Karen H. Wright

Ovarian smooth muscle in the human being, rabbit, and cat: Histochemical and electron microscopic study☆

Abstract The nature and distribution of smooth muscle cells in the ovaries of the cat, rabbit, and human being were investigated by both histochemical and electron microscopic studies. In the human ovary, the typical smooth muscle cells are abundant in both the theca externa of the follicle and the cortical stroma. In the rabbit ovary, smooth muscle cells are concentrated in the cortical stroma rather than in the follicle wall. In the cat ovary, the smooth muscle cells cannot be considered typical. The characteristic features of these cells are discussed, and the possible role of ovarian smooth muscle in the ovulatory mechanism is suggested.

Investigation of mammalian ovulation with an in vitro perfused rabbit ovary preparation

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.

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.

In Vitro Reversal of Indomethacin-Blocked Ovulation by Prostaglandin F2α

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.

Studies of Rabbit Ovarian Contractility Using Chronically Implanted Transducers

A chronically implanted, highly sensitive force transducer was used to study ovarian contractions in the rabbit. The transducer is implanted into the medulla of the ovary through its long axis. The leads are then drawn through the abdominal wall and directed subcutaneously to the back of the neck. The miniature pin connector is fixed into place beneath the skin to facilitate accessibility for repeated recordings, obviating the necessity for anesthesia or serial laparotomies. Intraovarian transducers were implanted in isolated rabbits. Serial recordings of ovarian contractions were made at weekly intervals for 3 weeks, following which ovulation was induced with 100 IU of human chorionic gonadotropin (HCG). Extended recordings were made from 3 through 13 hours after HCG administration to six animals. Recordings revealed increased ovarian contractile activity beginning just before the anticipated time of ovulation. The observations support those of previous acute experiments and demonstrate a relationship between ovarian contractile activity and the process of follicle rupture.

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.

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

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 (hCG) 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, hCG at a dose of 25, 10, 5, or 1IU was added to the perfusate of both ovaries. An additional group received no hCG. One ovary from each rabbit was treated with progesterone (1mg/150ml of perfusate) together with hCG. The contralateral ovary served as a control, receiving only hCG. The number of ovaries ovulating in each group varied directly with the amount of hCG added to the perfusate (four of five with 25IU, three of five with 10IU, two of five with 5IU, one of five with 1IU, and none of five without hCG). 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 hCG and ovulation in the in vitro model and that amounts of hCG as small as 1IU 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.

Influence of estrogen and progesterone treatment on ovarian contractility in the monkey.

Five female rhesus monkeys were treated with natural estrogens, 5 mg/day for three weeks, after which ovarian contractility was studied in vitro in one of the ovaries. Estrogen treatment was followed by progesterone, 25 mg/day for three weeks, after which the contractility of the remaining ovary was similarly measured. Responses to autonomic agents and prostaglandins were studied in both groups. Spontaneous ovarian contractility and ovarian contractile responsiveness to prostaglandins and norepinephrine were found to be enhanced after progesterone treatment. Cholinergic agonists had a stimulatory effect after progesterone and an inhibitory effect after estrogens. Our results suggest that ovarian contractile responsiveness is modified by the local steroid environment, perhaps through intracellular changes in cyclic AMP.

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.

The influence of ovarian denervation and nerve stimulation on ovarian contractions.

Two techniques were used to study the neuromuscular control of rabbit ovarian contractions. First, the ovary was denervated by stripping its artery. Spontaneous contractions were noted, as was a normal response to exogenously administered adrenergic agents. Second, the ovarian nerves were electrically stimulated in an in vitro perfused ovary system. Inhibition of ovarian contractions was noted, and the additive effects with adrenergic agents suggested a betamimetic effect of nerve stimulation in the estrous rabbit. After administration of exogenous human chorionic gonadotropin, the response was reversed. The significance of the contribution of a neuromuscular mechanism to the control of ovulation is discussed.