Search

An in vitro perfused ovary preparation was used to study the role of prostaglandin F2alpha (PGF2alpha) in follicle rupture. The administration of PGF2alpha alone has been shown to restore indomethacin-blocked ovulation in rabbits and monkeys. In the model used, ovulation consistently occurred when human chorionic gonadotropin (hCG) was given to the intact rabbit prior to ovarian removal. hCG-induced ovulation was blocked in both perfused and in situ control ovaries by indomethacin (10 mg/kg intravenously 6 hours after hCG) given to the intact animal. The addition of PGF2alpha (1 mg/200 ml) to the perfusion fluid restored ovulation in the isolated ovary as compared with the in situ ovary (P less than 0.005) and with the perfused, untreated ovary (P less than 0.01). Following removal and perfusion of both ovaries from rabbits treated with indomethacin, ovulation occurred following the addition of PGF2alpha to the perfusate, but did not occur without PGF2alpha (P less than 0.05). These data indicate that indomethacin can block ovulation and that ovulation can be restored by the addition of PGF2alpha to the perfusion system, further supporting the significance of PGF2alpha in the process of follicular rupture.

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. PGF2 alpha 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 PGF2 alpha-treated ovaries was lower than that of ovaries exposed to human chorionic gonadotropin (hCG, 100 IU). PGF2 alpha-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 hCG 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 hCG. On the basis of these experiments, PGs and histamine do not appear to be interdependent in their effects on the ovulatory process in vitro.

An increase in the content of histamine in the ovary following luteinizing hormone (LH) release and the inhibition of ovulation in the rabbit by antihistamines suggest that histamine may be involved in the ovulatory process. The effects of various doses of histamine and antihistamines on ovulation were investigated using the in vitro perfused rabbit ovary system. Histamine (100 ng/ml) added to the perfusate at hourly intervals induced ovulation, although at a rate below that observed following human chorionic gonadotropin (hCG) administration. Cimetidine (10 micrograms/ml), an H2 blocker, inhibited histamine-induced ovulation, while the H1 blocker, chlorpheniramine (66.7 micrograms/ml), failed to do so. Neither cimetidine nor chlorpheniramine was able to block ovulation following hCG (50 IU). In all experimental groups in which histamine was used to induce ovulation, both extruded ova and follicular oocytes remained in an immature stage and displayed little evidence of degeneration. In contrast, a high percentage of ova exposed to hCG were mature. Ovarian edema was increased in ovaries in which ovulation occurred, regardless of treatment. A linear correlation was noted between ovulatory efficiency and degree of ovarian edema. Histamine may be an intermediary in the mechanism of follicular rupture, but does not support ovum maturation. However, the inability of H1 and H2 antagonists to block hCG-induced ovulation raises questions regarding the role of histamine in the physiologic process of ovulation.

An in vitro perfused rabbit ovary preparation has been developed to study local factors responsible for the ovulatory process. This preparation enables: (1) isolation of the ovary from systemic influences, (2) direct serial observations of follicle development and ovulation, (3) addition to the perfusing medium of substances that may influence ovulation acting at the level of the ovary, and (4) correlation of the times of individual follicle rupture with ovarian contractile patterns. With this model the temporal requirements for human chorionic gonadotropin in the process of ovulation have been studied. The direct effects on ovulation of prostaglandin F3 alpha (PGF 2 alpha), the antihistamine chlorpheniramine, and calcium deprivation were also investigated. Chlorpheniramine and EDTA each led to inhibition of ovulation and depressed ovarian contractility. The use of the isolated in vitro ovary preparation permits characterization of the local requirements for ovulation.

The investigation was designed to study the influence of indomethacin on gonadotropin induced ovulation in the rhesus monkey. Six mature female monkeys were treated with HMG-HCG for at least 2 control ovulatory cycles at dosage levels adjusted to induced ovulation while avoiding superovulation. Ovulation was confirmed by observation of the ovaries for fresh ovulation points at laparotomy. Following establishment of an appropriate dosage schedule, treatment was begun with indomethacin (5 mg/kg/day) starting 5 days prior to HCG and continuing to the time of laparotomy. In a second treatment cycle, indomethacin was administered at a dose of 5 mg/kg b.i.d. together with the established dose of HMG-HCG. Ovarian inspection was carried out as in the control cycles. Venous blood was obtained on treatment days 4, 7, 10 and 11 for determination of serum estrone, estradiol and progesterone. Indomethacin administration resulted in ovulation inhibition at a dose of 10 mg/kg/day when ovulation inducing doses of gonadotropins were administered. Peripheral blood steroid levels suggest that follicle maturation and estrogen production was unimparied by indomethacin. These findings indicate that the ovarian synthesis of prostaglandin may be essential in the process of ovulation.

PGF2a has been proposed as a mediator of mammalian ovulation. To elucidate further the role of PGF2a in the process of ovulation, PGF and PGF2a metabolite were measured by radioimmunoassay in the perfusate of an in vitro perfused rabbit ovary preparation. Perfusion medium samples were collected over a 10 to 12 hour period from ovaries perfused with tissue culture M199 (total volume 150 ml, sample volume 3 ml) to which varying amounts of hCG had been added. [The PGF2a antisera a 40% cross reaction with PGF1a, hence total PGF was measured with this antisera.] Both PGF and PGF2a metabolite showed a linear increase with time and numbers of ovulations. PGF media accumulation was 575 pg/ovary/ovulation/hr and PGF2a metabolite accumulation was 367 pg/ovary/ovulation/hr. Medium prostaglandin content could be correlated with numbers of ovulations, ovulatory efficiency (number of ovulations/total follicles) but not total follicles. These data best fit a model of independent ovulatory units producing PGF2a without recruitment or interaction between them. We infer that PGF and PGF2a metabolites in this system can be used as a direct index of the ovulation process.

Ovaries removed from New Zealand White rabbits were perfused and exposed to gonadotropin in vitro. The ova ovulated in vitro (N = 56) were recovered and cultured and then transferred to the oviducts of six previously mated Dutch Belted hosts. Twelve of the resulting 36 offspring (33.3 percent) were white. In control matings between 12 Dutch Belted females (six randomly selected and the six hosts) and New Zealand White males, only one of 80 (1.2 percent) offspring was white. These data indicate that ova ovulated in vitro can be transferred to the oviduct of a host rabbit where they may be fertilized and after implantation may develop into viable embryos.

Prostaglandins (PGs) have been implicated in the mechanism of ovulation in several species through the use of PG synthesis inhibitors such as indomethacin. Studies of ovulation in the in vitro perfused rabbit ovary have aided in the delineation of the process of ovulation. This study was designed to determine the effects of indomethacin on follicle rupture and ovum development in the in vitro perfused rabbit ovary preparation. Indomethacin treatment (0.5 microgram/ml) significantly reduced the occurrence of ovulation in gonadotropin-treated ovaries. The percentage of in vitro ovulated ova and ova recovered from unruptured follicles during ovarian perfusion which achieved Metaphase II did not significantly differ between indomethacin-treated and control ovaries (hCG alone). However, increased degeneration of Metaphase II ova was associated with indomethacin treatment. In in vitro culture experiments this degeneration could be prevented by supplemental PGF2 alpha. These results indicate that inhibition of PG synthesis by indomethacin prevents follicle rupture but does not affect ovum maturation, thus providing further evidence that these two processes are distinct phenomena in vitro.

The role of calcium (Ca++) and magnesium (Mg++) in the ovulation process was studied using in vitro perfused rabbit ovaries. Ovaries were perfused with or without human chorionic gonadotropin (hCG) in Ca++/Mg++-free medium (M199) alone or combined with standard M199 to yield varying concentrations of Ca++ and/or Mg++. In all ovaries perfused with hCG, ovulatory efficiency was similar regardless of the concentration of Ca++ and/or Mg++. In ovaries perfused in Ca++/Mg++-free medium without hCG, ovulatory efficiency was similar to that in ovaries perfused with hCG. As Ca++/Mg++ levels were increased without hCG, ovulatory efficiency declined. Ovulation time was significantly accelerated in ovaries perfused in Ca++/Mg++-free medium with or without hCG. Most ovulated ova from ovaries perfused without hCG were immature. With hCG, degree of ovum maturity was directly related to ovulation time. Ovarian smooth muscle contractions were undetectable in 3 ovaries perfused in Ca++/Mg++-free M199 despite occurrence of ovulation. Smooth muscle contractions were recorded in 2 of 3 ovaries perfused in standard M199 with hCG. These results indicate: 1) Ca++/Mg++ exclusion results in rapid follicle rupture and immature ova; 2) oocyte maturation appears to be gonadotropin-dependent; 3) ovulation occurs in the absence of ovarian smooth muscle contractions during perfusion with Ca++/Mg++-free medium.

Six mature female rhesus monkeys were treated with HMG-HCG in control cycles at doses adjusted to induce ovulation while avoiding superovulation. Occurrence of ovulation was determined by observation of fresh ovulation points at laparotomy 48 to 120 hours following HCG. In subsequent cycles animals were treated with indomethacin (treatment days 4 through 10) together with the established dose of HMG-HCG. In 8 cycles indomethacin 5 mg/kg was given i.m. once daily; in 9 cycles 10 mg/kg i.m. was administered in 2 divided doses. Following this, PGF2alpha (3 mg t.i.d. s.c.) was administered for 3 days together with indomethacin 10 mg/kg and HMG-HCG, beginning on the day prior to HCG. Determinations of progesterone were performed by RIA on treatment days 4, 7, 10, and 11. Eleven of the 13 control cycles were ovulatory. With indomethacin 5 mg/kg/day, 5 of 8 cycles were ovulatory but ovulation was delayed in 2 instances. Of 9 cycles using indomethacin 10 mg/kg/day only 1 was ovulatory. When PGF2alpha was administered in susequent cycles along with indomethacin (10 mg/kg) and HMG-HCG, ovulation occurred in 13 of 19 cycles. These data suggest that local ovarian PGF2alpha may be essential in the mechanics of follicle rupture in gonadotropin-treated rhesus monkeys.

The in vitro perfused rabbit ovary preparation was used to examine the role of calcium in the ovulatory process. Two groups of rabbits were studied. In the first group, verapamil hydrochloride (10(-4) mol/L), a calcium channel blocker, was used together with human chorionic gonadotropin (50 IU) in the perfusate. Verapamil had no apparent effect on human chorionic gonadotropin-induced ovulation. Verapamil treatment, however, significantly reduced the percentage of ovulated ova that were mature (68.8%) in comparison to ovulated ova from human chorionic gonadotropin-treated control ovaries (95.0%). In a second experimental group, ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid (2.0 mmol/L), a calcium ion chelator, was included in the perfusate with gonadotropin. The ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid significantly reduced ovulatory efficiency (16.7% +/- 9.43%) in comparison to that of controls exposed to human chorionic gonadotropin alone (79.5% +/- 11.1%). In addition, ovulation occurred at an earlier time in ovaries perfused with ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid; however, only four ovulations occurred in these ovaries. These four ovulated ova were immature, probably reflecting the early time of ovulation. Furthermore, both verapamil and ethyleneglycol-bis(beta-aminoethyl ether)-n,n'-tetraacetic acid blocked ovarian smooth muscle contractions during ovarian perfusion. These data provide additional support for the concept that calcium dynamics influence the processes of ovulation and ovum maturation. Furthermore ovarian smooth muscle contractions do not appear to be essential for ovulation in this model.

During in-vitro perfusion of rabbit ovaries, ova that ovulated in response to hCG were recovered and cultured with spermatozoa capacitated in vivo. After culture for 30 h, 113 (48.8%) of the ova underwent cleavage and were not degenerate. Of these 113 zygotes, 75 were successfully transferred to the oviducts of 14 gonadotrophin-treated host rabbits. On Day 14 after transfer there were 14 implantation sites and 9 pregnancies proceeded to term. These data indicate that in-vitro ovulated ova can be fertilized in vitro and be used to establish normal pregnancies in a host rabbit.

Prolactin is best known for its effects on the breast, promoting mammary growth and lactation. In some species, including rat, mouse, hamster, sheep and rabbit, prolactin is necessary for the maintenance of the corpus luteum. Further, a relationship has been recognised between hyperpromus and or adenohypophysis. McNatty et al. have postulated a direct ovarian effect of prolactin, reporting that low prolactin levels were essential for progesterone production by preovulatory human granulosa cells cultured in vitro. However, high levels of prolactin decreased progesterone production. The present investigation sought to determine the effect of prolactin on ovulation using the in vitro perfused rabbit ovary preparation. We report that its effects are inhibitory.

The described method for selective sympathetic denervation of the in vivo rabbit ovary involved stripping the ovarian artery of its nerve bundles and adventitial tissue. The ovary was then entirely free of fluorescent-staining adrenergic nerves. This technique was used to study the effects of ovarian denervation on HCG-induced ovulation. After HCG was administered to 22 rabbits which had previously undergone unilateral ovarian denervation, the ovaries were observed for follicular maturation and rupture. Control ovaries demonstrated a mean of 5.6 stimulated follicles/ovary; denervated ovaries had a mean of 5.4. An average of 3.5 follicles/control ovary ruptured; an average of 3.1 follicles/denervated ovary ruptured. Furthermore, the time course of ovulation after HCG did not differ between denervated and intact ovaries. These results indicate that HCG-induced ovulation in the rabbit is not interrupted by ovarian denervation.

A system has been developed for the perfusion of the rabbit ovary in vitro. At laparotomy, the ovarian artery is cannulated and perfused with M 199 tissue culture medium containing insulin and heparin, then removed with its vascular pedicle intact. Perfusion at 37 degrees C is maintained by using a capillary oxygenator and Buchler roller pump. The functional integrity of the perfused ovary is confirmed by serial determinations of the perfusate pH, glucose and lactate concentrations, and by ovarian histology. This in vitro model was used to study the mechanism of ovulation. One group of isolated rabbits received human chorionic gonadotropin (50 IU, intravenously) and, 8 hours later, one ovary was removed and perfused; the contralateral ovary remained in situ, serving as an in vivo control. Serial observations for follicle development and rupture were made over the subsequent 7-hour interval. The occurrence of ovulation in vitro was documented by time-lapse photography. In each animal, comparisons made between the in vitro and in vivo ovary indicated that the rate and time of follicle maturation and ovulation were comparable. Ovulation occurred between 10 and 15 hours after administration of human chorionic gonadotropin in both preparations.

Ovulation may be achieved and studied in an isolated perfused rabbit ovary upon inclusion of human chorionic gonadotropin (hCG) in the perfusion fluid. The ultrastructural features of the rabbit ovarian follicle prior to ovulation in vitro were compared with those in vivo. The perifollicular vasculature was also examined in in vitro perfused rabbit ovaries during the preovulatory interval. Granulosa cells of the preovulatory follicle share many ultrastructural features in vivo and in vitro; however, only small amounts of smooth endoplasmic reticulum (sER) were observed in granulosa cells in vitro after hCG. Ovulation after hCG in the in vitro preparation tends to occur earlier (6 hours) than in vivo (12 hours). Thus, there may be insufficient time and/or gonadotropin exposure to permit full functional development of granulosa cells, as reflected by reduced amounts of sER. Degradation of collagen fibrils was less prominent in the theca externa and tunica albuginea in vitro than in in vivo. Perifollicular capillaries became dilated after hCG, but interendothelial gaps were not observed. Disappearance of surface epithelium in the apex of follicles was similar in vitro and in vivo.

Following unilateral ovarian denervation by ovarian artery stripping in female rabbits, ovulation and pregnancy were achieved within 3 days of denervation. Three weeks later, at repeat laparotomy, ovarian dimensions and the numbers of corpora lutea and pregnancies were noted. Fluorescent histochemical studies confirmed complete adrenergic denervation in five of the rabbits' ovaries. There was no significant difference in ovarian dimensions, numbers of corpora lutea and pregnancies, or the excess of corpora lutea over implantations when the intact control side was compared with the denervated side. The control ovaries demonstrated an average of 5 corpora lutea/ovary and an average of 3.4 conceptuses in the adjacent uterine horn, while the numbers on the denervated side were 5 and 3.2, respectively. Although these findings demonstrate that central neural efferent control is not essential for the occurrence of pregnancy after mating in the rabbit, the potential significance of ovarian neuromuscular mechanisms in these functions is discussed, and future studies are suggested.