6 39*4 23+2
100 100+5 85*3 130+8 -
100 10055 64k2 42+2 _ _
acid
15
TIME
INHIBITOR
(M)
Fig. 5. Effect of various inhibitors of arachidonic acid metabolism on acrosin release. Sperm cells (lO’/ml) were incubated in TALP containing 2 mM Ca2+ and 2 pM A23187, for 3 h with increased concentration of the inhibitor. The inhibitors used are: NDGA, (0); flufenamic acid, (0); propyl-gallate, (m), and butylated hydroxytoluene, (0). Each point represents the mean f S.E. of duplicates from three experiments.
correlation with acrosin release data (see Fig. 1). When the acrosome reaction was induced by PC12, the rate of CaZf accumulation was identical to the control without the phospholipid. There was no effect of the inhibitor NDGA on ca2+ accumulation when the acrosome reaction was induced by A23187 or PC12 (Fig. 7). The products of the lipoxygenase activity were quantitatively analyzed after incubating the cells with [‘4C]arachidonic acid and separating the products on thin-layer chromatography. The autoradiogram pattern revealed four migrated bands of products and a residual in the origin (Fig. 8).
(min)
Fig. 7. Effect of NDGA on calcium accumulation by the cells. For details, see legend to Fig. 6. The upper lines represent Ca2+ accumulation in the presence of 2 uM A23187 and the lower lines with 360 PM PC12. The closed circles are the controls and the open circles are in the presence of 0.1 mM NDGA.
The product with R, = 0.69 was identified as 15-HETE by normal phase, high-pressure liquid chromatography [16,19]. It should be pointed out that although there is some evidence for trace amounts of 12-HETE, we could not detect any traces of 5-HETE or 5-HPETE. Analysis of the autoradiogram (see Fig. 9) revealed that most of the products (90%) were located in the origin. From the pattern of the migrated products, the major product is with R, = 0.85, followed by a second product with R, = 0.81, followed by the band of 15-HETE. The highest levels of products were detected in cells that were incubated with Ca2+ plus A23187. Incubation in
A
A
B
arachidona te c
EO-
TIME (min) Fig. 6. Calcium accumulation by the cells. Sperm cells (lOs/ml) were incubated in medium containing 2 mM Ca2+ and 5 pCi/ml 45Ca2+. At time intervals, 0.1 ml aliquots were removed and vacuum-filtered. The radioactivity trapped on the washed filters was counted. The conditions are: control or 360 PM PC12, (0); 2 pM A23187, (m). Each point represents the mean + SE. of duplicates from five experiments, after subtracting the zero-time Ca2+ uptake of 7.6 rfr 1.3 nmol Ca/lO” cells (P < 0.005).
origin -b Fig. 8. Autoradiogram of lipoxygenase products. Sperm cells (5. lOs/ml) were incubated as described in Materials and Methods, in the presence of 2 mM Ca2+ and 2 nM A23187. (A) Control; (B) Standard arachidonic acid incubated without cells.
16 TABLE
II
Effect of ISHETE
and IS-HPETE
on acrosin release
from the cells
Sperm cells (lOn/ml) were incubated in TALP containing of incubation. The solvent of 15HETE and 15HPETE; three experiments. Concentration of NDGA is 0.1 mM. Incubation conditions
2 mM Ca*+ and 2 PM A23187. Acrosin released from the cells was determined after 3 h methanol/water, had no effect. Each point represents the meanf SE. of duplicates from
A23187
15HETE
(15-HPETE)
(2 PM)
&g/ml)
(pg/ml)
Control Control NDGA NDGA NDGA NDGA NDGA NDGA Control Control
1.0 _ 0.4 0.9 1.8 _ _ _
_
1.8 3.6 _ _
1.0
the presence of NDGA revealed 38%, 69%, 48% and 63% inhibition in products located in the origin, R, = 0.69, R, = 0.81 and R, = 0.85, respectively. We have tried to eliminate the inhibitory effect of NDGA on acrosome reaction, by adding 15-HETE, 15HPETE, or 1Slipoxygenase products into the incubation medium. The data in Table II indicate that 90% and 85% of acrosin release activity that was inhibited by NDGA is recovered by adding back 1.8 pgg/ml 15-HETE. By adding 0.4 or 0.9 pg/ml of 15HETE, the recovery is 51% and 66%, respectively. The addition of 15-HETE to the cell-suspension in absence of A23187, or in the presence of A23187, but without NDGA did not affect acrosin release.
ORIGIN
0.61
0.69
LIPOXYGENASE
ACTIVITY
PRODUCTS
0.65
(I+)
Fig. 9. Analysis of lipoxygenase products after thin-layer chromatography. See details in Materials and Methods and Fig. 8. Sperm cells were incubated under various conditions: 2 mM Ca*+ and 2 ,uM A23187, (white); 2 mM Ca’+, 2 PM A23187 and 0.1 mM NDGA, (light grey); 2 mM Ca*+, 2 PM A23187 and 20 pg/ml arachidonic acid, (circles); 2 mM Ca*+, 2 nM A23187, 20 /.rg/rnl arachidonic acid and 0.1 mM NDGA, (squares); 0.5 mM EGTA, (vertical lines) and 0.5 mM EGTA plus 0.1 mM NDGA, (dark grey). This is a representative experiment which has been repeated three times.
nmol BAEE’ (min~108cells)-’
B Inhibition
365 f 15 37Ok 18
_ _
lOOk 235 + 15 274k 17 340* 10 235 + 10 324 * 28 170*30 16Ok25
73 36 25 7 36 11 _ _
% Recovery _ _ 51 66 90 51 85 _
Discussion
The acrosome of bull spermatozoa is relatively small, therefore, it is difficult to determine quantitatively the occurrence of the acrosome reaction under the light microscope. In our recent paper, it was shown that determination of acrosin release from ram sperm is a reliable quantitative assay to follow the acrosome reaction [18]. The data here, which show the dependency of acrosin release upon extracellular Ca2+ concentration, the stimulation of acrosin release by known acrosome reaction inducers (A23187, PC12 and arachidonic acid) and good correlation with electron microscope study (data not shown), indicate that measurement of acrosin release from bull sperm is a reliable assay to determine the occurrence of the acrosome reaction. Inhibition of the acrosome reaction by the arachidonic acid metabolism inhibitors NDGA, ETYA, butylated hydroxytoluene or propyl gallate and the lack of inhibition by indomethacin indicate that the lipoxygenase, rather than the prostaglandin-synthetase, activity is involved in the mechanism of the acrosome reaction. It was reported elsewhere that acrosome reaction in hamster and guinea-pig sperm is inhibited by indomethacin [14,15] and can be stimulated by adding certain prostaglandins to the incubation medium [14,15,20]. These authors have suggested that prostaglandins stimulate the acrosome reaction by acting as Ca’+-ionophores and directly facilitating the transport of Ca2+ across the sperm-plasma membrane [21]. In the present study, when acrosome reaction was A23187, we did not induced by the Ca 2+-ionophore expected to see any inhibition by indomethacin, if prostaglandins act as Ca 2+-ionophores. Acrosome reaction induced by PC12 or arachidonic acid is also not
17 inhibited by indomethacin, thus, we thought that these two inducers may act as Ca*+-ionophores as well. The data which are concerned with Ca*+ transport (Fig. 6) show high increase in Ca’+ accumulation in the presence of the Ca2+ -tonophore . A23187 and no effect by PC12, indicating that PC12 is not acting as a Ca2+ionophore. In addition, if PC12 stimulates prostaglandin production, it is expected that an increase in Cal’ accumulation as a result of the ionophoric properties of prostaglandins would occur. We conclude that prostaglandin-synthetase activity is not involved in the mechanism of the acrosome reaction in bull spermatozoa. The 30% in~bition of the acrosome reaction found with 100 FM of the cycle-oxygenase inhibitor, flufenamic acid, is probably a non-specific effect, since indomethacin at this concentration shows about 35% stimulation in acrosin release. This stimulation by indomethacin is probably a result of the inhibition of the cycle-oxygenase pathway, which may cause an increase in lipoxygenase products through an increase in available substrate arachidonic acid. It was shown in human neutrophils that indomethacin increased the formation of the lipoxygenase products LTB4 as well as 5-, 12and 15-HETE [22]. In ram spermatozoa, 15-HETE was significantly increased (65%) as a result of the addition of 0.1 mM indomethacin [16]. As mentioned before, PC12 stimulates acrosin release but did not affect Ca*+ accumulation in the cells. Since we do not know the mechanism by which PC12 stimulates the Ca*+-dependent acrosin release, it is difficult to explain the absence of effect on Ca*+ accumulation. Since PC12 has a positive charge, it is possible that the stimulation of the acrosome reaction by PC12 can take place with a relatively low concentration of intracellular Ca*+. This also explains why PC12 shows little stimulation in acrosin release in absence of extracellular CaZ’. The lipoxygenase product with R, = 0.69 was identified as 15-HETE [16,19]. Incubation of sperm cells in the presence of extracellular CaZ+ and A23187, caused about 100% enhancement in the level of 1%HETE (see Fig. 9). The addition of NDGA caused a 69% reduction in 15-HETE and 73% inhibition in acrosin release rate. These data indicate that the metabolism of arachidonic acid via the lipoxygenase pathway plays an important role in the mechanism of the acrosome reaction. The fact that the inhibition of acrosin release by NDGA can be overcome by adding 15-HETE or 15-HPETE further support conclusion. The presence of 15-lipoxygenase and inhibition of 15-HETE production by NDGA has been shown in ram spermatozoa as well [16]. The failure of 15-HETE to stimulate acrosin release in the absence of A23187 and NDGA (see Table II), indicates that 15-HETE as well as high intracellular Ca2+ concentration, are both required for the occurrence of the acrosome reaction. This result also indicates that 15-HETE is not acting as Ca2+ ionophore.
The fact that NDGA inhibits the acrosome reaction in the presence of A23187, conditions by which Ca*+ can enter freely into the cells, indicate as well that the products of the lipoxygenase are not involved in Ca’+ entry into the cell, but rather in other steps of the acrosome reaction mechanism. The data indicate that metabolism of arachidonic acid via the lipoxygenase pathway is an important step in the acrosome reaction. Hydrolytic release of arachidonic acid from cell phospholipids can occur by phospholipase A, [23]. Several studies have suggested that sperm phospholipase A, is involved in the acrosome reaction [7,12,23-251. Since 15-HETE did not affect acrosin release in the presence of A23187, unless NDGA was present (Table II), we conclude that the cells are saturated with 15-HETE and/or other lipoxygenase products under these conditions. We suggest, that increased concentration of intracellular Ca2+, stimulates the phospholipase A, activity, which leads to the release of free arachidonic acid, which is further metabolized by lipoxygenase to 15HETE, 15-HPETE and other products. The mechanism involved in stimulation of membrane fusion by 15HETE is not clear, but the data indicate that intracellular Ca2+ is involved in this mechanism. Esterification of HETE compounds into neutrophil phospholipids can occur and it has been suggested that this event is important for the mechanism of degranulation [26]. It is possible that such esterification, in addition to neutralizing the negative charge of the outer acrosomal and the plasma membranes by Ca’+, are two essential events which lead to the acrosome reaction. It was shown recently in heart cells, that metabohtes of lipoxygenase activate G-protein-gated K’ channel [27,28]. It is possible that such an ion-channel activity is involved in the mechanism of the acrosome reaction and that this channel can be activated by the lipoxygenase products. In a recent study. Oliw and Sprecher [29] have suggested that (n - 6) lipoxygenase activity of human semen is associated with prostate excreted organelles called ‘prostasomes’ and not with the spermatozoa itself. They also showed that the level of 15-HETE is not affected by A23187 and Ca’“. Our data in bull sperm, which show 100% increase in 15HETE (Fig. 9X indicate that our bull-cell preparation differs from the human sperm preparation. The presence of ‘prostasomes’ can be detected by following phosphatase activity and by the electron microscope [30]. This study in our preparation (data not shown) reveals that our sperm preparation is free of ‘prostasomes’.
Acknowledgements We wish to thank D. Kalay and M. Bar-El of Hasherut, Artificial Insemination Center, Hafez-Haim, Israel, for the frozen bull sperm generously supplied by
18
them. We also thank Avrille Goldreich for the careful typing of this manuscript. References 1 Bedford, J.M. (1970) Biol. Reprod. (Suppl.) 2, 128-158. 2 Yanagimachi, R. (1981) in Fertilization and Embryomic Development in vitro (Mastroianni, L. Jr. and Biggers, J.D., eds.), pp. 82-182, Plenum Press, New York. 3 Russell, L., Peterson, R. and Freund, M. (1979) J. Exp. 2001. 208, 41-56. 4 Hall, A.K. and Behrman, H.R. (1982) in Prostaglandins (Lee, J.B., ed.), pp. l-38, Elsevier, New York. 5 Samuelsson, B. (1982) in Leukotrienes and other Lipoxygenase Products (Samuelsson, B. and Paoeletti, R., eds.), pp. l-17, Raven Press, New York. 6 Laychock, S.G. and Putney, J.W. Jr. (1982) in Cellular Regulation of Secretion and Release (Conn, M.P., ed.), pp. 53-105, Academic Press, New York. 7 Dravland, J.E. and Meizel, S. (1982) J. Androl. 3, 388-395. 8 Llanos, M.N., Lui, C.W. and Meizel, S. (1982) J. Exp. Zool. 221, 107-117. 9 Meizel, S. and Turner, K.O. (1983) FEBS Lett. 161, 315-318. 10 Llanos, M.N. and Meizel, S. (1983) Biol. Reprod. 28, 1043-1051. 11 Fleming, A.D. and Yanagimachi (1981) Gamete Res. 4, 253-273. 12 Ono, K., Yanagimachi, R., Huang, T.T.F. (1982) Dev. Growth Differ. 24, 305-310. R. (1982) J. Exp. Zool. 224, 259-263. 13 Ohzu, E. and Yanagimachi,
14 Meizel, S. and Turner, K.O. (1984) J. Exp. Zool. 231, 283-288. 15 Joyce, C.L., Nuzzo, N.A., Wilson, L. Jr. and Zaneveld, L.J.D. (1987) J. Androl. 8, 74-82. 16 Grossman, S., Schon, I., Sofer, Y., Magid, N. and Bartoov, B. (1986) Adv. Prostaglandin, Thromboxane Leuktriene Res. 16. 241-252. 17 Graham, J.K., Foote, R.H. and Parrish, J.J. (1986) Biol. Reprod. 35, 413-424. 18 Ben-Av, P., Rubinstein, S. and Breitbart, H. (1988) Biochim. Biophys. Acta 939, 214-222. 19 Funk, M.O., Issac, R. and Porter, N.A. (1976) Lipids 11, 113-117. 20 Aitken, R.J. and Kelly, R.W. (1985) J. Reprod. Fert. 73, 139-146. 21 Kirtland. S.J. and Baum, H. (1972) Nature 236, 47-49. 22 Docherty, J.C. and Wilson, T.W. (1987) B&hem. Biophys. Res. Commun. 148, 534-538. 23 Irvine, R.F. (1982) J. Biochem. 204, 3-16. 24 Thakkar, J.K., East, .I., Soyler, D. and Franson, R.C. (1983) Biochim. Biophys. Acta 754, 44-50. 25 Lui, M.N. and Meizel, S. (1979) J. Exp. Zoo]. 207, 173-185. 26 Stenson, W.F. and Parker, C.W. (1980) J. Immunol. 124, 2100-2104. 27 Kurachi, Y., Ito, H., Sugimoto, T.. Shimizu, T., Miki, I. and Ui, M. (1989) Nature 337, 555-557. 28 Kim, D., Lewis, D.L., Graziadei, L., Neer, E.J., Bar-Sag, D. and Clapham, D.E. (1989) Nature 337, 557-560. 29 Oliw, E.H. and Sprecher, H. (1989) Biochim. Biophys. Acta 1002, 283-291. 30 Breitbart, H. and Rubinstein, S. (1982) Arch. Androl. 9, 147-157.
BBALIP
Biochrmica
et Biaphywu
Acta, 1043 (1990) 12- 18 Elsevier
53329
Role of lipoxygenase in the mechanism of acrosome reaction in mammalian spermatozoa Y. Lax, S. Grossman,
S. Rubinstein,
Health Sciences Research Center, Department
ofLife Science, Bar-Ilan Uniuersity, Ramut-Gun (Israel)
(Received
Key words:
Acrosome
reaction;
Acrosin;
N. Magid and H. Breitbart
5 October
Calcium:
1989)
Lipoxygenase;
Calcium
ionophore;
(Spermatozoa)
acrosome reaction (AR) in bull spermatozoa was induced by the Ca*+ -ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca*+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 PM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20,40 or 80 pg/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 pg/ml, respectively. Under identical conditions, 100 PM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 19HETE, the classical product of 15lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa. The
Introduction The acrosome reaction in mammalian spermatozoa essential to fertilization and occurs in sperm that have first undergone a series of events known as capacitation [1.2]. The Ca’+-dependent breakdown and fusion of the outer acrosomal membrane and the overlying plasma membrane are major events in the acrosome reaction and result in exocytosis of soluble acrosome content [1,3]. The acrosome reaction seems to be required for both the penetration of the zona pellucida by sperm and
Abbreviations: AR, acrosome reaction; BHT, butylated hydroxytoluene: I5-HETE, 15-hydroxy-5,8,11,13-eicosatetraenoic acid: NDGA. nordihydroguaiaretic acid; PC12, dilauroylphosphatidylcholine: 15-HPETE, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid: BAEE. benzoylarginine ethyl ester; ETYA, eicosatetraynoic acid. Correspondence: H. Breitbart, Health Sciences Research Center, Department of Life Science, Bar-Ban University, Ramat-Gran 52100.
I wael 0005.2760/90/$03.50
“, 1990 Elsevier Science Publishers
B.V. (Biomedical
the fusion with the oocyte plasma membrane. In somatic cells, metabolism of arachidonic acid via the cyclooxygenase pathway leads to prostaglandins, prostacyclins and thromboxanes [4] and metabolism involving the lipoxygenase pathway leads to hydroperoxyand hydroxyeicosatetraenoic acids and to leukotrienes [5]. The products of the oxidative metabolism of arachidonic acid appear to be important for exocytosis in somatic cells [6]. Studies by some investigators [7-131 suggest that phospholipase A, and the products from its hydrolysis of membrane phospholipids (lysophospholipids and arachidonic acid) are involved in the hamster and guinea-pig sperm acrosome reaction. Inhibitors of cycle-oxygenase and lipoxygenase pathways inhibit the acrosome reaction of hamster and guinea-pig sperm and products of these enzymes stimulate the acrosome reaction when added to capacitated sperm [14,15]. It was shown that 15-lipoxygenase is involved in arachidonic acid metabolism in ram tests [16]. The data in the present study indicate, to our knowledge for the Division)
13 first time, the involvement of 15lipoxygenase in the mechanism of acrosome reaction in bovine spermatozoa. This work was presented in preliminary form at the 21st Meeting of the American Society for the Study of Reproduction (1988). Materials and Methods Sperm preparation. Frozen bull sperm cells were thawed at 37” C by taking one frozen capsule (5 +10’ cells) into 0.875 ml of medium comprising 150 mM NaCl, 10 mM histidine (pH 7.4). The cells were washed by three centrifugations at 780 X g, at 25 o C for 10 min. The washed cells were resuspended in TALP medium [17] to reach a final concentration of 1 . lo* cells/ml. Acrosin release and calcium uptake. Incubation of cells, acrosin activity and 45Ca accumulation in the cells were essentially performed as described by us earlier
“0
1.0
2.0
30
40
5.0
mM Co’2 Fig. 1. Effect of extracellular Ca2+ concentration on acrosin release. Sperm cells (lO’/ml) were incubated in TALP medium which contained increased concentrations of Ca2+. Acrosin release, induced by 2 pM A23187, was determined after 1 h (0) or 3 h (0) of incubation, Each point represents the mean f S.E. of duplicates from three experiments (P < 0.005).
W31. Determination of lipoxygenase products. Sperm cells (5 . 108/ml) were incubated for 3 h at 37” C in TALP medium containing 5.0 PCi [l-‘4C]arachidonic acid spec. act. (56 mCi/mmol). The reaction was stopped by adding 10% formic acid to get pH 4.0, then extracted with twice the sample volume of diethyl ether. The organic phases were combined, dried over anhydrous Na,SO, and evaporated under reduced pressure. The residue was subsequently taken in a small volume of ether and dried under N,. The residue was dissolved in 0.1 ml of diethyl ether and separated by thin-layer chromatography using petroleum ether/diethyl ether/ acetic acid (100 : 50 : 1, v/v) as solvents [16]. The plates were dried and autoradiographed with Curix RP-2 Agfa film with an exposure time of lo-14 days. The film was developed and the spots in each lane were scraped, dissolved in scintillation fluid and counted in Contron liquid scintillation spectrometer. Materials. A23187 (Calbiochem) was dissolved in dimethylformamide/ethanol (3 : 1, v/v). PC12 liposomes (Sigma) were prepared as described earlier [17]. Arachidonic acid (Sigma) was dissolved in chloroform/ methanol (2 : 1, v/v) and kept at - 80” C. Before use, the solvent was removed by N, and the residue was dissolved in TALP to get 0.5 mg/ml. NDGA, BHT, 15-HETE and indomethacin were purchased from Sigma, 15HPETE and ETYA from Cayman, flufenamic acid from Aldrich and radiochemicals from Amersham. Control tubes always contained equivalent volume of solvent.
ency of acrosin release on extracellular Ca*+ concentrations. It can be seen that the optimal Ca*+ concentration for acrosin release is between 2 and 5 mM. It has been shown before that phospholipids like phosphatidylcholinedilauroyl (PC12) can stimulate the occurrence of the acrosome reaction in bull spermatozoa as detected by staining and by penetration into zona-free hamster eggs [17]. In the present study, the acrosome reaction was determined by following acrosin release from the cells; it was thus essential to see whether this phospholipid can stimulate the release of acrosin. It can be seen in Fig. 2 that acrosin release is enhanced by increased concentrations of PC12. Acrosome reaction in hamster sperm is stimulated by arachidonic acid [14]. In Fig. 3 it is shown that
500 ln
z
I
0
400-
I
,E =
300-
/;p---_
The acrosome reaction in washed bull spermatozoa was induced by Ca2+ and the Ca2+-ionophore A23187. The acrosome reaction is defined in the literature as a Ca2+-dependent process. In Fig. 1 we show the depend-
,a,
--._ 1
p
ti $ 5 E c
IQ zoo-
j/ /‘/
looI/ 0
Results
7
*@.c
/-
//
. . .0 ~,,,,.,,....,..~.....~.~~ ,.,.........'i' I I I I
2
3
4
TIME (h) Fig. 2. Effect of PC12 on acrosin release. Sperm cells (lO*/ml) were incubated in TALP medium in the presence of three concentrations of PC12 liposomes and 2 mM Ca2+. Acrosin released from the cells was determined during 4 h of incubation. The symbols are: control without PC12, 0; 90 nM PC12, 0; 180 pM PC12, 0 and 360 nM PC12, H. n = 3.
14 1000
I
I
I 1
=” s
I
750-
w$ ,;
500-
:
I I 2
I 3
L
I
1
IO*
0
TIME (h) Fig. 3. Effect of arachidonic acid on acrosin release. Sperm cells (lox/ml) were incubated in TALP medium containing 20 pg/ml arachidonic acid. Acrosin released from the cells was determined during 3 h of incubation in the presence of 2 mM Ca*+, (m), or in the absence of Ca2+ plus 0.5 mM EGTA, (0). n = 4.
arachidonic acid stimulate acrosin release from bull sperm. No indication of AR could be found with 20 pgg/ml linoleic acid (data not shown). The effects of PC12 and arachidonic acid on acrosin release, are both Ca’+-dependent (data not shown). PC12 causes small stimulation in acrosin release in absence of extracellular Ca’+. In order to find out whether arachidonic acid metabolism is involved in the mechanism of acrosome reaction, we studied the effect of lipoxygenase and cycle-oxygenase inhibitors. Nordihydroguaiaretic acid (NDGA), which inhibits the two pathways, but is more specific to the lipoxygenase pathway causes inhibition of acrosin release that was induced under various conditions (Fig. 4). NDGA (10 PM) causes 32%, 43%, 45% and 28% inhibition in the presence of Ca2+ alone, Ca2+ plus A23187, PC12 or arachidonic acid, respectively. Eicosatetraynoic acid (ETYA), which is an analogue of arachidonic acid, inhibits acrosin release that was induced by A23187 or arachidonic acid (Table I). When AR is induced by arachidonic acid, the system is more sensitive to inhibition by ETYA in comparison to the A23187 induced cells. In the presence of 2 pg/ml ETYA there is 0% or 36% inhibition of AR induced by A23187 or arachidonic acid, respectively. When 20 pg/ml ETYA was used, there is 17% or 58% inhibition of AR induced by A23187 or arachidonic acid, respectively. ETYA itself could not induce acrosin release under conditions in which arachidonic acid does. Indomethacin, an inhibitor of the cycle-oxygenase activity, showed no inhibition of the acrosome reaction induced by A23187, PC12 or by arachidonic acid. In fact, with IOOpM indomethacin, we can see 30-35% stimulation of acrosin release in the presence of A23187 or pC12. The effect of propyl gallate and butylated hydroxy-
I
10-S
10-4
NDGA (Ml Fig. 4. Effect of NDGA concentrations on acrosin release. Sperm cells (lOx/ml) were incubated in TALP containing 2 mM Ca*+ for 3 h, with increased concentrations of NDGA. Acrosin release was induced by: Ca*+ only, (0); 2 pM A23187, (0); 360 PM PC12, (0) and 20 pg/ml arachidonic acid, (m). Each point represents the mean + SE. of duplicates from three to five experiments (P i 0.005 for 10e4. 10e5 M NDGA and P i 0.025 for 10Uh M MDGA).
toluene, which inhibit the lipoxygenase and cyclooxygenase pathways, and flufenamic acid, which inhibits the cycle-oxygenase, on acrosin release is demonstrated in Fig. 5. Very small inhibition, if any, was found with butylated hydroxytoluene. Propyl-gallate and flufenamic acid at 100 PM cause 40% and 30% inhibition, respectively. In our recent paper, we had shown an increase in calcium accumulation in ram spermatozoa under capacitation conditions [18]. The data in the present paper (Fig. 6) show an increase in Ca2+ uptake with time. In the presence of the Ca ’ ‘-ionophore, A231 87, Ca2+ accumulation is about twice as high in comparison to the control without ionophore. These results are in good
TABLE
I
Effeci of indomethocin und ETYA
on acrosin release
Sperm cells (lOa/ml) were incubated in TALP containing 2 mM Ca*+ for 3 h with increased concentrations of indomethin. Acrosin release was induced by 2 PM A23187,0.36 mM PC12 or 20 pg/ml arachidonic acid. Each point represents the mean * SE. of duplicates from three experiments. The 100% acrosin activity is 395, 500 and 750 nmol BAEE/min per 10’ cells in the presence of A23187, PC12 or arachidonic acid, respectively. Inhibitor
added
None Indomethacin Indomethacin Indomethacin ETYA ETYA ETYA ETYA
Acrosin
(1 PM) (10 pM) (100 PM) (2 (20 (40 (80
pg/ml) pg/ml) pg/ml) pg/ml)
activity
(8)
A23187
PC12
arachidonic
100 89+ 4 88i 5 135514 107+ 5 83*6 39*4 23+2
100 100+5 85*3 130+8 -
100 10055 64k2 42+2 _ _
acid
15
TIME
INHIBITOR
(M)
Fig. 5. Effect of various inhibitors of arachidonic acid metabolism on acrosin release. Sperm cells (lO’/ml) were incubated in TALP containing 2 mM Ca2+ and 2 pM A23187, for 3 h with increased concentration of the inhibitor. The inhibitors used are: NDGA, (0); flufenamic acid, (0); propyl-gallate, (m), and butylated hydroxytoluene, (0). Each point represents the mean f S.E. of duplicates from three experiments.
correlation with acrosin release data (see Fig. 1). When the acrosome reaction was induced by PC12, the rate of CaZf accumulation was identical to the control without the phospholipid. There was no effect of the inhibitor NDGA on ca2+ accumulation when the acrosome reaction was induced by A23187 or PC12 (Fig. 7). The products of the lipoxygenase activity were quantitatively analyzed after incubating the cells with [‘4C]arachidonic acid and separating the products on thin-layer chromatography. The autoradiogram pattern revealed four migrated bands of products and a residual in the origin (Fig. 8).
(min)
Fig. 7. Effect of NDGA on calcium accumulation by the cells. For details, see legend to Fig. 6. The upper lines represent Ca2+ accumulation in the presence of 2 uM A23187 and the lower lines with 360 PM PC12. The closed circles are the controls and the open circles are in the presence of 0.1 mM NDGA.
The product with R, = 0.69 was identified as 15-HETE by normal phase, high-pressure liquid chromatography [16,19]. It should be pointed out that although there is some evidence for trace amounts of 12-HETE, we could not detect any traces of 5-HETE or 5-HPETE. Analysis of the autoradiogram (see Fig. 9) revealed that most of the products (90%) were located in the origin. From the pattern of the migrated products, the major product is with R, = 0.85, followed by a second product with R, = 0.81, followed by the band of 15-HETE. The highest levels of products were detected in cells that were incubated with Ca2+ plus A23187. Incubation in
A
A
B
arachidona te c
EO-
TIME (min) Fig. 6. Calcium accumulation by the cells. Sperm cells (lOs/ml) were incubated in medium containing 2 mM Ca2+ and 5 pCi/ml 45Ca2+. At time intervals, 0.1 ml aliquots were removed and vacuum-filtered. The radioactivity trapped on the washed filters was counted. The conditions are: control or 360 PM PC12, (0); 2 pM A23187, (m). Each point represents the mean + SE. of duplicates from five experiments, after subtracting the zero-time Ca2+ uptake of 7.6 rfr 1.3 nmol Ca/lO” cells (P < 0.005).
origin -b Fig. 8. Autoradiogram of lipoxygenase products. Sperm cells (5. lOs/ml) were incubated as described in Materials and Methods, in the presence of 2 mM Ca2+ and 2 nM A23187. (A) Control; (B) Standard arachidonic acid incubated without cells.
16 TABLE
II
Effect of ISHETE
and IS-HPETE
on acrosin release
from the cells
Sperm cells (lOn/ml) were incubated in TALP containing of incubation. The solvent of 15HETE and 15HPETE; three experiments. Concentration of NDGA is 0.1 mM. Incubation conditions
2 mM Ca*+ and 2 PM A23187. Acrosin released from the cells was determined after 3 h methanol/water, had no effect. Each point represents the meanf SE. of duplicates from
A23187
15HETE
(15-HPETE)
(2 PM)
&g/ml)
(pg/ml)
Control Control NDGA NDGA NDGA NDGA NDGA NDGA Control Control
1.0 _ 0.4 0.9 1.8 _ _ _
_
1.8 3.6 _ _
1.0
the presence of NDGA revealed 38%, 69%, 48% and 63% inhibition in products located in the origin, R, = 0.69, R, = 0.81 and R, = 0.85, respectively. We have tried to eliminate the inhibitory effect of NDGA on acrosome reaction, by adding 15-HETE, 15HPETE, or 1Slipoxygenase products into the incubation medium. The data in Table II indicate that 90% and 85% of acrosin release activity that was inhibited by NDGA is recovered by adding back 1.8 pgg/ml 15-HETE. By adding 0.4 or 0.9 pg/ml of 15HETE, the recovery is 51% and 66%, respectively. The addition of 15-HETE to the cell-suspension in absence of A23187, or in the presence of A23187, but without NDGA did not affect acrosin release.
ORIGIN
0.61
0.69
LIPOXYGENASE
ACTIVITY
PRODUCTS
0.65
(I+)
Fig. 9. Analysis of lipoxygenase products after thin-layer chromatography. See details in Materials and Methods and Fig. 8. Sperm cells were incubated under various conditions: 2 mM Ca*+ and 2 ,uM A23187, (white); 2 mM Ca’+, 2 PM A23187 and 0.1 mM NDGA, (light grey); 2 mM Ca*+, 2 PM A23187 and 20 pg/ml arachidonic acid, (circles); 2 mM Ca*+, 2 nM A23187, 20 /.rg/rnl arachidonic acid and 0.1 mM NDGA, (squares); 0.5 mM EGTA, (vertical lines) and 0.5 mM EGTA plus 0.1 mM NDGA, (dark grey). This is a representative experiment which has been repeated three times.
nmol BAEE’ (min~108cells)-’
B Inhibition
365 f 15 37Ok 18
_ _
lOOk 235 + 15 274k 17 340* 10 235 + 10 324 * 28 170*30 16Ok25
73 36 25 7 36 11 _ _
% Recovery _ _ 51 66 90 51 85 _
Discussion
The acrosome of bull spermatozoa is relatively small, therefore, it is difficult to determine quantitatively the occurrence of the acrosome reaction under the light microscope. In our recent paper, it was shown that determination of acrosin release from ram sperm is a reliable quantitative assay to follow the acrosome reaction [18]. The data here, which show the dependency of acrosin release upon extracellular Ca2+ concentration, the stimulation of acrosin release by known acrosome reaction inducers (A23187, PC12 and arachidonic acid) and good correlation with electron microscope study (data not shown), indicate that measurement of acrosin release from bull sperm is a reliable assay to determine the occurrence of the acrosome reaction. Inhibition of the acrosome reaction by the arachidonic acid metabolism inhibitors NDGA, ETYA, butylated hydroxytoluene or propyl gallate and the lack of inhibition by indomethacin indicate that the lipoxygenase, rather than the prostaglandin-synthetase, activity is involved in the mechanism of the acrosome reaction. It was reported elsewhere that acrosome reaction in hamster and guinea-pig sperm is inhibited by indomethacin [14,15] and can be stimulated by adding certain prostaglandins to the incubation medium [14,15,20]. These authors have suggested that prostaglandins stimulate the acrosome reaction by acting as Ca’+-ionophores and directly facilitating the transport of Ca2+ across the sperm-plasma membrane [21]. In the present study, when acrosome reaction was A23187, we did not induced by the Ca 2+-ionophore expected to see any inhibition by indomethacin, if prostaglandins act as Ca 2+-ionophores. Acrosome reaction induced by PC12 or arachidonic acid is also not
17 inhibited by indomethacin, thus, we thought that these two inducers may act as Ca*+-ionophores as well. The data which are concerned with Ca*+ transport (Fig. 6) show high increase in Ca’+ accumulation in the presence of the Ca2+ -tonophore . A23187 and no effect by PC12, indicating that PC12 is not acting as a Ca2+ionophore. In addition, if PC12 stimulates prostaglandin production, it is expected that an increase in Cal’ accumulation as a result of the ionophoric properties of prostaglandins would occur. We conclude that prostaglandin-synthetase activity is not involved in the mechanism of the acrosome reaction in bull spermatozoa. The 30% in~bition of the acrosome reaction found with 100 FM of the cycle-oxygenase inhibitor, flufenamic acid, is probably a non-specific effect, since indomethacin at this concentration shows about 35% stimulation in acrosin release. This stimulation by indomethacin is probably a result of the inhibition of the cycle-oxygenase pathway, which may cause an increase in lipoxygenase products through an increase in available substrate arachidonic acid. It was shown in human neutrophils that indomethacin increased the formation of the lipoxygenase products LTB4 as well as 5-, 12and 15-HETE [22]. In ram spermatozoa, 15-HETE was significantly increased (65%) as a result of the addition of 0.1 mM indomethacin [16]. As mentioned before, PC12 stimulates acrosin release but did not affect Ca*+ accumulation in the cells. Since we do not know the mechanism by which PC12 stimulates the Ca*+-dependent acrosin release, it is difficult to explain the absence of effect on Ca*+ accumulation. Since PC12 has a positive charge, it is possible that the stimulation of the acrosome reaction by PC12 can take place with a relatively low concentration of intracellular Ca*+. This also explains why PC12 shows little stimulation in acrosin release in absence of extracellular CaZ’. The lipoxygenase product with R, = 0.69 was identified as 15-HETE [16,19]. Incubation of sperm cells in the presence of extracellular CaZ+ and A23187, caused about 100% enhancement in the level of 1%HETE (see Fig. 9). The addition of NDGA caused a 69% reduction in 15-HETE and 73% inhibition in acrosin release rate. These data indicate that the metabolism of arachidonic acid via the lipoxygenase pathway plays an important role in the mechanism of the acrosome reaction. The fact that the inhibition of acrosin release by NDGA can be overcome by adding 15-HETE or 15-HPETE further support conclusion. The presence of 15-lipoxygenase and inhibition of 15-HETE production by NDGA has been shown in ram spermatozoa as well [16]. The failure of 15-HETE to stimulate acrosin release in the absence of A23187 and NDGA (see Table II), indicates that 15-HETE as well as high intracellular Ca2+ concentration, are both required for the occurrence of the acrosome reaction. This result also indicates that 15-HETE is not acting as Ca2+ ionophore.
The fact that NDGA inhibits the acrosome reaction in the presence of A23187, conditions by which Ca*+ can enter freely into the cells, indicate as well that the products of the lipoxygenase are not involved in Ca’+ entry into the cell, but rather in other steps of the acrosome reaction mechanism. The data indicate that metabolism of arachidonic acid via the lipoxygenase pathway is an important step in the acrosome reaction. Hydrolytic release of arachidonic acid from cell phospholipids can occur by phospholipase A, [23]. Several studies have suggested that sperm phospholipase A, is involved in the acrosome reaction [7,12,23-251. Since 15-HETE did not affect acrosin release in the presence of A23187, unless NDGA was present (Table II), we conclude that the cells are saturated with 15-HETE and/or other lipoxygenase products under these conditions. We suggest, that increased concentration of intracellular Ca2+, stimulates the phospholipase A, activity, which leads to the release of free arachidonic acid, which is further metabolized by lipoxygenase to 15HETE, 15-HPETE and other products. The mechanism involved in stimulation of membrane fusion by 15HETE is not clear, but the data indicate that intracellular Ca2+ is involved in this mechanism. Esterification of HETE compounds into neutrophil phospholipids can occur and it has been suggested that this event is important for the mechanism of degranulation [26]. It is possible that such esterification, in addition to neutralizing the negative charge of the outer acrosomal and the plasma membranes by Ca’+, are two essential events which lead to the acrosome reaction. It was shown recently in heart cells, that metabohtes of lipoxygenase activate G-protein-gated K’ channel [27,28]. It is possible that such an ion-channel activity is involved in the mechanism of the acrosome reaction and that this channel can be activated by the lipoxygenase products. In a recent study. Oliw and Sprecher [29] have suggested that (n - 6) lipoxygenase activity of human semen is associated with prostate excreted organelles called ‘prostasomes’ and not with the spermatozoa itself. They also showed that the level of 15-HETE is not affected by A23187 and Ca’“. Our data in bull sperm, which show 100% increase in 15HETE (Fig. 9X indicate that our bull-cell preparation differs from the human sperm preparation. The presence of ‘prostasomes’ can be detected by following phosphatase activity and by the electron microscope [30]. This study in our preparation (data not shown) reveals that our sperm preparation is free of ‘prostasomes’.
Acknowledgements We wish to thank D. Kalay and M. Bar-El of Hasherut, Artificial Insemination Center, Hafez-Haim, Israel, for the frozen bull sperm generously supplied by
18
them. We also thank Avrille Goldreich for the careful typing of this manuscript. References 1 Bedford, J.M. (1970) Biol. Reprod. (Suppl.) 2, 128-158. 2 Yanagimachi, R. (1981) in Fertilization and Embryomic Development in vitro (Mastroianni, L. Jr. and Biggers, J.D., eds.), pp. 82-182, Plenum Press, New York. 3 Russell, L., Peterson, R. and Freund, M. (1979) J. Exp. 2001. 208, 41-56. 4 Hall, A.K. and Behrman, H.R. (1982) in Prostaglandins (Lee, J.B., ed.), pp. l-38, Elsevier, New York. 5 Samuelsson, B. (1982) in Leukotrienes and other Lipoxygenase Products (Samuelsson, B. and Paoeletti, R., eds.), pp. l-17, Raven Press, New York. 6 Laychock, S.G. and Putney, J.W. Jr. (1982) in Cellular Regulation of Secretion and Release (Conn, M.P., ed.), pp. 53-105, Academic Press, New York. 7 Dravland, J.E. and Meizel, S. (1982) J. Androl. 3, 388-395. 8 Llanos, M.N., Lui, C.W. and Meizel, S. (1982) J. Exp. Zool. 221, 107-117. 9 Meizel, S. and Turner, K.O. (1983) FEBS Lett. 161, 315-318. 10 Llanos, M.N. and Meizel, S. (1983) Biol. Reprod. 28, 1043-1051. 11 Fleming, A.D. and Yanagimachi (1981) Gamete Res. 4, 253-273. 12 Ono, K., Yanagimachi, R., Huang, T.T.F. (1982) Dev. Growth Differ. 24, 305-310. R. (1982) J. Exp. Zool. 224, 259-263. 13 Ohzu, E. and Yanagimachi,
14 Meizel, S. and Turner, K.O. (1984) J. Exp. Zool. 231, 283-288. 15 Joyce, C.L., Nuzzo, N.A., Wilson, L. Jr. and Zaneveld, L.J.D. (1987) J. Androl. 8, 74-82. 16 Grossman, S., Schon, I., Sofer, Y., Magid, N. and Bartoov, B. (1986) Adv. Prostaglandin, Thromboxane Leuktriene Res. 16. 241-252. 17 Graham, J.K., Foote, R.H. and Parrish, J.J. (1986) Biol. Reprod. 35, 413-424. 18 Ben-Av, P., Rubinstein, S. and Breitbart, H. (1988) Biochim. Biophys. Acta 939, 214-222. 19 Funk, M.O., Issac, R. and Porter, N.A. (1976) Lipids 11, 113-117. 20 Aitken, R.J. and Kelly, R.W. (1985) J. Reprod. Fert. 73, 139-146. 21 Kirtland. S.J. and Baum, H. (1972) Nature 236, 47-49. 22 Docherty, J.C. and Wilson, T.W. (1987) B&hem. Biophys. Res. Commun. 148, 534-538. 23 Irvine, R.F. (1982) J. Biochem. 204, 3-16. 24 Thakkar, J.K., East, .I., Soyler, D. and Franson, R.C. (1983) Biochim. Biophys. Acta 754, 44-50. 25 Lui, M.N. and Meizel, S. (1979) J. Exp. Zoo]. 207, 173-185. 26 Stenson, W.F. and Parker, C.W. (1980) J. Immunol. 124, 2100-2104. 27 Kurachi, Y., Ito, H., Sugimoto, T.. Shimizu, T., Miki, I. and Ui, M. (1989) Nature 337, 555-557. 28 Kim, D., Lewis, D.L., Graziadei, L., Neer, E.J., Bar-Sag, D. and Clapham, D.E. (1989) Nature 337, 557-560. 29 Oliw, E.H. and Sprecher, H. (1989) Biochim. Biophys. Acta 1002, 283-291. 30 Breitbart, H. and Rubinstein, S. (1982) Arch. Androl. 9, 147-157.
