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Gonadotropin-releasing hormone (GnRH) has been shown to inhibit ovulation in gonadotropin-primed hypophysectomized rats and steroid production in cultured rat granulosa cells. To determine if similar effects of GnRH can be observed in another species, the extracorporeal perfused rabbit ovary was utilized. Two groups of rabbit ovaries were exposed to GnRH in a pulsatile fashion at two dose levels (Group I, 2.56 X 10(-8) M; Group II, 2.56 X 10(-7) M). Contralateral ovaries were not perfused with GnRH. Human chorionic gonadotropin (hCG) was added to the perfusate of all ovaries 30 min after the onset of perfusion. Ovulation occurred in all ovaries exposed to hCG in the presence or absence of GnRH. Ovulatory efficiency was similar in both the experimental and control groups. No statistical difference could be determined in the time of ovulation, stage of maturity of oocytes, or percent of degeneration of ovulated or follicular oocytes. Progesterone production was not inhibited in the GnRH-treated ovaries. In contrast to observations in the rat, GnRH does not exhibit a direct inhibitory effect on ovulation or steroid production in the rabbit.

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.

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.

Perifollicular vasculature undergoes significant morphologic changes as ovulation approaches. These vascular changes were observed in in vitro perfused and in situ rabbit ovaries by means of scanning electron microscopy of microcorrosion casts. Casts were made in in situ unstimulated ovaries, in situ ovaries stimulated with human chorionic gonadotropin, in vitro perfused unstimulated ovaries, and in vitro perfused ovaries after an ovulation-inducing dose of human chorionic gonadotropin, prostaglandin F2 alpha, histamine, or norepinephrine. Dilated vessels, extravasation of resin from weakened vessels, and filling defects at the apex of the follicle were observed in in situ ovaries 9 to 12 hours after stimulation and in in vitro perfused ovaries 4 to 6 hours after human chorionic gonadotropin. In vitro perfused ovaries stimulated with prostaglandin F2 alpha or histamine demonstrated dilated capillaries with extravasation of the resin and filling defects at the apex of large follicles. Norepinephrine-stimulated ovaries showed incomplete filling of vessels, although some large follicles showed extravasation of resin. The occurrence of dilated vessels, extravasation of resin, and filling defects is indicative of preovulatory vascular changes in both in situ and in vitro perfused ovaries, regardless of the ovulatory stimulus.

The effects of aminoglutethimide phosphate (AGP) on ovulation, ovum maturation, fertilizability, and steroid production were studied with the use of an isolated perfused rabbit ovary preparation. AGP (10(-3) or 10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused in medium alone. Thirty minutes later human chorionic gonadotropin (hCG) (50 IU) was added to the perfusate of all ovaries. No difference was observed in time of ovulation or ovulatory efficiency between controls and AGP-treated ovaries. The degree of ovum maturity and degeneration was also comparable in the two groups. Progesterone and estradiol production were significantly reduced by AGP treatment. A second experiment examined fertilizability of ova ovulated in vitro after perfusion with 10(-3) M AGP. AGP significantly reduced the rate of normal fertilization as observed 12 h after insemination. The percentage of inseminated ova with evidence of degeneration was greater in ova from AGP-treated ovaries than in those from controls, however, this difference was not significant. The study indicates that AGP affects neither hCG-induced ovulation nor meiotic resumption; however, fertilizability of ova from ovaries treated with AGP is impaired. These data suggest that the intrafollicular steroid environment may participate in cytoplasmic maturation of ovulated ova.

The effects of clomiphene citrate (CC) on ovulation and ovum maturation were studied using the isolated perfused rabbit ovary. CC (10(-5) M) added to the perfusate with human chorionic gonadotropin (50 IU) did not affect ovulatory efficiency, ovulation time, oocyte maturation, or degeneration of ovulated ova and follicular oocytes. During perfusion without human chorionic gonadotropin, the percentage of follicular oocytes with germinal vesicle breakdown was significantly increased in response to CC (10(-5) M or 10(-7) M); a greater percentage of follicular oocytes was degenerated. Estradiol (100 ng/ml) added to the perfusate reversed the effect of CC on degeneration of follicular oocytes. Of follicular oocytes from ovaries perfused with CC, 79.3% were degenerated; in contrast, 25% were degenerated in ovaries treated with CC plus estradiol. These data suggest that CC has a direct ovarian effect and that ovum degeneration associated with CC may be related to an antiestrogenic action.

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.

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.

This study examined whether the addition of estradiol (E2) to the perfused rabbit ovary would reverse the deleterious effects of clomiphene citrate (CC) on early embryonic development. Ovaries were perfused with CC (10(-5) M) or CC + E2 (1 to 1000 ng/ml). Human chorionic gonadotropin (hCG, 50 IU) was added to the perfusate of each ovary. In vitro ovulated ova in cumulus were retrieved and inseminated in vitro. E2 significantly increased the percentage of ovulated ova achieving (1) the 2-cell stage at 36 hours, (2) the morula stage by 84 hours, and (3) the blastocyst stage at 132 hours. The percentage of inseminated ova showing evidence of degeneration was reduced in ovaries treated with E2. These data suggest that CC may exert an antiestrogenic effect on the intrafollicular oocyte, which interferes with postfertilization development.

An isolated perfused rabbit ovary preparation was used to determine the effects of cyanoketone, a potent inhibitor of 3 beta-hydroxysteroid dehydrogenase, on ovulation, ovum maturation and fertilizability, and steroid production. In the first experiment, cyanoketone (10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused with medium alone. Thirty minutes after the onset of perfusion, hCG (50 IU) was added to the perfusate of both ovaries. The ovulatory efficiency of ovaries treated with cyanoketone plus hCG (82.3 +/- 4.6%) was similar to that of ovaries treated with hCG alone (84.8 +/- 4.4%). No difference was observed in the degree of ovum maturity or degeneration between control and cyanoketone-treated ovaries. Progesterone and estradiol production were significantly reduced by cyanoketone treatment; concentrations in the perfusate of ovaries treated with cyanoketone were 9.7% and 8.0% of the control values, respectively, 2 h after exposure to hCG. The concentration of 17-hydroxypregnenolone was not affected by cyanoketone treatment. Exposure to cyanoketone resulted in a significant (P less than 0.005) reduction in the fertilizability of ova ovulated and fertilized in vitro. In the second experiment, the percentage of ova that showed evidence of normal fertilization was significantly (P less than 0.025) increased in ovaries perfused with cyanoketone plus estradiol (64.5%) compared to that in ovaries perfused with cyanoketone alone (32.4%). In the third experiment, the addition of progesterone to the perfusate did not affect fertilizability of ovulated ova in ovaries perfused with cyanoketone plus estradiol. These results suggest that the presence of estradiol in the ovarian steroid environment may be essential for fertilizability of ova, but not for the processes of ovulation or meiotic maturation.