Intraovarian Pressure Changes During Ovulation in Rabbits*†

FERTILITY AND STERILITY Copyright 1976 The American Fertility Society

Vol.

27,

No.2, February 1976 Printed in U.S.A.

INTRAOV ARIAN PRESSURE CHANGES DURING OVULATION IN RABBITS*t PRAMUAN VIRUTAMASEN, M.D., YUTHANA SMITASIRI, B.Sc., AND ANNA-RIITTA FUCHS, M.Sc.t

Laboratory for Reproductive Biology and Physiology, Department of Obstetrics and Gynecology, Chulalongkorn University Medical School, Bangkok, Thailand, and Biomedical Division, The Population Council, The Rockefeller University, New York, New York 10021

The process by which follicular rupture is brought about at ovulation remains enigmatic. Several possible mechanisms have been considered. 1 Enzymatic action is indicated by light and electron microscopic studies which demonstrate a thinning of the wall of the Graafian follicle at the follicular stigma. 2 • 3 A dispersion of collagen fibers has been observed at the apex of the follicle, 4 but no increase in collagenase activity or in the activity of other proteolytic enzymes was detected in the follicles by biochemical means. It has also been proposed that rupture results from increased follicular pressure caused by the contraction of perifollicular muscular fibers, 5 but measurement of intrafollicular pressure did not show any significant increase during maturation and near ovulation. 6 • 7 Besides the perifollicular muscle fibers, which do not seem to be present in all species, smooth muscle cells in ovarian cortical stroma have been demonstrated in all species thus far studied by both light and electron microscopy. 8 ' 12 In addition, fluorescence microscopy combined with electron microscopy has revealed a Received March 28, 1975. *Supported by grants from the Ford Foundation (to P. Virutamasen) and The Population Council (to Chulalongkorn University). t Presented in part at the Annual Meeting of the Society for the Study of Reproduction, Ottawa, Ont., Canada, August 1974. tReprint requests: Anna-Riitta Fuchs, M.Sc., The Population Council, The Rockefeller University, New York, N.Y. 10021.

close approximation of ovarian adrenergic nerve terminals with these smooth muscle-like cells, 13 • 14 suggesting that the ovarian contractile elements might be controlled by the sympathetic nervous system. Rocerto et al.1 5 were the first to demonstrate that the ovary is capable of spontaneous contractile activity in vitro, and that it responds to catecholamines added to the bath. Several in vivo experiments have since been carried out with anesthetized animals, 16 - 18 as well as with human ovaries in situ. 19• 20 These observations suggest that ovarian steroids may modify the responsiveness of the ovarian contractile elements to various smooth muscle-stimulating agents, and that human chorionic gonadotropin (hCG) may influence the ovarian contractility pattern at the time of anticipated ovulation. Although the results from these acute experiments in vivo and in vitro have contributed much to our present knowledge of the factors that may alter or induce ovarian contractile activity in various species, 16- 18 • 21 -24 they have not yet provided an answer to the question of whether ovarian contractility increases at the time of ovulation. The purpose of the present investigation was to provide a technique for chronic recording of ovarian contractile activity in otherwise intact, unanesthetized animals, and to gain information on ovarian contractility during actual ovulation. Rabbits were used as the experimental ani-

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INTRAOVARIAN PRESSURE DURING OVULATION

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FIG. 1. Balloon-tipped catheter used for recording intraovarian pressure changes in chronic preparations of otherwise intact rabbits. The device consists of a fenestrated polyethylene tube covered with thin latex tubing, tied in place by 6-0 nylon suture. It is inserted into the ovary by means of a straight needle and a piece of silk, by simply pulling it through the ovarian stroma.

mals because their ovaries are large enough to accommodate a small pressurerecording device and because ovulation can be timed accurately in this species.

sule and the ovarian ligament with 6-0 nylon sutures. This was done during a laparotomy performed under pentobarbitone anesthesia. The balloon was filled with 0.02 to 0.04 ml of water, according to the size of the ovary. Blanching of the MATERIALS AND METHODS ovary indicated when the balloon was too lntraovarian pressure changes were distended and interfered with the ovarian monitored by means of a small balloon, blood supply. Figure 2 shows the balloon made by tying a piece of thin latex tubing in place. The other end of the catheter over the perforated end of a polyethylene was then sealed with heat, pushed tube (PE 20; 0.015 inch inner diameter, through the muscle ofthe flank, tunneled 0.043 inch outer diameter). The latex subcutaneously to the back of the neck, tubing was approximately 10 mm long and and exteriorized there through a puncture was tied with 6-0 nylon suture, at both of the skin. The abdomen was closed in ends, around the catheter, as is seen in two layers with catgut and the animals Figure 1. This balloon-tipped catheter is were allowed to recover from the operation. similar to that used by Coutinho and Recordings could then be made by connectMaia 19 to record contractility of the human ing the catheter to Statham pressure ovary, only smaller in dimension. The transducers (PA-23AC) and a Grass model balloon was placed within the ovarian 7P polygraph. During actual recordings, stroma by pulling it through the longi- the animals were kept in restricting cages tudinal axis by means of a straight needle. but were otherwise kept in their usual It was then anchored to the ovarian cap- cages.

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FIG. 2. The pressure-recording device in place inside the ovary.

Altogether, 25 mature New Zealand rabbits of mixed breed, weighing between 3.5 and 4.5 kg, were used. In some of the rabbits, another balloon was placed within the lumen of the midampullary portion of the ipsilateral fallopian tube, and, in some rabbits, a balloon was placed in both ovaries.

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RESULTS

The ovaries of all 25 rabbits studied exhibited regular spontaneous contractions. Figure 3 illustrates the types of intraovarian pressure changes that occurred in mature, nonpregnant rabbits with ovaries containing medium-sized and large follicles. These changes consisted of

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FIG. 3. Simultaneous recordings of intrauterine pressure from both ovaries of a rabbit having many medium-sized and large follicles but no corpora lutea.

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INTRAOVARIAN PRESSURE DURING OVULATION

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FIG. 4. Simultaneous recordings of intraovarian and intratubal pressure changes from the midampullary portion of the ipsilateral fallopian tube. The upper tracing of each pair is from the ovary, the lower tracing from the tube.

periodically occurring series of rapid pressure fluctuations that were often associated with an elevated base line pressure. The length of such active periods varied from 1.8 to 3.8 minutes (average, 2. 7 minutes ± 0.15 SE). The intervals between these periods varied from 11 to 32 minutes, averaging 22.7 minutes ± 2.0 SE. During the periods of contractile activity, the intraovarian pressure sometimes increased as much as 15 mm Hg,

5

but the average pressure increase was approximately 6 mm Hg (6.0 ± 0.52 SE). As can be seen in the rabbit in which recordings were obtained from both ovaries (Fig. 3), the contractions appeared asynchronously in each ovary and the stimulus for the contractions, therefore, appeared to arise locally and not to be systemically mediated. Figure 4 shows contractions obtained from the ovary and the ipsilateral fallo-

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FIG. 5. The effect of intravenously injected norepinephrine (NE), PGF""' and oxytocin (OT) on intraovarian and intratubal pressure, recorded from a rabbit with normal estrous ovaries.

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FIG. 6. Continuous monitoring ofintraovarian pressure changes in a rabbit receiving 100 IU of hCG intravenously at the beginning of the second tracing. Recordings from before the injection of the gonadotropin until 9 hours after the injection are shown.

pian tube simultaneously. The contractions of each tissue occurred quite independently in all six rabbits studied in this way. Figure 5 shows the responses of both ovary and tube to intravenously injected norepinephrine (5 p.,g), prostaglandin F 2" (PGF:za, 25 and 50 p.,g), and oxytocin (250 mU). The ovary responded to all three agents with an increase in intraovarian pressure; the tube showed a contractile response to norepinephrine and PGF 2", but not to oxytocin.

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In 11 rabbits, recordings were made continuously for 24 hours after an ovulatory dose (100 IU) of hCG had been injected intravenously. Figures 6, 7, and 8 show recordings obtained from one of these rabbits. Each line represents intraovarian pressure changes during each subsequent hour after the injection. There was no immediate effect of the injection, and ovarian activity continued unaltered for several hours, as is seen in Figure 6. At 7 to 8 hours after the injection, ovarian contractile activity started

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FIG. 7. Continuous monitoring ofintraovarian pressure changes in the rabbit shown in Figure 6, 10 to 17 hours after the injection of 100 IU of hCG intravenously.

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INTRAOVARIAN PRESSURE DURING OVULATION

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FIG. 8. Continuous monitoring of intraovarian pressure changes in the rabbit shown in Figures 6 and 7. Recordings show tracings obtained 17 to 25 hours after the injection of 100 IU of hCG intravenously, as well as 3 and 5 days after ovulation.

to increase in that the intervals between bursts of activity became progressively shorter. At 10 to 12 hours after the injection, the ovaries were continuously active and there were no quiescent intervals (Fig. 7). No increase in the amplitude of contractions was observed about the expected time of ovulation in any of the rabbits studied. The state of increased activity persisted for 5 to 6 hours before it gradually diminished, becoming again periodic, and at about 24 hours it was similar to that observed before the injection of hCG (Fig. 8). By day 3, activity had further diminished but, by day 5 or 6,

the activity was again of an intensity similar to that of the preinjection period. In 12 rabbits, the changes in intraovarian pressure were recorded after natural mating. Ovulation had occurred in all of them, as verified by laparotomy 5 to 7 days later, when prom~nent corpora lutea were seen in all ovaries. The changes in ovarian contractile activity were similar to those observed after injection of 100 IU ofhCG, but were somewhat less pronounced. In the mated group, ovarian activity never became quite continuous, as it did in the hCGinjected group, but the duration of the

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FIG. 9. Changes in ovarian contractility following mating, expressed as the duration of quiescent intervals between series of contractions. Values are means of 12 rabbits, calculated on an hourly basis, starting from mating. Bars represent standard errors of the mean; the broken line is the mean value for the control period before mating; the hatched area represents ± 2 SEM.

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February 1976

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FIG. 10. Changes in ovarian contractility following mating, expressed as the duration of the series of contractions that occurred periodically. Values are means of 12 rabbits calculated on an hourly basis, starting from mating. Bars represent standard' errors of the mean; the broken line is the value for the control period, prior to mating; the hatched area represents ± 2 SEM.

active periods increased and the intervals between them were significantly shortened in comparison with control periods. The results were quantitated by measuring the intervals from one series of contractions to the next, calculated on an hourly basis for all 12 rabbits. Figure 9 shows the duration of quiescent intervals in 12 rabbits before mating and in the 24 subsequent hours after mating. Each point represents the mean of all intervals within 1 hour. The broken line shows the mean value for the control period and the shaded area represents ± 2 SEM. From the 8th hour onward, these intervals were significantly shortened, but returned to control values approximately 15 hours after mating. Figure 10 shows the duration of the active periods. Each point represents the mean of all periods during 1 hour. Again, the broken line indicates the mean value for the control period and the shaded area represents ± 2 SEM. The increase in duration became evident 5 hours after mating and persisted with minor fluctuations for the next 20 hours. As in the hCG-treated animals, the amplitude of the contractions did not change significantly during this period nor did the frequency of the rapid pressure changes during the active periods change significantly at any time after ovulation.

DISCUSSION

The timing of ovulation was not ascertained in the present experiments, but others have shown that ovulation in rabbits takes place in the time range of 91h to 13 hours after an injection of luteinizing hormone, 25 which is in agreement with observations in rabbits examined after coitus-induced ovulations. 26 The midpoint of ovulation in both instances occurred between 101h and 10% hours after the ovulatory stimulus. Coitus did not always induce ovulation, but in the experimental group the presence of fresh corpora lutea on day 5 or 6 after mating indicated that none of the animals failed to ovulate in response to copulation. Thus, the presence of the transducer in the ovarian stroma did not interfere with the action of the endogenous gonadotropins, and there is no reason to believe that the transducer inhibited the action of the exogenous hormone in the hCG-injected group. In intact animals, all does receiving similar doses of hCG ovulated. 27 The possibility that the presence of the transducer altered the time sequence of ovulation cannot be excluded, but it seems quite unlikely in view of the normal appearance of the ovaries at the end of the experiment. These studies demonstrate that, in the period surrounding the interval of anticipated ovulation, the ovary exhibits signif-

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INTRAOVARIAN PRESSURE DURING OVULATION

icantly increased contractile activity. Apparently, the whole ovary contracts in a pulsatile fashion, resulting in increased intraovarian pressure at intervals of roughly 20 minutes, which become significantly shorter some hours before and during ovulation. Such contractions must result in a squeezing and pulling action at the periphery, where the follicles are located. In association with the thinning of the follicular wall, and the decrease in the breaking strength of the preruptured follicles, 28 these contractions of the underlying stroma could lead to the rupture of the follicles at the point of least resistance, the apex, at normal intrafollicular pressures. There is good temporal correlation with this increase in activity with the increase in ovarian PGF 2'>' content following hCG injections observed by both Armstrong and co-workers 29 and LeMaire et al.3° Since PGF 2'>' is a potent stimulator of ovarian contractility, it is tempting to postulate that the increase in follicular PGF2'>' that occurs before ovulation stimulates ovarian contractility which, in turn, promotes follicular rupture and expulsion of the ovum. Grinwich et al.3 1 could, in fact, demonstrate that indomethacin, given as late as 5 hours after hCG injection, apparently prevented the extrusion of the ovum without affecting luteinization of the granulosa cells that occurred in response to the injected gonadotropin. The effects of indomethacin treatment on the contractility of rabbit ovaries were studied in in vitro preparations and in acute in vivo experiments.32 The results clearly indicated that PGF 2'>' enhanced ovarian contractility in indomethacin-treated rabbits, while the results regarding the effect of indomethacin treatment on spontaneous ovarian contractility are equivocal and inconclusive. Work is in progress to use our chronic preparations to study the effect of indomethacin on ovarian contractile activity following mating and hCG injections.

195

SUMMARY

Contractile elements are found in the ovaries of many species, but it has not been possible to ascertain whether these elements are of importance in the process of ovulation. In this report, we describe changes in intraovarian pressure recorded continuously in vivo in unanesthetized rabbits under normal conditions and under the influence of intravenously injected human chorionic gonadotropin (hCG), as well as following the ovulatory stimulus of normal copulation. The recordings were made by means of small latex balloons (0.02- to 0.04-ml volume) attached to indwelling catheters, inserted into the ovarian stroma, and secured with 6-0 nylon sutures. All 24 rabbits studied showed changes in intraovarian pressure indicative of ovarian contractile activity. The intraovarian pressure changes followed a characteristic pattern which was different from the changes in intratubal pressure, recorded simultaneously from the lumen of the ipsilateral fallopian tube, indicating that the contractions of both organs occurred independently. In normal animals, before an ovulatory stimulus was applied, the ovarian contractility pattern consisted of a series of rapid contractions (average amplitude, 6 mm Hg; average frequency; 8 per minute) occurring with intervals of quiescence lasting from 11 to 36 minutes. The base line tonus was frequently elevated during these series of contractions. Mating or an injection of hCG had no immediate effect on intraovarian pressure but, 6 to 8 hours after the stimulus was applied, ovarian contractile activity increased significantly in all rabbits. This enhanced activity persisted for several hours before returning to initial levels approximately 15 to 18 hours after mating or the hCG injection. This demonstration of increased contractile activity about the time of ovulation suggests that ovarian contractions participate in the process offollicular rup-

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ture and the extrusion of ova at ovulation. Prostaglandin F 2, , norepinephrine, and oxytocin were effective in inducing ovarian contractions. Acknowledgments. We appreciate the technical assistance of Ong-Aij Louhawat and Pricha Chumrearnchantavong. REFERENCES 1. Blandau RJ: The mechanisms of ovulation. In Ovulation, Edited by RB Greenblatt. Philadelphia, JB Lippincott, 1966, p 1 2. Espey LL: Ultrastructure of the apex of the rabbit Graafian follicle during the ovulatory process. Endocrinology 81:267, 1967 3. Parr EL: Rupture of ovarian follicles. J Reprod Fertil [Suppl) 22:1, 1975 4. Anderson JW, Yatvin MB: Metabolic and ultrastructural changes in the frog ovarian follicle in response to pituitary stimulation. J Cell Bioi 46:491, 1970 5. Lipner HJ, Maxwell BA: Hypothesis concerning the role of follicular contractions in ovulation. Science 131:1737, 1960 6. Blandau RJ, Rumery RE: Measurement of intrafollicular pressure in ovulatory and pre-ovulatory follicles of the rat. Fertil Steril 14:330, 1963 7. Espey LL, Lipner H: Measurement of intrafollicular pressures in the rabbit ovary. Am J Physiol 205:1067, 1963 8. Guttmacher MS, Guttmacher AF: Morphological and physiological studies on the musculature of the mature Graafian follicle of the sow. Bull Johns Hopkins Hosp 32:394, 1921 9. Oswaldo-Decima L: Smooth muscle in the ovary of the rat and monkey. J Ultrastruct Res 29: 218, 1970 10. O'Shea JD: An ultrastructural study of smoothmuscle-like cells in the theca externa of ovarian follicles in the rat. Anat Rec 167:127, 1970 11. Okamura H, Virutamasen P, Wright KH, Wallach EE: Ovarian smooth muscle in the human being, rabbit and cat. Histochemical and electron microscopic study. Am J Obstet Gynecol 112:183, 1972 12. Burden HW: Ultrastructural observations on ovarian perifollicular smooth muscle in the cat, guinea pig and rabbit. Am J Anat 133:125, 1972 13. Owman C, Sjoberg N-0: Adrenergic nerves in the female genital tract of the rabbit. With remarks on cholinesterase-containing structures. Z Zellforsch Mikrosk Anat 74:182, 1966 14. Rosengren E, Sjoberg N-0: The adrenergic nerve supply to the female reproductive tract of the cat. Am J Anat 121:271, 1967 15. Rocerto T, Jacobowitz D, Wallach EE: Observations of spontaneous contractions of the cat ovary in vitro. Endocrinology 84:1336, 1969

February 1976

16. Virutamasen P, Wright KH, Wallach EE: Effects of catecholamines on ovarian contractility in the rabbit. Obstet Gynecol 39:225, 1972 17. Virutamasen P, Wright KH, Wallach EE: Effects of prostaglandins E 2 and F 'P on ovarian contractility in the rabbit. Fertil Steril 23:675, 1972 18. Virutamasen P, Wright KH, Wallach EE: Monkey ovarian contractility-its relationship to ovulation. Fertil Steril 24:763, 1973 19. Coutinho EM, Maia H: Effects of gonadotropins on motility of human ovary. Nature [New Bioi) 235:94, 1972 20. Coutinho EM, Maia H, Maia H Jr: Ovarian contractility. In Physiology and Genetics of Reproduction, Edited by EM Coutinho, F Fuchs. New York, Plenum Press, 1974, p 127 21. Walles B, Edvinsson L, Nybell G, Owman C, Sjoberg N-0: Amine-induced influence on spontaneous ovarian contractility in the guinea pig and the cat. Fertil Steril 25:602, 1974 22. Okamura H, Okazaki T, Nakajima A: Effects of neurotransmitters and prostaglandins on human ovarian contractility. Obstet Gynecol 44: 720, 1974 23. Diaz-Infante A, Virutamasen P, Connaughton JF, Wright KH, Wallach EE: In vitro studies of human ovarian contractility. Obstet Gynecol 44:830, 1974 24. Diaz-Infante A, Wright KH, Wallach EE: Influence of estrogen and progesterone treatment on ovarian contractility in the monkey. Fertil Steril 26:101, 1975 25. Harper MJK: Ovulation in the rabbit: the time of follicular rupture and expulsion of the eggs, in relation to injection of luteinizing hormone. J Endocrinol26:307, 1963 26. Walton A, Hammond J: Observations on ovulation in the rabbit. Br J Exp Bioi 6:190, 1929 27. Fox RR: Coitus as ovulation stimulant in the laboratory rabbit. Proc Soc Exp Bioi Med 128: 639, 1968 28. Rondell P: Biophysical aspects of ovulation. Bioi Reprod [Suppl) 2:64, 1970 29. Armstrong DT, Grinwich DL, Moon YS: Inhibition of ovulation in rabbits by intrafollicular injection of indomethacin and prostaglandinantiserum. Life Sci 14:129, 1974 30. LeMaire WJ, Yang NST, Behrman HR, Marsh JM: Pre-ovulatory changes in the concentration of prostaglandins in rabbit Graafian follicles. Prostaglandins 3:367, 1973 31. Grinwich DL, Kennedy TG, Armstrong DT: Dissociation of ovulatory and steroidogenic actions of luteinizing hormone in rabbits with indomethacin, an inhibitor of prostaglandin biosynthesis. Prostaglandins 1:69, 1972 32. Diaz-Infante A, Wright KH, Wallach EE: Effects of indomethacin and prostaglandin F 'P on ovulation and ovarian contractility in the rabbit. Prostaglandins 5:567, 1974