- Email: [email protected]
Comparison of the effect of nonsteroidal and steroidal antiinflammatory agents on prostaglandin production during ovulation in the rabbit
PROSTAGLANDINS COMPARISON OF THE EFFECT OF NONSTEROIDAL AND STEROIDAL ANTIINFLAMMATORY AGENTS ON PROSTAGLANDIN PRODUCTION DURING OVULATION IN THE RABBIT Lawrence L. Espey Department of Biology Trinity University San Antonio, Texas 78284 ABSTRACT The nonsteroidal antiinflammatory agents diclofenac, fenoprofen and aspirin were tested to determine how well they inhibit the preovulatory elevation in prostaglandin (PG) production in rabbit follicles in comparison to indomethacin. In addition, the steroidal antiinflammatory agent dexamethasone and the antipyretic agent acetaminophen were tested. The agents were administered 8 h after the ovulatory process was stimulated by hCG (50 I.U./kg). At 10 h after hCG (i.e., at the expected time of ovulation) control follicles had PGF and PGE levels of 370.0 and 582.6 pg/mg of follicle, respectively. Diclofenac inhibited PG production the most-reducing PGF and PGE to 22.8 and 53.6 pg/mg, respectively. Indomethacin reduced the PGF and PGE levels to 27.4 and 76.6 pg/mg, respectively. Fenoprofen was less effective, reducing the PGF and PGE to 77.8 and 222.4 pg/mg, respectively. Aspirin reduced the PGF and PGE to 123.4 and 174.6 pg/mg, respectively. Dexamethasone and acetaminophen did not inhibit PG production. Ovulation was completely inhibited by diclofenac and indomethacin, partially inhibited by fenoprofen, and unaffected by aspirin, acetaminophen, or dexamethasone. The results suggest that any potent nonsteroidal antiinflammatory agent can inhibit ovulation provided it adequately reduces PG production; whereas steroidal antiinflammatory agents are ineffective. The antiinflammatory agent must completely abolish the preovulatory elevation in PGs in mature follicles in order to totally inhibit ovultion.
KEY WORDS:
ovulation prostaglandin diclofenac indomethacin
JULY 1983VOL. 26 NO. 1
antiinflammatoryagents fenoprofen dexamethasone
71
PROSTAGLANDINS
INTRODUCTION A decade aoo it was first demonstrated that indomethacin, a nonsteroidaT antiinflammatory agent, can inhibit ovulation (l-3). Indomethacln acts by suppressing the production of prostaglandins (PGs) in mature follicles (4-5). More recently it has been demonstrated that a number of different nonsteroidal antiinflammatory agents can inhibit ovulation in the rabbit (6). However, steroidal antiinflammatory agents do not prevent ovulation, even when they are given in massive, multiple doses. The purpose of the present study was to determine the effect of several nonsteroidal antiinflammatory agents (other than indomethacin) on PG production in ovulatory rabbit follicles. In addition, an analysis was made of the production of follicular PGs following the treatment of the animals with a potent steroidal antiinflammatory agent, dexamethasone. The results showthat nonsteroidal antiinflammatory agents, diclofenac sodium and fenoprofen calcium, inhibit the production of prostaglandin E (PGE) and prostaglandin F (PGF) in ovulatory follicles, but the steroidal agent, dexamethasone, does not. METHODS Animals, A total of 60 New Zealand White rabbits were used in this m Rabbits were selected as the experimental animal because they conveniently ovulate at I.0h (* 1 h) after receiving human chorionic gonadotropin (hCG). Each rabbit was selected on the basis of whether its vaginal orifice was red (as opposed to whitish or deep purple), because this color is more indicative of normal estrus. Animals that had ovaries with adhesions, tumors, or immature follicles were excluded from the data tabulations. Ovulation Induction. Ovulation was induced by hCG (CG-5, Sigma Chemical Company, St. Louis, MO) at a dose of 50 I.U./kg, intravenously (iv). To obtain ovaries for experimental analysis, the rabbits were anesthetized with sodium pentobarbital and a laparotomy was performed. Routinely, only one of the ovaries was removed for radioimmunoassay (RIA). The other ovary was left in situ and the animal was allowed to recover from anesthesia. ApEo%%ely lo-15 h after the expected time of ovulation (i.e., approximately 20-25 h after hCG dosage), the animals were anesthetized again and a second laparotomy was performed to determine whether ovulation had occurred on the remaining ovary. In those instances when a specific drug inhibited ovulation, the ovaries were examined under a dissecting microscope to be certain that none of the follicles had ruptured. Indomethacin (I-7378, Sigma Chemical Company, St. Louisq)Do;ag;s. lc ofenac sodium (CIBA-GEIGY Corporation, Summit, NJ) and finoprifen calcium (Eli Lilly Research Laboratories, Indianapolis, IN) were all administered iv at a dosage of 10 mg/kg (10 mg suspended in 1.0 ml of 0.1 M Sorensen's phos hate buffer-pH 8.0). Aspirin (Bayer) and acetaminophen (TylenolP were administered iv at a dosage
72
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS of 50 mg/kg (100 mg suspended in 1.0 ml of 0.1 M NaP04 and 0.1 M Sorensen's buffer, respectively). Dexamethasone-Zl-acetate (D-1881, Sigma Chemical Company, St. Louis, MO) was administered intramuscularly at a dosage of 20 mg/kg (50 mg dissolved in 1.0 ml of 100% EtOH). Prosta landin Radioimmunoassay. Immediately followinq their excishies for RIA were placed in a paraffin dish and anchored with straight pins. Three mature follicles were dissected free from the stromal tissue of each ovary and transferred to a preweighed 2.0 ml glass homogenizer tube (S73, Tri-R Instruments, Inc., Rockville Center, NY), which contained 1.0 ml of 0.1 M acetate buffer (pH 4.5) and 2O.ug of indomethacin. (The indomethacin was dissolved in DMSO at a concentration of 2.0 mg/ml and added by microburet to the acetate buffer,) The homogenizer tube was then reweighed in order to calculate the weight of the three follicles. After weighing, the follicles were homogenized for 1 min with a glass pestle connected to a 12,000 RPM stirrer (Model S63, Tri-R Instruments, Inc., Rockville Center, NY) set at medium speed. The interval of time between excision of the ovaries and completion of the homogenization was approximately 10 min. The homogenate was transferred to a polyethylene tube, capped and stored at -300C until a complete series of experimental samples could be collected and assayed simultaneously for PG. (In no instance were the samples stored for longer than 3 weeks.) The RIAs were performed according to procedures which have been described by Harper, et al (7-8). Although the antibodies were produced by innoculating rabbits with prostaglandins E2 and F2*, the results are expressed simply as PGE and PGF since the antibodies cross-react essentially 100% with prostaglandins El and Fla, respectively. The results are expressed as pg of PG per mg of follicle (wet weight). (Data were analyzed using the Student's ttest for a difference between two independent means.) RESULTS Controls. Prior to treatment with hCG, the PG levels in mature folliclesfrom estrus rabbits were 51.0 f 10.76 and 105.0 f 27.87 for PGF and PGE, respectively (Fig. 1). At 10 h after hCG (i.e., at the expected time of ovulation) the PG levels were 370.0 f 49.06 and 582.6 f 120.44 for PGF and PGE, respectively. These values reflect a 7.3-fold (p 4 .OOl) and a 5.5-fold (p 4 .Ol) increase in PGF and PGE, respectively, during the ovulatory process in rabbits. The ovulatory efficiency of the large follicles on the "second" ovaries of the hCG-treated controls was 65% (t 16.0%) (Fig. 1). Prosta landin Levels After Treatment with Nonsteroidal AntientsTheZV&ent test agents were given am I;fla*. a ter t e anima s received hCG because this is the optimum time to administer indomethacin to inhibit ovulation in the rabbit (9). Indomethacin caused the PGF and PGE levels to decrease to 27.4 k 7.56 (p r: .OOl) and 76.6 f 22.06 (p < .Ol) pg/mg follicle, respectively, at 10 h after hCG (i.e., at the expected time of ovulation), and ovulation was completely inhibited (Fig. 1).
JULY
1983 VOL. 26 NO. 1
73
PROSTAGLANDINS Fig, 1. PG level at 10 h after hCG (i.e., at the time of ovulation) when anttinflammatory agents were given at 8 h after hCG. Bars marked "N" indicate the PG level in normal follicles from unstimulated estrus rabbits, Fracttons at the base of each bar indicate anlmals ovulated / total animals. Percentages at the base of each bar indicate the ovulation efficiency of the total number of mature follicles In each group of animals. I
PGF I
-PPGE------_,
600
_. _--
__-_-_------- --_ La r--w -N
----m-t--
,___-.
N
N
,_m_--
a-
0
015 on
o/5 0%
215
515
14%
72!d
/* 2%
_.
w-w
1
-em
5/5 65%
015 0%
O/5 0%
215 14%
5/5 729
i/5 59 -
ANTI-INFLAMMATORY AGENTS (administered 8 hours after hCG)
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS In comparison, diclofenac sodium was equally (if not slightly more) effective in reducing PGF (p < .OOl) and PGE (p < .OOl) below the control level at the expected time of ovulation, This agent also inhibited ovulation completely. Fenoprofen calcium was somewhat less effective in reducing PGF (P < .OOl) and PGE (p 4 .05) below the control level even though it was given at the same dosage as diclofenac. This agent only partially inhibited ovulation, with 14% of the mature follicles rupturing. Aspirin was essentially as effective as fenoprofen calcium in reducing PGF (p 4 .Ol) and PGE (p 4 .02). However, this antipyretic, antiinflammatory agent did not inhibit ovulation-72% of the mature follicles ruptured. Acetaminophen, an antipyretic agent like aspirin, had negligible effect on PGF (p > .50) and PGE (p > .20), and it did not inhibit ovulation.
the PGF level was 361.8 f 51.54 and the PGE level was 622.6 f 163.22 (Fig. 1). Since there was the possibility that dexamethasone might be more effective if administered earlier during the ovulatory process, this steroidal antiinflarrmatoryagent was also given at 1 h after hCG and at 10 h before hCG. When dexamethasone was given at 1 h after hCG, the PGF and PGE levels were 391.8 i 53.72 and 789.4 f 173.81, respectively, at the time of ovulation. When dexamethasone was given at 10 h before hCG, the PGF and PGE levels were 348.6 i 86.92 and 692.6 f 222.20, respectively, at the expected time of ovulation. Thus, regardless of the time at which it was administered, dexamethasone-21-acetate did not reduce the level of PG in the follicles. DISCUSSION The results demonstrate that, like indomethacin, other nonsteroidal antiinflammatory agents such as diclofenac sodium and-o-fencalcium inhibit ovulation by inhibiting PG synthesis in mature ovarian follicles. This information suggests that any potent nonsteroidal antiinflammatory agent should inhibit ovulation in the same manner. It has been reported that aspirin, which is a weak nonsteroidal antiinflammatory agent, inhibits ovulation in the rml, 10). However, I have found that, when aspirin is given in large doses, either intragastrically (6). or iv ( as in the present study), it Behrman, et al (ll), have does not inhibit ovulation in the rabbit. suggested that aspirin blocks ovulation in rats at the hypothalamic level, rather than by acting at the ovarian level. If this is indeed the case, then aspirin would not have been expected to inhibit ovulation in the present study since it was administered 8 h after the rabbits received hCG.
JULY
1983 VOL. 26 NO. 1
75
PROSTAGLANDINS
Still, the data show that when aspirin is administered at a dosage 5X that of fenoprofen calcium it significantly (p < .Ol) inhibited PGF. Thus, aspirin clearly has at least a moderate inhibitory effect on PG production in ovulatory follicles, However, the aspirin treatment did not inhibit ovulation, whereas fenoprofen showed partial inhibition of ovulation. Apparently, what this means is that the PGF level must drop somewhere below 100 pg/mg of follicle in order for ovulation to be at least partially inhibited, And, only when the PGF level dropped below 50 pg/mg of follicle (as occurred following diclofenac and indomethacin treatment), is ovulation completely inhibited. Therefore, like PG inhibition, ovulation is not an "all-or-none" phenomenon. It has been suggested that PGF is more important than PGE for ovulation (12-15). In this regard, it may be relevant that fenoprofen, which reduced the PGF level slightly more than aspirin (p < .20), also inhibited ovulation; whereas, aspirin, which reduced the PGE level as much or more than fenoprofen (p 7 .50), failed to inhibit ovulation. Thus, it appears that the degree of inhibition of ovulation more closely parallels the!degree of inhibition of PGF. However, the differences are relatively minor, and therefore the interpretation that PGF is more important should be made with reservation. In descending order of effectiveness, PG synthesis was inhibited by diclofenac sodium, indomethacin, fenoprofen calcium and aspirin. The results show the same descending order of effectiveness with regard to the ability of these nonsteroidal antiinflammatory agents to inhibit ovulation. And, this order is fdentical to their antiovulatory activity when they are administered intragastrically at 1 h after hCG (6). It is also the same order of strength with which these agents inhibit acute inflammatory reactions (6). Thus, the results provide additm support for the hypothesis (16) that the mechanism of ovulation involves an inflammatory reaction-especially since PGs are well-known mediators of inflammation (17). In contrast, the potent steroidal antiinflammatory agent, dexamethasone, did not inhibit either PG synthesis or ovulation, no matter whether it was given at 2 h, 9 h, or 20 h before the expected time of follicular rupture. This failure of dexamethasone to inhibit ovulation is not too surprising because the available literature suggests that glucocorticoids have more of an effect on chronic inflammation (18-19), whereas the ovulatory process resembles an acute inflammatory reaction (16). In fact, in some instances, glu=ticoids such as dexamethasone stimulate the enzyme systems which lead to PG synthesis in inflammation (18). This action may explain their inability to inhibit ovulation, even when they are given in multiple doses for up to 3 days (6). In conclusion, potent nonsteroidal antiinflammatory agents inhibit follicular PG production and ovulation, but steroidal antiinflammatory agents do not. The results suggest that ovulation can be more readily inhibited by agents that inhibit acute inflaimiatory reactions.
76
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS ACKNOklLEDGPlENTS I appreciate the precision with which Catherine Norris, M.A., with the assistance of Elizabeth Hemnick, B.A., conducted the RIAs at the Center for Research in Reproductive Biology, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio. This study was supported by NIH Grant HD14539 to Trinity University and by NIH Grant P30-HD10202 to The University of Texas Health Science Center at San Antonio. REFERENCES
1. 2. 3.
4.
5. 6. 7.
8. 9. 10. 11.
Orczyk, G. P. and H. R. Behrman. Ovulation Blockade by Aspirin or Indomethacin - In vivo Evidence for a Role of Prostaglandin Prostaglandins 1: 3, 1972. in Gonadotrophin S'&%?ci%. Armstrong, D. T. and D. L. Grinwich. BlockaTe of Spontaneous and LH-Induced Ovulation in Rats by Indomethacin, an Inhibitor of Prostaglandin Biosynthesis. Prostaglandins 1: 21, 1972. O'Grady, J. P., B. V. Caldwell, F. J. Auletta, and L. Speroff. The Effects of an Inhibitor of Prostaglandin Synthesis (Indomethacin) on Ovulation, Pregnancy, and Pseudopregnancy in the Rabbit. Prostaglandins 1: 97, 1972. Armstrong, D. T., Y. S. Noon, and J. Zamecnik. Evidence for a Role of Ovarian Prostaglandins in Ovulation. In: Gonadotropins and Gonadal Function (N. R. Moudgal, ed.), Acazmic Press, NY, 1973, p. 345. Yang, N. S. T., J. M. Marsh, and W. J. LeMaire. Prostaglandin Changes Induced by Ovulatory Stimuli in Rabbit Graafian Follicles. The Effect of Indomethacin. Prostaglandins 4: 395, 1973. Survey of AntiEspey, L. L., V. I. Stein, and J. Dumitrescu. Inflammatory Agents and Related Drugs as Inhibitors of Ovulation in the Rabbit. Fertil. Steril. 38: 238, 1982. Harper, M. J. K., G. Valenzuela,and B. G. Hodgson. Accelerated Ovum Transport in Rabbits Induced by Endotoxin. I. Changes in Prostaglandin Levels and Reversal of Endotoxin Effects. Prostaglandins 15: 43, 1978. Harper, M. J. K., C. J. Norris, W. E. Friedrichs, and A. Moreno. Poly 1:C Accelerates Ovum Transport in the Rabbit by a Prostaglandin-Mediated Mechanism. J. Reprod. Fertil. 63: 81, 1981. Espey, L. L. Optimum Time for Administration of Edomethacin to Inhibit Ovulation in the Rabbit. Prostaglandins 23: 329, 1982. Parr, E. L. Histological Examination of the Rat Karian Follicle Wall Prior to Ovulation. Biol. Reprod. 11: 483, 1974. Effect of SynBehrman, H. R., G. P. Orczyk, and R. 0. peep. thetic Gonadotrophin-releasing Hormone (Gn-RH) on Ovulation Blockade by Aspirin and Indomethacin. Prostaglandins 1: 245, 1972.
JULY
1983 VOL. 26 NO. 1
77
PROSTAGLANDINS 12. Wallach, E, E,, R, Bronson, Y, Hamada, K. H, Wright, and V. C. Stevens, Effectiveness of Prostaglandin F2& in Restoration of HMG-HCG Induced Ovulation in Indomethacin-TreatedRhesus Monkeys, Prostaglandins 10: 129, 1975. and E. E. Wallach. In Vitro Reversal 13. Hamada. Y., K. KWrioht. of Indomethacin-BlockedOvulation by Prostaglandin F2oc. Fertil. Steril. 30: 702, 1978. 14. Downey, E R. and L. Ainsworth. Reversal of Indomethacin Blockade of Ovulation in Gilts by Prostaglandins. Prostaglandins-19: 17, 1980. 15. Armstrong, D. T. Prostaglandins and Follicular Function, J. Reprod. Fertil. 62: 283, 1981. 16. Espey, L. L. OvTjTationas an Inflammatory Reaction - A Hypothesis. Biol. Reprod. 22: 73, 1980. 17. Vane, J. R. Prostaglan?%s as Mediators of Inflammation. Adv. Prosta. Thromb. Res. 2: 791, 1976. 18. Dannenberg, A. M., Jr: The Antiinflammatory Effects of Glucocortlcosteroids.A Brief Review of the Literature. Inflammation 3: 329, 1979. 19. Tahey, J. V., P. M. Guyre, and A. Munck. Mechanisms of Antiinflammatory Actions of Glucocorticoids. Adv. Inflam. Res. 2: 21, 1981.
Editor: Harold R. Behrman
78
Received: 2-21-83
JULY
Accepted: 4-18-83
1983VOL.26NO.l
KEY WORDS:
ovulation prostaglandin diclofenac indomethacin
JULY 1983VOL. 26 NO. 1
antiinflammatoryagents fenoprofen dexamethasone
71
PROSTAGLANDINS
INTRODUCTION A decade aoo it was first demonstrated that indomethacin, a nonsteroidaT antiinflammatory agent, can inhibit ovulation (l-3). Indomethacln acts by suppressing the production of prostaglandins (PGs) in mature follicles (4-5). More recently it has been demonstrated that a number of different nonsteroidal antiinflammatory agents can inhibit ovulation in the rabbit (6). However, steroidal antiinflammatory agents do not prevent ovulation, even when they are given in massive, multiple doses. The purpose of the present study was to determine the effect of several nonsteroidal antiinflammatory agents (other than indomethacin) on PG production in ovulatory rabbit follicles. In addition, an analysis was made of the production of follicular PGs following the treatment of the animals with a potent steroidal antiinflammatory agent, dexamethasone. The results showthat nonsteroidal antiinflammatory agents, diclofenac sodium and fenoprofen calcium, inhibit the production of prostaglandin E (PGE) and prostaglandin F (PGF) in ovulatory follicles, but the steroidal agent, dexamethasone, does not. METHODS Animals, A total of 60 New Zealand White rabbits were used in this m Rabbits were selected as the experimental animal because they conveniently ovulate at I.0h (* 1 h) after receiving human chorionic gonadotropin (hCG). Each rabbit was selected on the basis of whether its vaginal orifice was red (as opposed to whitish or deep purple), because this color is more indicative of normal estrus. Animals that had ovaries with adhesions, tumors, or immature follicles were excluded from the data tabulations. Ovulation Induction. Ovulation was induced by hCG (CG-5, Sigma Chemical Company, St. Louis, MO) at a dose of 50 I.U./kg, intravenously (iv). To obtain ovaries for experimental analysis, the rabbits were anesthetized with sodium pentobarbital and a laparotomy was performed. Routinely, only one of the ovaries was removed for radioimmunoassay (RIA). The other ovary was left in situ and the animal was allowed to recover from anesthesia. ApEo%%ely lo-15 h after the expected time of ovulation (i.e., approximately 20-25 h after hCG dosage), the animals were anesthetized again and a second laparotomy was performed to determine whether ovulation had occurred on the remaining ovary. In those instances when a specific drug inhibited ovulation, the ovaries were examined under a dissecting microscope to be certain that none of the follicles had ruptured. Indomethacin (I-7378, Sigma Chemical Company, St. Louisq)Do;ag;s. lc ofenac sodium (CIBA-GEIGY Corporation, Summit, NJ) and finoprifen calcium (Eli Lilly Research Laboratories, Indianapolis, IN) were all administered iv at a dosage of 10 mg/kg (10 mg suspended in 1.0 ml of 0.1 M Sorensen's phos hate buffer-pH 8.0). Aspirin (Bayer) and acetaminophen (TylenolP were administered iv at a dosage
72
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS of 50 mg/kg (100 mg suspended in 1.0 ml of 0.1 M NaP04 and 0.1 M Sorensen's buffer, respectively). Dexamethasone-Zl-acetate (D-1881, Sigma Chemical Company, St. Louis, MO) was administered intramuscularly at a dosage of 20 mg/kg (50 mg dissolved in 1.0 ml of 100% EtOH). Prosta landin Radioimmunoassay. Immediately followinq their excishies for RIA were placed in a paraffin dish and anchored with straight pins. Three mature follicles were dissected free from the stromal tissue of each ovary and transferred to a preweighed 2.0 ml glass homogenizer tube (S73, Tri-R Instruments, Inc., Rockville Center, NY), which contained 1.0 ml of 0.1 M acetate buffer (pH 4.5) and 2O.ug of indomethacin. (The indomethacin was dissolved in DMSO at a concentration of 2.0 mg/ml and added by microburet to the acetate buffer,) The homogenizer tube was then reweighed in order to calculate the weight of the three follicles. After weighing, the follicles were homogenized for 1 min with a glass pestle connected to a 12,000 RPM stirrer (Model S63, Tri-R Instruments, Inc., Rockville Center, NY) set at medium speed. The interval of time between excision of the ovaries and completion of the homogenization was approximately 10 min. The homogenate was transferred to a polyethylene tube, capped and stored at -300C until a complete series of experimental samples could be collected and assayed simultaneously for PG. (In no instance were the samples stored for longer than 3 weeks.) The RIAs were performed according to procedures which have been described by Harper, et al (7-8). Although the antibodies were produced by innoculating rabbits with prostaglandins E2 and F2*, the results are expressed simply as PGE and PGF since the antibodies cross-react essentially 100% with prostaglandins El and Fla, respectively. The results are expressed as pg of PG per mg of follicle (wet weight). (Data were analyzed using the Student's ttest for a difference between two independent means.) RESULTS Controls. Prior to treatment with hCG, the PG levels in mature folliclesfrom estrus rabbits were 51.0 f 10.76 and 105.0 f 27.87 for PGF and PGE, respectively (Fig. 1). At 10 h after hCG (i.e., at the expected time of ovulation) the PG levels were 370.0 f 49.06 and 582.6 f 120.44 for PGF and PGE, respectively. These values reflect a 7.3-fold (p 4 .OOl) and a 5.5-fold (p 4 .Ol) increase in PGF and PGE, respectively, during the ovulatory process in rabbits. The ovulatory efficiency of the large follicles on the "second" ovaries of the hCG-treated controls was 65% (t 16.0%) (Fig. 1). Prosta landin Levels After Treatment with Nonsteroidal AntientsTheZV&ent test agents were given am I;fla*. a ter t e anima s received hCG because this is the optimum time to administer indomethacin to inhibit ovulation in the rabbit (9). Indomethacin caused the PGF and PGE levels to decrease to 27.4 k 7.56 (p r: .OOl) and 76.6 f 22.06 (p < .Ol) pg/mg follicle, respectively, at 10 h after hCG (i.e., at the expected time of ovulation), and ovulation was completely inhibited (Fig. 1).
JULY
1983 VOL. 26 NO. 1
73
PROSTAGLANDINS Fig, 1. PG level at 10 h after hCG (i.e., at the time of ovulation) when anttinflammatory agents were given at 8 h after hCG. Bars marked "N" indicate the PG level in normal follicles from unstimulated estrus rabbits, Fracttons at the base of each bar indicate anlmals ovulated / total animals. Percentages at the base of each bar indicate the ovulation efficiency of the total number of mature follicles In each group of animals. I
PGF I
-PPGE------_,
600
_. _--
__-_-_------- --_ La r--w -N
----m-t--
,___-.
N
N
,_m_--
a-
0
015 on
o/5 0%
215
515
14%
72!d
/* 2%
_.
w-w
1
-em
5/5 65%
015 0%
O/5 0%
215 14%
5/5 729
i/5 59 -
ANTI-INFLAMMATORY AGENTS (administered 8 hours after hCG)
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS In comparison, diclofenac sodium was equally (if not slightly more) effective in reducing PGF (p < .OOl) and PGE (p < .OOl) below the control level at the expected time of ovulation, This agent also inhibited ovulation completely. Fenoprofen calcium was somewhat less effective in reducing PGF (P < .OOl) and PGE (p 4 .05) below the control level even though it was given at the same dosage as diclofenac. This agent only partially inhibited ovulation, with 14% of the mature follicles rupturing. Aspirin was essentially as effective as fenoprofen calcium in reducing PGF (p 4 .Ol) and PGE (p 4 .02). However, this antipyretic, antiinflammatory agent did not inhibit ovulation-72% of the mature follicles ruptured. Acetaminophen, an antipyretic agent like aspirin, had negligible effect on PGF (p > .50) and PGE (p > .20), and it did not inhibit ovulation.
the PGF level was 361.8 f 51.54 and the PGE level was 622.6 f 163.22 (Fig. 1). Since there was the possibility that dexamethasone might be more effective if administered earlier during the ovulatory process, this steroidal antiinflarrmatoryagent was also given at 1 h after hCG and at 10 h before hCG. When dexamethasone was given at 1 h after hCG, the PGF and PGE levels were 391.8 i 53.72 and 789.4 f 173.81, respectively, at the time of ovulation. When dexamethasone was given at 10 h before hCG, the PGF and PGE levels were 348.6 i 86.92 and 692.6 f 222.20, respectively, at the expected time of ovulation. Thus, regardless of the time at which it was administered, dexamethasone-21-acetate did not reduce the level of PG in the follicles. DISCUSSION The results demonstrate that, like indomethacin, other nonsteroidal antiinflammatory agents such as diclofenac sodium and-o-fencalcium inhibit ovulation by inhibiting PG synthesis in mature ovarian follicles. This information suggests that any potent nonsteroidal antiinflammatory agent should inhibit ovulation in the same manner. It has been reported that aspirin, which is a weak nonsteroidal antiinflammatory agent, inhibits ovulation in the rml, 10). However, I have found that, when aspirin is given in large doses, either intragastrically (6). or iv ( as in the present study), it Behrman, et al (ll), have does not inhibit ovulation in the rabbit. suggested that aspirin blocks ovulation in rats at the hypothalamic level, rather than by acting at the ovarian level. If this is indeed the case, then aspirin would not have been expected to inhibit ovulation in the present study since it was administered 8 h after the rabbits received hCG.
JULY
1983 VOL. 26 NO. 1
75
PROSTAGLANDINS
Still, the data show that when aspirin is administered at a dosage 5X that of fenoprofen calcium it significantly (p < .Ol) inhibited PGF. Thus, aspirin clearly has at least a moderate inhibitory effect on PG production in ovulatory follicles, However, the aspirin treatment did not inhibit ovulation, whereas fenoprofen showed partial inhibition of ovulation. Apparently, what this means is that the PGF level must drop somewhere below 100 pg/mg of follicle in order for ovulation to be at least partially inhibited, And, only when the PGF level dropped below 50 pg/mg of follicle (as occurred following diclofenac and indomethacin treatment), is ovulation completely inhibited. Therefore, like PG inhibition, ovulation is not an "all-or-none" phenomenon. It has been suggested that PGF is more important than PGE for ovulation (12-15). In this regard, it may be relevant that fenoprofen, which reduced the PGF level slightly more than aspirin (p < .20), also inhibited ovulation; whereas, aspirin, which reduced the PGE level as much or more than fenoprofen (p 7 .50), failed to inhibit ovulation. Thus, it appears that the degree of inhibition of ovulation more closely parallels the!degree of inhibition of PGF. However, the differences are relatively minor, and therefore the interpretation that PGF is more important should be made with reservation. In descending order of effectiveness, PG synthesis was inhibited by diclofenac sodium, indomethacin, fenoprofen calcium and aspirin. The results show the same descending order of effectiveness with regard to the ability of these nonsteroidal antiinflammatory agents to inhibit ovulation. And, this order is fdentical to their antiovulatory activity when they are administered intragastrically at 1 h after hCG (6). It is also the same order of strength with which these agents inhibit acute inflammatory reactions (6). Thus, the results provide additm support for the hypothesis (16) that the mechanism of ovulation involves an inflammatory reaction-especially since PGs are well-known mediators of inflammation (17). In contrast, the potent steroidal antiinflammatory agent, dexamethasone, did not inhibit either PG synthesis or ovulation, no matter whether it was given at 2 h, 9 h, or 20 h before the expected time of follicular rupture. This failure of dexamethasone to inhibit ovulation is not too surprising because the available literature suggests that glucocorticoids have more of an effect on chronic inflammation (18-19), whereas the ovulatory process resembles an acute inflammatory reaction (16). In fact, in some instances, glu=ticoids such as dexamethasone stimulate the enzyme systems which lead to PG synthesis in inflammation (18). This action may explain their inability to inhibit ovulation, even when they are given in multiple doses for up to 3 days (6). In conclusion, potent nonsteroidal antiinflammatory agents inhibit follicular PG production and ovulation, but steroidal antiinflammatory agents do not. The results suggest that ovulation can be more readily inhibited by agents that inhibit acute inflaimiatory reactions.
76
JULY 1983 VOL. 26 NO. 1
PROSTAGLANDINS ACKNOklLEDGPlENTS I appreciate the precision with which Catherine Norris, M.A., with the assistance of Elizabeth Hemnick, B.A., conducted the RIAs at the Center for Research in Reproductive Biology, Department of Obstetrics and Gynecology, The University of Texas Health Science Center at San Antonio. This study was supported by NIH Grant HD14539 to Trinity University and by NIH Grant P30-HD10202 to The University of Texas Health Science Center at San Antonio. REFERENCES
1. 2. 3.
4.
5. 6. 7.
8. 9. 10. 11.
Orczyk, G. P. and H. R. Behrman. Ovulation Blockade by Aspirin or Indomethacin - In vivo Evidence for a Role of Prostaglandin Prostaglandins 1: 3, 1972. in Gonadotrophin S'&%?ci%. Armstrong, D. T. and D. L. Grinwich. BlockaTe of Spontaneous and LH-Induced Ovulation in Rats by Indomethacin, an Inhibitor of Prostaglandin Biosynthesis. Prostaglandins 1: 21, 1972. O'Grady, J. P., B. V. Caldwell, F. J. Auletta, and L. Speroff. The Effects of an Inhibitor of Prostaglandin Synthesis (Indomethacin) on Ovulation, Pregnancy, and Pseudopregnancy in the Rabbit. Prostaglandins 1: 97, 1972. Armstrong, D. T., Y. S. Noon, and J. Zamecnik. Evidence for a Role of Ovarian Prostaglandins in Ovulation. In: Gonadotropins and Gonadal Function (N. R. Moudgal, ed.), Acazmic Press, NY, 1973, p. 345. Yang, N. S. T., J. M. Marsh, and W. J. LeMaire. Prostaglandin Changes Induced by Ovulatory Stimuli in Rabbit Graafian Follicles. The Effect of Indomethacin. Prostaglandins 4: 395, 1973. Survey of AntiEspey, L. L., V. I. Stein, and J. Dumitrescu. Inflammatory Agents and Related Drugs as Inhibitors of Ovulation in the Rabbit. Fertil. Steril. 38: 238, 1982. Harper, M. J. K., G. Valenzuela,and B. G. Hodgson. Accelerated Ovum Transport in Rabbits Induced by Endotoxin. I. Changes in Prostaglandin Levels and Reversal of Endotoxin Effects. Prostaglandins 15: 43, 1978. Harper, M. J. K., C. J. Norris, W. E. Friedrichs, and A. Moreno. Poly 1:C Accelerates Ovum Transport in the Rabbit by a Prostaglandin-Mediated Mechanism. J. Reprod. Fertil. 63: 81, 1981. Espey, L. L. Optimum Time for Administration of Edomethacin to Inhibit Ovulation in the Rabbit. Prostaglandins 23: 329, 1982. Parr, E. L. Histological Examination of the Rat Karian Follicle Wall Prior to Ovulation. Biol. Reprod. 11: 483, 1974. Effect of SynBehrman, H. R., G. P. Orczyk, and R. 0. peep. thetic Gonadotrophin-releasing Hormone (Gn-RH) on Ovulation Blockade by Aspirin and Indomethacin. Prostaglandins 1: 245, 1972.
JULY
1983 VOL. 26 NO. 1
77
PROSTAGLANDINS 12. Wallach, E, E,, R, Bronson, Y, Hamada, K. H, Wright, and V. C. Stevens, Effectiveness of Prostaglandin F2& in Restoration of HMG-HCG Induced Ovulation in Indomethacin-TreatedRhesus Monkeys, Prostaglandins 10: 129, 1975. and E. E. Wallach. In Vitro Reversal 13. Hamada. Y., K. KWrioht. of Indomethacin-BlockedOvulation by Prostaglandin F2oc. Fertil. Steril. 30: 702, 1978. 14. Downey, E R. and L. Ainsworth. Reversal of Indomethacin Blockade of Ovulation in Gilts by Prostaglandins. Prostaglandins-19: 17, 1980. 15. Armstrong, D. T. Prostaglandins and Follicular Function, J. Reprod. Fertil. 62: 283, 1981. 16. Espey, L. L. OvTjTationas an Inflammatory Reaction - A Hypothesis. Biol. Reprod. 22: 73, 1980. 17. Vane, J. R. Prostaglan?%s as Mediators of Inflammation. Adv. Prosta. Thromb. Res. 2: 791, 1976. 18. Dannenberg, A. M., Jr: The Antiinflammatory Effects of Glucocortlcosteroids.A Brief Review of the Literature. Inflammation 3: 329, 1979. 19. Tahey, J. V., P. M. Guyre, and A. Munck. Mechanisms of Antiinflammatory Actions of Glucocorticoids. Adv. Inflam. Res. 2: 21, 1981.
Editor: Harold R. Behrman
78
Received: 2-21-83
JULY
Accepted: 4-18-83
1983VOL.26NO.l