Rosemary Santulli

Microvasculature of preovulatory follicles: Comparison of in situ and in vitro perfused rabbit ovaries following stimulation of ovulation

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 effect of prostaglandin synthesis inhibition by indomethacin on ovulation and ovum maturation in the in vitro perfused rabbit ovary

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.

Direct ovarian effect of clomiphene citrate in the rabbit

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.

Examination of the role of calcium in ovulation in the in vitro perfused rabbit ovary with use of ethyleneglycol-bis(β-aminoethyl ether)-n,n′-tetraacetic acid and verapamil+++

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.

Prostaglandin F2a, an ovulatory intermediate in the in vitro perfused rabbit ovary model☆

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.

The relationship between prostaglandins and histamine in the ovulatory process as determined with the in vitro perfused rabbit ovary**Supported by National Institutes of Health grant HD-05948, The Connelly Foundation, and The Mitchell and Lillian Duberstein Foundation.

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.

Ultrastructure of ovarian follicles in in vitro perfused rabbit ovaries: response to human chorionic gonadotropin and comparison with in vivo observations**Supported by NIH grant HD-05948, the Connelly Foundation, and the Mitchell and Lillian Duberstein Foundation.

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.