The Effects of Progesterone and Human Chorionic Gonadotropin on Ovulation in the in Vitro Perfused Rabbit Ovary*†

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FERTILITY AND SFERIUTY Copyright © 1979 The American Fertility Society

Vol. 32, No.3, September 1979 Printed in U.SA.

THE EFFECTS OF PROGESTERONE AND HUMAN CHORIONIC GONADOTROPIN ON OVULATION IN THE IN VITRO PERFUSED RABBIT OVARY*t

YASUO HAMADA, M.D.:f: KAREN H. WRIGHT, M.S. EDWARD E. WALLACH, M.D.§ Department of Obstetrics and Gynecology, Pennsylvania Hospital and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19107.

The in vitro perfused rabbit ovary preparation was used to study the effect of progesterone on follicle rupture and to determine the minimum level of human chorionic gonadotropin (heG) required to induce ovulation in vitro. Both ovaries from an intact female rabbit were removed and simultaneously placed in separate perfusion systems. In four groups of five rabbits each, heG at a dose of 25, 10, 5, or 1 IU was added to the perfusate of both ovaries. An additional group received no heG. One ovary from each rabbit was treated with progesterone (1 mgl150 ml of perfusate) together with heG. The contralateral ovary served as a control, receiving only heG. The number of ovaries ovulating in each group varied directly with the amount of heG added to the perfusate (four of five with 25 IU, three of five with 10 IU, two of five with 5IU, one of five with 1 IU, and none offive without heG). In each group the number of ovulations per ovary did not increase significantly by the addition of progesterone to the perfusate. These data suggest that a dose-response relationship can be established between heG and ovulation in the in vitro model and that amounts of heG as small as 1 IU can produce ovulation in an in vitro perfused rabbit ovary preparation. Although exogenous progesterone did not appear to influence ovulation in this model, these data do not exclude the possibility that progesterone exerts a direct facilitatory effect on ovulation. Fertil Steril 32:335, 1979

Initiation of the ovulatory process by gonadotropins released from the hypophysis is an accepted phenomenon. However, our knowledge of the sequence of events which occurs between gonadotropin stimulation of the ovary and release of the ovum is currently incomplete. The in vitro- perfused rabbit ovary! is an ideal

model for studying the sequence of events leading to follicle rupture. The system permits isolation of the ovary from systemic influences, continual observation of the ovary during the time interval surrounding ovulation, addition to the perfusion fluid of various agents to be studied for their effects on ovulation, and the collection of the effluent from the ovary for measurement of substances produced by the ovary. This model was used in the present experiment (1) to determine the minimum dose of human chorionic gonadotropin (hCG) required to bring about ovulation in the in vitro perfused rabbit ovary and (2) to determine the effect ofprogesterone, added to the perfusate together with hCG, on the number of ovulations and the time of occurrence of ovulation in vitro. Previous experiments in our laboratory2 have shown that the addition of 100 IU ofhCG directly to the perfusate is capable of inducing ovulation in

Received October 16, 1978; revised April 3, 1979; accepted April 6, 1979. *Supported by National Institutes of Health Grant HD05948 and the Mitchell and Lillian Duberstein Foundation. tPresented at the Thirty-Fourth Annual Meeting of The American Fertility Society, March 29 to April 1, 1978, New Orleans, La. :f:Connelly Foundation Fellow in Reproductive Biology, Pennsylvania Hospital. Present address: Department of Obstetrics and Gynecology, Keio University, Tokyo, Japan. §Reprint requests: Edward E. Wallach, M.D., Department of Obstetrics and Gynecology, Pennsylvania Hospital, Eighth and Spruce Streets, Philadelphia, Pa. 19107.

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90% of ovaries placed in the perfusion system. Dose-response studies have not previously been carried out using this method. When hCG is administered intravenously to the rabbit the percentage of rabbits ovulating in vivo has been shown to vary directly with the amount ofhCG; a dose of 5 units was the minimum dose of hCG required to induce ovulation. 3 Although preovulatory follicles apparently secrete progesterone,4'9 the role of progesterone in the ovulatory process is not well understood. Ronde1l 10 has postulated that steroids synthesized by the ovary, particularly progesterone, are essential intermediaries in the normal ovulatory process. Progesterone may stimulate ovulation by exerting a direct effect on the ovary. Multiple ovulations can be induced in hamster ovaries explanted to medium containing progesterone. 1 t. 12 In addition, preovulatory changes in porcine follicles can be initiated in vitro by the addition of progesterone to the incubation medium. 13

ovaries received a uniform amount of progesterone, 1 mg/150 ml of perfusate. hCG was added to the perfusate of both ovaries in the following doses: group 1, 25 IU of hCG/150 ml; group 2, 10 IU of hCG/150 ml; group 3, 5 IU ofhCG/150 ml; group 4, 1 IUofhCG/150 ml. In group 5, which served as an additional control, no hCG was added to the perfusion fluid. Hourly observations were made for ovulation points in both the experimental and control perfused ovaries. Follicles were considered ruptured only when an extruded cumulus oophorus containing an ovum was observed on the surface of the ovary. Each experiment was concluded 12 hours following hCG administration. At the conclusion of the experiment, each ovary was fixed in Bouin's solution and prepared for histologic examination. The numbers of ovaries ovulating in each group were compared using the test. The number of ovulation points per ovary and the number of ovulation points per ovulating ovary were compared using Student's t-test.

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MATERIALS AND METHODS

Sexually mature New Zealand White female rabbits were isolated for a minimum of 3 weeks under controlled temperature and light and fed Purina rabbit chow and water ad libitum. Rabbits were anesthetized with sodium pentobarbital (32 mg/kg intravenously). The cannulation technique and perfusion system have been described in detail previously. 1 At the time oflaparotomy, the ovarian artery is cannulated following ligation of all its branches. The ovary with its vein and artery and supporting adipose tissue is placed in the perfusion chamber and perfusion is begun within 5 minutes of removal. The perfusion system consists of a pulsatile pump which circulates perfusion fluid through an oxygenator to the tissue contained in a perfusion chamber. The perfusion fluid, 150 ml of tissue culture medium 199, is supplemented with heparin sulfate (20 units/liter); regular insulin (200 units/liter); streptomycin (50 mg/liter); and penicillin (75 mg/liter). The perfusate enters the ovary through the ovarian artery cannula and is collected from the unligated ovarian vein. The perfusion fluid is recycled; therefore, the ovary is continually exposed to hCG and progesterone. At the onset of perfusion, hCG was added to the fluid supplying the control ovary. Simultaneously, hCG together with progesterone was added to the perfusion fluid of the contralateral experimental ovary. Five groups consisting of five animals each were studied. In all groups the experimental

RESULTS

The number of ovaries ovulating in every group increased in proportion to each increment in the amount ofhCG added to the perfusate. One unit of hCG resulted in ovulation in one offive ovaries; 5 units, two of five; 10 units, three of five; 25 units, four offive (Table 1). The only increase in number of ovaries ovulating with the addition of progesterone was observed at the 5-unit dose level of hCG; however, this difference was insignificant (Table 1). Thus, the addition of supplemental progesterone (1 mg/150 ml of perfusate) together with hCG did not significantly influence the number of ovaries ovulating. Ovulation did not occur in any preparation without hCG regardless of whether or not progesterone was included in the perfusate. Comparisons (Table 1) were made of the numbers of ovulation points occurring in ovulating ovaries among all groups. The addition of progesterone to the perfusate was associated with an increase in the number of ovulation points per ovulating ovary; however, this difference was not significant. The data were also examined with regard to the total number of ovulations observed in all ovaries. A greater number of ovulations occurred in ovaries in which progesterone had been included in the perfusate; this increase was also insignificant. The ratio of ruptured follicles observed at the

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Vol. 32, No.3

EFFECTS OF PROGESTERONE AND hCG ON OVULATION IN VITRO

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conclusion of the experiment to mature follicles observed at the time of laparotomy was also compared in all groups. The ratio provides an index of the over-all influence of any specific variable on the final stages of follicular maturation in vitro. The number of mature follicles proceeding to rupture increased in proportion to the amount ofhCG included in the perfusate. The inclusion of supplemental progesterone in the perfusate further enhanced the number of mature follicles ultimately achieving rupture (Table 1); this difference was not significant . Ovulations occurred between the 2nd and 11th hour following hCG administration. The amount ofhCG included in the perfusate did not affect the time of occurrence of ovulation. Most of the ovulations were clustered during the interval from 4 to 6 hours after hCG (hCG-only: mode = 4th hour, median = 4th hour; hCG and progesterone; mode = 4th hour, median = 6th hour). Ovulation occurred earlier than when hCG was administered to the intact animal prior to ovarian removal and perfusion begun 4 hours after hCG (mode = 8th hour, median = 9th hour)2 The addition of supplemental progesterone neither accelerated nor delayed the occurrence of ovulation. Histologic examination of sections from the perfused ovaries did not reveal any abnormalities in follicle development or tissue viability secondary to the perfusion.

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DISCUSSION

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Local progesterone elaborated by the ovary has been implicated as a significant factor in the mechanism of ovulation. Experiments conducted on rats have provided evidence indicating that either preovulatory production of progesterone by the ovary or progesterone administration can enhance ovulation in vivo. 6 , 14, 15 Baranczuk and Fainstat,l1, 12 using an organ culture of the hamster ovary as an in vitro model, have also suggested that progesterone contributes to the terminal follicular events resulting in ovulation. These authors have considered progesterone to be the ultimate hormonal mediator of ovulation in the follicle matured and prepared by the appropriate sequence of hormonal stimuli. 12 Measurements of ovarian vein progesterone in the rabbit after mating demonstrated an increase prior to follicular rupture. 7 Mills and Savard 8 were able to detect an increase in synthesis of progesterone in rabbit ovaries isolated shortly after mating. Wu et a1. 9 also reported increased progesterone secretion in

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the estrous rabbit with levels peaking at 4 hours following hCG administration. The estrous rabbit, which normally exhibits ovulation in response to coitus, will ovulate in response to progesterone after pretreatment with estrogen. 16 Progesterone also facilitates coitalinduced ovulation or ovulation following either cervical stimulation or copper acetate administration. 16 Progesterone has been reported by Takahashi et al. 17 to enhance ovulation in the hypophysectomized rat. In those experiments, progesterone induced ovulation in follicles previously exposed to endogenous luteinizing hormone which had been released just prior to hypophysectomy. Ovulation of the mature follicle is preceded by increased distensibility of the follicular wal1. 13 In porcine follicles these changes can be induced in vitro by the addition of progesterone to the incubation medium. Luteinizing hormone and cyclic adenosine 3':5'-monophosphate are also able to induce the appropriate changes in distensibility. The effects of these agents on distensibility may be blocked by adding cyanoketone, an inhibitor of 3~-hydroxysteroid dehydrogenase, to the incubation medium. The addition of progesterone to the medium overcomes the cyanoketone block. Rondell 10 has interpreted these results as suggesting that progesterone may be directly responsible for the final stages of follicular rupture. The results of the present investigation fail to demonstrate that exogenous progesterone enhances ovulation. These data do not, however, exclude the possibility that locally produced progesterone acting directly on the ovary may foster rupture of mature follicles. Observations that progesterone administration facilitates ovulation do not prove conclusively that spontaneous ovulation is a direct result of the action of progesterone. The incorporation of specific progesterone antiserum in a similar preparation isolated from systemic influences would be helpful to substantiate ovarian progesterone production as a prerequisite for ovulation. Determinations of serial progesterone levels in the perfusate during the course of an experiment may also provide further information regarding the influence of progesterone on ovulation. It is quite possible that local production of progesterone is sufficient to enhance ovulation of mature follicles in this system and that exogenous progesterone in the perfusate is superfluous. The results of the other aspect of this study demonstrate a dose-response relationship between the amount of hCG added to the perfusate and the

September 1979

number of ovulations in the in vitro system. The 80% ovulation rate in vitro using 50 IU of hCG parallels that in our previous experiments!' 2 and is similar to the ovulatory rate in in vivo controls.l Ovulation occurred with as small a dose as 1 IU of hCG. These data parallel in vivo observations by Edgren and Carter. 3 Using intravenous hCG at 40, 20,10,5, and 2.5 IU dose levels, ovulation occurred in 100%, 80%, 50%, 20%, and animals, respectively. This linear relationship was upheld in our in vitro studies. The lower doses of hCG capable of achieving ovulation in the in vitro model may relate to higher effective circulating concentrations and lack of hCG clearance from this system. Measurement of hCG in the perfusate has indicated that virtually all of the hCG is still present 12 hours after its addition to the perfused rabbit ovary preparation. (Double-antibody radioimmunoassay for ,8-hCG was kindly performed by Sheldon Schlaff, M.D.) Although progesterone appeared to enhance ovulation at each dose level of hCG, this change was not significant. Furthermore, there is no evidence to suggest that progesterone in the absence of gonadotropins can bring about ovulation in vivo or in vitro.

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Acknowledgments. The authors gratefully acknowledge the excellent technical assistance of Anne T. Barrett, Thomas J. Henry, and Judy Reitman. REFERENCES 1. Lambertsen CJ Jr, Greenbaum DF, Wright KH, Wallach

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EE: In vitro studies of ovulation in the perfused rabbit ovary. Ferlil Steril 27: 178, 1976 Wallach EE, Wright KH, Hamada Y: Investigation of mammalian ovulation using an in vitro perfused rabbit ovary preparation. Am J Obstet Gynecol 132:728, 1978 Edgren RA, Carter DL: Failure of various steroids to block gonadotropin-induced ovulation in rabbits. J Endocrinol 24:525, 1962 Resko JA, Koering MJ, Goy RW, Phoenix CH: Preovulatory progestins: observations on their source in rhesus monkeys. J Clin Endocrinol Metab 41:120, 1975 Norman RL, Greenwald GS: Effect of phenobarbital, hypophysectomy and x-irradiation on preovulatory progesterone levels in the cyclic hamster. Endocrinology 89:598, 1971 Meyer RK, Karavolas HJ, Klausing M, Norgard DW: Blood progesterone and pregnenolone levels during phenobarbital (PB) block of PMS-induced ovulation in immature rats. Endocrinology 88:983, 1971 Hilliard J, Eaton LW, Estradiol-17{3, progesterone and 200'-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. II. From mating through implantation. Endocrinology 89:522, 1971 Mills TM, Savard K: Steroidogenesis in ovarian follicles isolated from rabbits before and after mating. Endocrinology 92:788, 1973

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9. Wu CH, Blasco L, Flickinger GL, Mikhail G: Ovarian function in the preovulatory rabbit. BioI Reprod 17:304, 1977 10. Rondell P: Role of steroid synthesis in the process of ovula~ tion. BioI Reprod 10:199, 1974 11. Baranczuk RJ, Fainstat T: In vitro ovulation from adult hamster ovary. Am J Obstet GynecoI124:517, 1976 12. Baranczuk RJ, Fainstat T: Progesterone-induced ovulation of the hamster ovary in vitro.J Endocrinol 70:317, 1970 13. Rondell P: Follicular processes in ovulation. Fed Proc 29:1875,1970 14. Kobayashi F, Hara K, Miyake T: Facilitation by progesterone of ovulating hormone release in sodium pentobarbital-blocked proestrous rats. Endocrinol Jap 20:175, 1973

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15. Sridharan BN, Meyer RK, Karavolas HJ: Induction of ovulation by pregn-5-ene-3,20-dione and progesterone in immature rats treated with PMSG and phenobarbital. J Reprod Fertil 39:259,1974 16. Sawyer CH, Everett JW: Stimulatory and inhibitory effects of progesterone on the release of pituitary ovulating hormone in the rabbit. Endocrinology 66:644, 1959 17. Takahashi M, Ford JJ, Yoshinaga K, Greep RO: Induction of ovulation in hypophysectomized rats by progesterone. Endocrinology 95:1322, 1974