The relationship between prostaglandins and histamine in the ovulatory process as determined with the in vitro perfused rabbit ovary*

FERTILITY AND STERILITY Copyright 0 1985 The American Fertility Society

Vol. 43, No.4, April 1985 Printed in U.s A.

The relationship between prostaglandins and histamine in the ovulatory process as determined with the in vitro perfused rabbit ovary*

Hirokatsu Kitai, M.D.t Yoshimune Kobayashi, M.D.t Rosemary Santulli, RA.:j: Karen H. Wright, M.S.:j: Edward E. Wallach, M.D.:j:§ Pennsylvania Hospital and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

The process of follicle rupture has been described as an inflammatory reaction in which prostaglandins (PGs) and/or histamine may be involved. With an in vitro perfused rabbit ovary preparation, experiments were carried out for determination of whether a relationship exists among PGs, histamine, and ovulation. PGF2cx alone was capable of inducing ovulation when added to the perfusion fluid at 1, 10, and 100 ng/ ml. Effectiveness in achieving ovulation varied directly with the dosage; however, the ovulatory efficiency of PGF2cx -treated ovaries was lower than that of ovaries exposed to human chorionic gonadotropin (heG, 100 IU). PGF2cx -induced ovulation could not be blocked by the H2 receptor antagonist, cimetidine. The PG synthesis inhibitor, indomethacin, did not prevent histamine-induced ovulation. Ovulation induced by heG was partially blocked by the administration of indomethacin; however, the concomitant administration of cimetidine was not associated with further reduction in ovulation. In all but one experimental group, the majority of ovulated ova did not progress beyond the intact germinal vesicle stage unless the ovaries had been exposed to heG. On the basis of these experiments, PGs and histamine do not appear to be interdependent in their effects on the ovulatory process in vitro. Fertil Steril43:646, 1985

Received July 2, 1984; revised and accepted November 27, 1984. *Supported by National Institutes of Health grant HD05948, The Connelly Foundation, and The Mitchell and Lillian Duberstein Foundation. tConnelly Foundation Fellow in Reproductive Biology, Pennsylvania Hospital. :j:Department of Obstetrics and Gynecology, Pennsylvania Hospital and the University of Pennsylvania School of Medicine. §Reprint requests: Edward E. Wallach, M.D., Department of Gynecology and Obstetrics, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205. 646

Kitai et al. PGs and histamine in ovulation

The process of follicle rupture has been compared to an inflammatory reaction possibly mediated by prostaglandins (PGs) and/or histamine. l The involvement of PGs in the ovulatory process has been studied in several species. PGs E and F (PGE and PGF) both increase in rabbit Graafian follicles as ovulation approaches. 2 PGF enhances gonadotropin-stimulated ovulation both in vivo and in vitro.3 Indomethacin, a PG synthesis inhibitor, blocks ovulation in several species. 4 - 8 Ovulation can subsequently be restored by supplemental PGF 2u . 9 , 10 Ovarian histamine has also Fertility and Sterility

been shown to be released following luteinizing hormone stimulation. 11 Histamine can induce ovulation in the in vitro perfused rabbit ovary.12 The H2 receptor antagonist, cimetidine, can block histamine-induced ovulation. 12 PGF2a has been shown to overcome the inhibitory effect on ovulation exerted by the HI blocker, chlorpheniramine. 13 In view of the effects of PGs and histamine on the mechanics of the ovulatory process, it would be of interest to determine the interaction of these two agents in bringing about follicle disruption. The purpose of this study was to determine whether histamine and PGF2a are interrelated or are sequentially involved in the local ovarian events leading to ovulation.

MATERIALS AND METHODS

Sexually mature New Zealand White female rabbits were used in all experiments. The rabbits were isolated for a minimum of 3 weeks prior to laparotomy under controlled light and temperature with free access to Purina rabbit chow and water. At laparotomy, the rabbits were anesthetized with sodium pentobarbital (32 mg/kg) intravenously. Anastomotic connections to the ovary were ligated, and the ovarian artery was cannulated. Both ovaries were removed and placed in separate perfusion chambers. Ovaries were perfused with tissue culture medium 199 (M.A. Bioproducts, Walkersville, MD) supplemented with insulin, heparin, penicillin, and streptomycin. Details of the cannulation technique and specifications of the perfusion system have been previously described. 13-15 The first series of experiments was designed to determine whether PGF2a alone was capable of inducing ovulation. Four groups of six rabbits each were designated. PGF2a (The Upjohn Company, Kalamazoo, MI) was added to the perfusate of one ovary at the onset of ovarian perfusion at a concentration of 0.1 ng/ml, 1 ng/ml, 10 ng/ml, or 100 ng/ml. Human chorionic gonadotropin (hCG; 100 IU/150 ml) was added to the perfusate of the contralateral ovary. The second series of experiments was designed to answer the following question: Is PGF2a -induced ovulation mediated by histamine? PGF2a, 100 ng/ml, was added to the perfusate of both ovaries. This dose of PGF 2a was determined from the first series of experiments as the most effective for induction of ovulation in vitro. Following Vol. 43, No.4, April 1985

the addition of PGF 2o" 1.5 mg of cimetidine (Smith Kline Beckman, Philadelphia, PA), an H2 receptor antagonist, was added hourly in 0.15 ml of medium to the perfusate of the experimental ovary. This dose of cimetidine (10 f,Lg/mllhour) was previously found to be effective in blocking histamine-induced ovulation. 12 The contralateral ovary, which served as a control, was treated with 0.15 ml of medium at the same time intervals. A third series of experiments was designed to answer the following question: Is histamine-induced ovulation mediated by PGs? An ovulationinducing dose (100 ng/ml) of histamine was added to the perfusate of both ovaries at the onset of perfusion and at hourly intervals thereafterP The experimental ovary was perfused with the PG synthesis inhibitor, indomethacin (500 ng/ ml), at the onset of the experiment (Merck Sharp & Dohme Research Laboratories, West Point, PA). The contralateral control ovary was perfused with only the vehicle for indomethacin (M199). The fourth set of experiments was designed to answer the following question: Is hCG-induced ovulation mediated by both histamine and PGs? At the onset of perfusion, hCG (100 IUl150 ml) and indomethacin (500 ng/ml) were added to the perfusate of both ovaries. One ovary of the two was also perfused with cimetidine (10 f,Lg/ml) , added at hourly intervals. The control ovary was perfused with vehicle which was also added hourly. In all experiments ovarian perfusion was carried out for 12 hours. Ovaries were continually observed for follicular growth and ovulation. When ovulation occurred, the extruded ovum, enclosed in its cumulus, was recovered from the surface of the ovary and examined microscopically to determine the stage of maturity. Ovulated ova were classified as (1) germinal vesicle intact (GV stage); (2) metaphase I (MI)-nucleus absent and no observable polar body; or (3) metaphase II (MII)-first polar body extruded. Ova were also examined for signs of degeneration, including necrosis, cytolysis, vacuolation, and loss of spherical shape. The degree of ovarian edema was determined by calculating the percentage difference between wet weight, obtained at the conclusion of one experiment, and oven-dried weight, obtained after drying in a vacuum oven at 80°C for 24 hours. Statistical analysis was carried out using Student's t-test or chi-square analysis with Yates' corrections. Kitai et al. PGs and histamine in ovulation

647

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RESULTS Ovulation occurred in all ovaries exposed to heG; the ovulatory efficiency, which ranged from 69.7% to 81.0%, was similar to that observed previously with this in vitro system. 15 The term "ovulatory efficiency" refers to the percentage of mature follicles (> 1.5 mm in diameter) observed at the onset of perfusion which proceed to rupture by the time of conclusion of each experiment. PGF 20: was effective in inducing ovulation when used at the 1.0 ng/ml (four of six ovaries), 10 ng/ml (four of six ovaries), and 100 ng/ml (five of six ovaries) levels but failed to achieve ovulation at a dose of 0.1 ng/ml. Ovulatory efficiency increased with higher doses of PGF 2 0: (Fig. 1). At each dose level of PGF 2 0:, ovulatory efficiency was, however, significantly lower than that observed in the contralateral heG-treated ovaries (P < 0.01; Fig. 1). In the second series of experiments, ovulation occurred in all ovaries (six of six) treated with PGF 2a (l00 ng/ml) in combination with cimetidine (10 J.Lg/mllhour) as well as in the contralateral control ovaries (six of six); which were perfused with PGF 2 0: alone (100 ng/ml). Ovulatory efficiency was similar for both groups. In the third experiment, histamine (100 ng/ml) induced ovulation in five of six ovaries with an ovulatory efficiency of 54.0%. Indomethacin (500 ng/ml) failed to block histamine-induced ovulation. In 648

Kitai et aI. PGs and histamine in ovulation

the fourth experiment, a combination of indomethacin and cimetidine had no greater inhibitory effect on heG-induced ovulation than did indomethacin alone (Fig. 1). The time interval to ovulation was not altered by 10 or 100 ng/ml of PGF 2 0:, as compared with heG-treated ovaries. However, ovulation occurred earlier in those ovaries treated with PGF 2a at a dose of 1.0 ng/ml than in the contralateral heG-treated ovaries (P < 0.001). In the second, third, and fourth series of experiments, the blockers (cimetidine and indomethacin), used individually or in combination in an attempt to inhibit PGF 2 o:> histamine, or heG-induced ovulation, respectively, did not affect the latent time from onset of treatment to ovulation. Indomethacin-treated ovaries, however, showed significantly delayed (P < 0.05) heG-induced ovulation when compared with ovaries treated with heG alone in the first experimental series (Table 1). The majority of ovulated ova, examined for stage of maturity (Fig. 2), did not progress beyond the GV intact stage following treatment with PGF 2 0: (series l) or treatment with PGF 2 0: and cimetidine (series lI). In contrast, > 70% of ova in Table 1. Time Interval to Ovulation in Relationship to Specific Treatment Treatment group

Interval to ovulation mean ± standard error (no. of ovulations) hrs

Series I PGF 2a (0.1 ng/mll heG (100 IV)

No ovulation 7.12 ± 0.50 (24)a

PGF 2a (1 ng/mll heG (100 IV)

3.55 ± 0.45 (10)b 6.26 ± 0.36 (28)b

PGF 2a (10 ng/mll heG (100 IV)

6.26 ± 1.03 (11) 7.09 ± 0.62 (23)

PGF 2a (100 ng/mll

4.07 ± 0.38 (16) 5.17 ± 0.47 (35)

heG (100 IV) Series II PGF 2a (100 ng/mll PGF 2a (100 ng/mll/cimetidine (10 f-Lg/mll Series III Histamine (100 ng/mll Histamine (100 ng/mll/ indomethacin (500 ng/mll Series IV heG (100 IV)/indomethacin (500 ng/mll heG (100 IV)/indomethacin (500 ng/mll/cimetidine (10 f-Lg/mll

4.88 ± 0.80 (19) 6.26 ± 0.83 (18) 6.09 ± 0.60 (19) 8.09 ± 1.18 (9) 9.05 ± 0.63 (16)a 8.31 ± 0.78 (13)

aSignificant at P < 0.05. bSignificant at P < 0.001.

Fertility and Sterility

DISCUSSION

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each group of hCG-treated ovaries progressed to at least the MI stage of development (P < 0.005). In the third series, after histamine or histamine/ indomethacin treatment, a greater percentage of ova progressed beyond the GV intact stage. This difference, however, was not significant. In the final series, in which both groups received hCG, the percentage of mature ova was increased. Indomethacin alone or in combination with cimetidine did not appear to influence the effect of hCG on ovum maturation. Examination of ovulated ova for signs of degeneration showed no significant differences in the percentage of degenerated ova between groups within each series of experiments (Fig. 3). Exposure to histamine and, to a greater extent, to indomethacin (series III and IV) increased the percentage of degenerated ova in comparison with all other treatment groups. The percentage of ovarian water content was consistently higher in those ovaries exposed to hCG than in those treated with PGF2a 9 alone. The degree of ovarian edema appears to be associated with ovulatory efficiency, as previously reported. 15 The mean figures and the standard error of the mean calculated for dry weight/wet weight x 100 were as follows for PGF2a : 14.6 ± 0.8, 13.0 ± 1.1, and 15.3 ± 1.5 at doses of 100 ng/ml, 1 ng/ml, and 0.1 ng/ml, respectively. The comparable figures for the contralateral control ovaries treated with hCG were 11.8 ± 0.9, 10.6 ± 0.6, and 12.6 ± 0.7. Percent dry weight per wet weight is the inverse expression of ovarian edema. Vol. 43, No.4, April 1985

The sequence of ovulatory events at the ovarian level initiated by the gonadotropin surge has received considerable attention. Several factors in. ovarian contractility, enzyme activity ' cludmg perifollicular changes, steroid production, PGs: and histamine, have been implicated in the mechanism offollicle rupture and ovum maturation. In this study we explored the interrelationship between histamine and PGs with regard to ovulation and ovum maturation. With the use of an in vitro system for studying ovulation in the rabbit ovary, PGF2a was capable of inducing ovulation at three doses with the highest ovulatory efficiency observed at 100 ng/ ml. Ovulatory efficiency was lower than that observed with standard ovulation-inducing doses of hCG (100 or 50 IU) but was greater than that with a minimal dose (5 IU) ofhCG. 8 The failure of PGF2a to induce ovulation with an efficiency comparable to hCG suggests that other components within the preovulatory follicular environment may be required for the follicle to attain full ovulatory potential. In the series of events that precede ovulation, we questioned whether PG production leads to histamine release or whether histamine release leads to PG production. We also investigated whether blockade of both PGs and histamine simultaneously can result in complete inhibition of hCG-induced ovulation. To answer these questions we made an attempt to block PGF2a -induced ovulation with the H2 antagonist, cimetiI 100

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649

dine, to block histamine-induced ovulation with the PG synthesis inhibitor, indomethacin, and to block hCG-induced ovulation with a combination of both indomethacin and cimetidine. Interestingly, cimetidine was unable to block PGF2a,-induced ovulation; histamin~induced ovulation could not be blocked by indomethacin; and hCG-induced ovulation, partially inhibited by indomethacin, was not further reduced by supplementation with cimetidine. From these and previously reported data, hCGinduced ovulation has been observed to be inhibited by indomethacin. Cimetidine or chlorpheniramine, either alone or in combination, does not block hCG-induced ovulation in the perfused rabbit ovaryP Cimetidine, together with indomethacin, does not further suppress indomethacin-inhibited ovulation. Histamine-induced ovulation can be blocked by cimetidine but not by chlorpheniramine or indomethacin. The sequential events that follow the gonadotropin surge include the production of PGs which may exert a direct role in the ovulatory process. That PGs alone can induce follicle rupture and that hCG-induced ovulation can be blocked by inhibition of PG synthesis serve as evidence for this concept. On the other hand, histamine does not appear to be essential in the mechanism of ovulation. Neither the H1 nor the H2 antagonist is effective in inhibiting hCG-induced ovulation, although follicle rupture induced by histamine alone can be inhibited by the H2 blocker, cimetidine. 12 It has been suggested that histamine-induced disruption of the follicle wall may be artifactual because of morphologic characteristics distinct from those observed in hCG-induced ovulation. 12 Furthermore, the failure of ova to undergo GV breakdown and further maturation following exposure to PGF2.. and histamine indicates that gonadotropin stimulation must elicit factors other than histamine and PGF2.. which are essential for ovum maturation. Other agents have also been shown to evoke follicle rupture without ovum maturation in this system. 16, 17 It is of interest that the latent period from onset of perfusion with the stimulating agent to time of ovulation did not differ significantly among hCG, PGF2.. , and histamine treatment groups. Therefore, the time required for ovum maturation was present in all three conditions; but with PGF2.. and histamine, the appropriate stimulus was apparently deficient. In the present series of experiments, ovum exposure to histamine and/or in650

Kitai et at. PGs and histamine in ovulation

domethacin correlated with ovum degeneration, an observation which has been reported previously.15 PGs may exert a protective influence on the ovum and thus prevent degeneration, as indicated in earlier studies. In contrast, histamine may have a deleterious effect on ovum development. Based on these experiments, although PGs and histamine are each capable of leading to follicle rupture, no sequence of production or interrelationship in their activity could be detected. Acknowledgments. We gratefully acknowledge the technical assistance of Thomas Henry and Maria Calzonetti. We would also like to thank Smith Kline Beckman, Philadelphia, Pennsylvania, and The Upjohn Company, Kalamazoo, Michigan, for graciously supplying agents necessary for the completion of these studies.

REFERENCES 1. Espey LL: Ovulation as an inflammatory reaction-a hypothesis. BioI Reprod 22:73, 1980 2. LeMaire WJ, Yang NST, Behrman HH, Marsh JM: Preovulatory changes in the concentration of prostaglandins in rabbit Graafian follicles. Prostaglandins 3:367, 1973 3. Hamada Y, Bronson RA, Wright KH, Wallach EE: Ovulation in the perfused rabbit ovary: the influence of prostaglandins and prostaglandin inhibitors. BioI Reprod 17:58, 1977 4. Wallach EE, de la Cruz A, Hunt J, Wright KH, Stevens YC: The effect of indomethacin on HMG-HCG induced ovulation in the rhesus monkey. Prostaglandins 9:645, 1975 5. O'Grady JP, Caldwell BY, Auletta FJ, Speroff L: The effects of an inhibitor of prostaglandin synthesis (indomethacin) on ovulation, pregnancy, and pseudopregnancy in the rabbit. Prostaglandins 1:97, 1972 6. Lau IF, Saksena SK, Chang MC: Prostaglandins F and ovulation in mice. J Reprod Fertil 40:467, 1974 7. Mori T, Kohda H, Kinoshita Y, Ezaki Y, Morimoto M, Nishimura T: Inhibition by indomethacin of ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin. J Endocrinol 84:333, 1980 8. Kobayashi Y, Santulli R, Wright KH, Wallach EE: The effect of prostaglandin synthesis inhibition by indomethacin on ovulation and ovum maturation in the in vitro perfused rabbit ovary. AmJ Obstet GynecoI141:53, 1981 9. Wallach EE, Hamada Y, Bronson RA, Wright KH, Stevens YC: Effectiveness of prostaglandin F 2a in restoration of HMG-HCG-induced ovulation in the indomethacintreated rhesus monkey. Prostaglandins 10:129, 1975 10. Hamada Y, Wright KH, Wallach EE: In vitro reversal of indomethacin-blocked ovulation by prostaglandin F 2a . Fertil Steril 30:702, 1978 11. Szego CM, Gitin ES: Ovarian histamine depletion during acute hyperaemic response to luteinizing hormone. Nature 201:682, 1964

Fertility and Sterility

12. Kobayashi Y, Wright KH, Santulli R, Kitai H, Wallach EE: Effect of histamine and histamine blockers on the ovulatory process in the in vitro perfused rabbit ovary. BioI Reprod 28:385, 1983 13. Wallach EE, Wright KH, Hamada Y: Investigation of mammalian ovulation with an in vitro perfused rabbit ovary preparation. Am J Obstet Gynecol 132:728, 1978 14. Lambertsen CJ Jr, Greenbaum DF, Wright KH, Wallach EE: In vitro studies of ovulation in the perfused rabbit ovary. Fertil Steril 27:178, 1976 15. Kobayashi Y, Wright KH, Santulli R, Wallach EE: Ovulation and ovum maturation in the rabbit ovary perfused in vitro. BioI Reprod 24:483, 1981

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16. Kobayashi Y, Sjoberg N-O, Walles B, Owman CH, Wright KH, Santulli R, Wallach EE: The effect of adrenergic agents on the ovulatory process in the in vitro perfused rabbit ovary. Am J Obstet Gynecol 145:857, 1983 17. Kobayashi Y, Kitai H, Santulli R, Wright KH, Wallach EE: The influence of a calcium- and magnesium-free environment on in vitro ovulation in the rabbit. Fertil Steril (Abstr) 39:396, 1983

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