Prostaglandin E(1) inhibits abnormal follicle rupture and restores ovulation in indomethacin-treated rats

In the presence of indomethacin, an inhibitor of prostaglandin (PG) synthesis, the gonadotropin surge induces abnormal follicle rupture at the basolateral follicle sides, thus preventing effective ovulation in rats. This study was undertaken to analyze whether exogenous prostaglandin administration can overcome the antiovulatory action of indomethacin. Cycling rats were treated with vehicle (olive oil) or indomethacin (1 mg/rat) on the morning of proestrus. Rats treated with indomethacin were injected with different doses (50, 250, or 500 micro g/rat) of PGE(1), PGE(2), PGF(2alpha), or vehicle (saline) at 1900 h in proestrus. The ovulatory response was analyzed on the morning of estrus by evaluating follicle rupture and the location of the oocytes in serially sectioned ovaries. The number of oocytes in the oviducts was also counted in rats treated with the highest prostaglandin doses. In indomethacin-treated rats, most newly formed corpora lutea showed abnormal follicle rupture at the basolateral sides. In addition, invasion of the ovarian stroma and blood and lymphatic vessels by granulosa cells and follicular fluid was observed. Prostaglandins of the E series, and especially PGE(1), inhibited abnormal follicle rupture and restored ovulation, although the number of oocytes in the oviducts were significantly decreased. PGF(2alpha) was only partially effective in inhibiting abnormal follicle rupture and restoring ovulation. These data suggest that prostaglandins of the E series, and particularly PGE(1), play a crucial role in ovulation by determining the targeting of follicle rupture at the apex, thus allowing release of oocytes to the periovarian space.     

A possible dual mechanism of the anovulatory action of antiprogesterone RU486 in the rat

The purpose of these experiments was to investigate the mechanism of the anovulatory action of antiprogesterone RU486 (RU486) in rats by studying its effects on follicular growth, secretion of gonadotropins and ovarian steroids, and ovulation. Rats with 4-day estrous cycles received injections (s.c.) of either 0.2 ml oil or 0.1, 1, or 5 mg of RU486 at 0800 and 1600 h on metestrus, diestrus, and proestrus. At the same times, they were bled by jugular venipuncture to determine serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17 beta-estradiol (E), and progesterone (P). On the morning of the day after proestrus, ovulation and histological features of the ovary were recorded. Rats from each group were killed on each day of ovarian cycle to assess follicular development. Rats treated similarly were decapitated at the time of the ovulatory LH surge and blood was collected to measure LH. The serum levels of LH increased and those of FSH decreased during diestrus in rats treated with RU486. Neither E nor P levels differed among the groups. Treatment with RU486 caused both a blockade of the ovulation and an increase in ovarian weight in a dose-dependent manner. At the time of the autopsy (the expected day of ovulation), rats treated with 1 mg RU486 had ovaries presenting both normal and post-ovulatory follicles and unruptured luteinized follicles. Rats treated with 5 mg RU486 presented post-ovulatory follicles without signs of luteinization. The number of follicles undergoing atresia increased in rats treated with RU486. Rats treated with 5 mg RU486 exhibited a significant decrease in ovulatory LH release. The mechanism by which RU486 produces the ovulatory impairment in rats seems to be dual: first, by inducing inadequate follicular development at the time of the LH surge and second, by reducing the amount of ovulatory LH released. The physiological events-decreased basal FSH secretion and follicular atresia-that result from use of RU486 cannot be elucidated from these experiments and should be investigated further.     

Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat

The role of progesterone in the regulation of the preovulatory surge in gonadotropins and ovulation was examined in this study by use of a potent antagonist of progesterone, RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha- [prop-1-ynyl]estra-4,9-diene-3-one). The immature rat primed with pregnant mare's serum gonadotropin (PMSG) and the cycling adult animal were the models used to verify the role of progesterone. When RU 486 (200 micrograms/rat) was given as a single dose on the morning of proestrus, there was a significant reduction in the preovulatory surge levels of gonadotropins and ovulation in both animal models. Serum progesterone levels in both models at the time of death on the evening of proestrus were unaltered upon treatment with RU 486. RU 486 did not have any effect on gonadotropin levels in immature rats 7 days after castration. These results show that the actin of RU 486 on the preovulatory gonadotropin surge is due to an antagonism of the action of progesterone on the hypothalamic-pituitary axis. Thus, a role for progesterone in modulating the preovulatory surge of gonadotropins and, consequently, ovulation is strongly suggested.     

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immature rats show ovulatory defects similar to those in adult rats lacking prostaglandin and progesterone actions

Gonadotropin-primed immature rats (GPIR) constitute a widely used model for the study of ovulation. Although the equivalence between the ovulatory process in immature and adult rats is generally assumed, the morphological and functional characteristics of ovulation in immature rats have been scarcely considered. We describe herein the morphological aspects of the ovulatory process in GPIR and their response to classical ovulation inhibitors, such as the inhibitor of prostaglandin (PG) synthesis indomethacin (INDO) and a progesterone (P) receptor (PR) antagonist (RU486). Immature Wistar rats were primed with equine chorionic gonadotropin (eCG) at 21, 23 or 25 days of age, injected with human chorionic gonadotropin (hCG) 48 h later, and sacrificed 16 h after hCG treatment, to assess follicle rupture and ovulation. Surprisingly, GPIR showed age-related ovulatory defects close similar to those in adult rats lacking P and PG actions. Rats primed with eCG at 21 or 23 days of age showed abnormally ruptured corpora lutea in which the cumulus-oocyte complex (COC) was trapped or had been released to the ovarian interstitum, invading the ovarian stroma and blood and lymphatic vessels. Supplementation of immature rats with exogenous P and/or PG of the E series did not significantly inhibit abnormal follicle rupture. Otherwise, ovulatory defects were practically absent in rats primed with eCG at 25 days of age. GPIR treated with INDO showed the same ovulatory alterations than vehicle-treated ones, although affecting to a higher proportion of follicles. Blocking P actions with RU486 increased the number of COC trapped inside corpora lutea and decreased ovulation. The presence of ovulatory defects in GPIR, suggests that the capacity of the immature ovary to undergo the coordinate changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age.     

Effects of indomethacin on ovarian leukocytes during the periovulatory period in the rat

We have investigated the effects of indomethacin (IM), a non-steroidal anti-inflammatory drug, and the role of prostaglandins on the accumulation of leukocytes in the rat ovary during the periovulatory period. Adult cycling rats were injected sc with 1 mg of IM in olive oil or vehicle on the morning of proestrus. Some animals were killed at 16:00 h in proestrus. On the evening (19:00 h) of proestrus, IM-treated rats were injected with 500 micrograms of prostaglandin E1 in saline or vehicle. Animals were killed at 01:30 and 09:00 h in estrus. There was an influx of macrophages, neutrophils, and eosinophils into the theca layers of preovulatory follicles, and of neutrophils and eosinophils into the ovarian medulla from 16:00 h in proestrus to 01:30 h in estrus. All these changes, except the accumulation of neutrophils in the theca layers of preovulatory follicles, were blocked by IM treatment. At 09:00 h in estrus, large clusters of neutrophils were observed in IM-treated rats, around abnormally ruptured follicles. The accumulation of leukocytes was not restored by prostaglandin supplementation, despite the inhibition of abnormal follicle rupture and restoration of ovulation in these animals. These results suggest that different mechanisms are involved in leukocyte accumulation in the ovary during the periovulatory period, and that the inhibitory effects of IM on the influx of leukocytes are not dependent on prostaglandin synthesis inhibition.     

Estradiol-17 beta reduces number of ovulations in adult rats: direct action on the ovary?

The specific role of estrogen and other steroids in folliculogenesis is unclear since both inhibitory and stimulatory effects have been described. We reported that atresia of the preovulatory follicle was induced when estradiol-17 beta (E2) or progesterone was administered peripherally in rhesus monkeys, presumably due to a direct effect at the ovarian level. The present study was designed to determine if a similar direct action of E2 and other steroids occurs in rats. Minicapsules of Silastic containing E2, progesterone or dihydrotestosterone in amounts of 12.5% to 100% mixed with cholesterol, were placed unilaterally under the ovarian bursa on the morning of metestrus in rats having 4-day cycles. At autopsy on the morning of estrus, the number of oocytes ovulated from treated and untreated contralateral ovaries was determined. Ovaries treated with E2 averaged 3.1 +/- 0.4 oocytes while untreated ovaries in the same animals averaged 6.4 +/- 0.4 oocytes (P less than 0.001 by paired t test, n = 20). Results were similar for all amounts of E2 used and serum levels of E2 were not elevated at autopsy by this local treatment. Cholesterol alone did not alter the number of oocytes. Results of similar experiments with progesterone and dihydrotesterone were less conclusive than for E2. In additional trials, ovaries were treated with E2 as above, and preovulatory follicles were explanted on the morning of proestrus to determine their steroidogenic capability in vitro. Follicles from treated ovaries released somewhat less E2 and progesterone into luteinizing hormone (LH)-free medium than follicles from untreated ovaries, but not when LH was added to the medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unilateral ovariectomy, flutamide treatment and HCG reverse the anovulatory action of antiprogesterone RU486 in rat.

Administration of antiprogesterone RU486 (4 mg/day) from estrus through proestrus to cyclic rats blocked ovulation. Moreover, RU486 increased basal serum concentrations of LH, PRL, testosterone and estradiol, while it decreased basal serum concentration of FSH. Both unilateral ovariectomy and antiandrogen flutamide treatment, as well as an ovulatory injection of HCG in the proestrus afternoon partially reversed, the ovulatory blockade of RU486. These results indicate that both the decreased FSH concentration and the increased testosterone concentration, as well as the reduced ovulatory LH release are responsible for the anovulatory effects of RU486.     

The relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated immature rat.

To clarify the mechanisms by which progesterone acts as a mediator in the ovulatory process, ovulation rate and proteolytic enzyme activities were investigated in immature 22-day-old rats treated with PMSG/hCG, RU486 (10 mg/kg), synthetic antiprogesterone, and RU486 (10 mg/kg) + progesterone (10 mg/kg). The number of ova was significantly decreased when RU486 (10 mg/kg) was given from 2 h before to 4 h after the hCG injection. In addition, its inhibitory action on ovulation was reversed by exogenous progesterone (10 mg/kg) at 2 or 4 h after the hCG injection. Serum progesterone and estradiol concentrations in the rats treated with RU486 did not show any significant differences compared to controls. The proteolytic enzyme activities were measured by using the synthetic substrates alpha-N-benzoyl-DL-Arg-beta naphthylamide (BANA) and dinitrophenyl peptide (DNP). Activities were significantly increased after hCG injection in the control group during 8-9 h for BANA hydrolase and 7-10 h for DNP peptidase. The preovulatory increase of these activities was totally suppressed by RU486 with hCG. After administration of progesterone (10 mg/kg) following hCG and RU486 injection, the elevation of proteolytic, enzyme activities in the preovulatory phase was effectively reversed, and levels became similar to those in the control group. These results suggest that progesterone plays an indispensable role in the first 4 h of the ovulatory process by regulating proteolytic enzyme activities.     

Changes in the ovarian dynamics and endocrine profiles in goats treated with a progesterone antagonist during the early luteal phase of the estrous cycle

The aim of the present study was to determine the physiological role of endogenous progesterone in the regulation of ovarian dynamics, gonadotropin and progesterone secretion during the early luteal phase in the goat. Cycling Shiba goats received subcutaneously a vehicle (control group, n=5) or 50 mg of RU486 (RU486 group, n=4) daily from 1 to 7 days after ovulation (day 0) determined by transrectal ultrasonography. Ovarian dynamics were monitored by the ultrasonography and blood samples were collected daily until the subsequent ovulation for analysis of progesterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion. Blood samples were also collected at 10 min intervals for 6 h on day 3 and day 7 for the analysis of pulsatile patterns of LH and FSH. The LH pulse frequency was significantly (P<0.05) higher in the RU486 group than in the control group on day 7 (4.8+/-1.1 pulses/6 h versus 1.2+/-0.4 pulses/6 h). The shape of the FSH pulses was unclear on day 3 and day 7 in both groups and the overall means of FSH concentration for 6 h on day 3 and day 7 were not significantly different between the RU486 and the control groups. The pattern of daily FSH concentrations showed a wave-like fluctuation in both groups. There was no significant difference in the inter-peak intervals of the wave-like pattern of daily FSH secretion between the RU486 and the control groups (4.1+/-0.6 days versus 4.5+/-0.6 days). The maximum diameter of the largest follicle that grew from day 1 to day 7 in the RU486 group tended to be greater than that in control goats (6.4+/-0.8 mm versus 5.0+/-0.8 mm, P=0.050), whereas no significant difference was detected in the size of the corpus luteum and progesterone concentrations between the control and RU486 groups on almost all days during the treatment period. These results indicate that the rise of the progesterone concentration suppresses the pulsatile LH secretion and follicular growth, whereas progesterone has no physiological role in the regulation of FSH secretion and luteal function during the early luteal phase of the estrous cycle in goats.     

Antiprogestagen RU486 prevents the LH-dependent decrease in the serum concentrations of inhibin in the rat

Summary 1. In the rat, the LH-dependent ovarian progesterone rise mediates several actions of the primary surge of LH on the ovary. This experiment was aimed at elucidating the effects of the antiprogestagen RU486 on the LH-dependent decrease in both the serum concentrations and the ovarian content of inhibin.2. All rats in this experiment were treated with an antagonist of LHRH (1 mg/200 µl saline at 0800 h in proestrus) to supress the endogenous release of LH. One group of rats received 32 µg LH/250 µl saline at 1200 h in proestrus. Other group was given 4 mg RU486/200 µl oil at 0800 h in proestrus. The third group was injected with both RU486 and LH. Rats from the control group were injected with 250 µl saline and 200 µl oil. Animals were decapitated at 1700 h in proestrus and trunk blood and ovaries collected to determine the serum concentrations of LH, FSH, progesterone, 17ß-estradiol and inhibin as well as the ovarian content of inhibin.3. The ovulatory dose of LH in LHRHa-treated rats decreased both the serum concentrations and the ovarian content of inhibin and increased the serum concentrations of FSH. The administration of RU486 blocked the effect of LH on the serum concentrations of inhibin but not that on the ovarian content of inhibin.4. Since the antiprogestagen RU486 blocked the effect of LH on the serum concentrations of inhibin, we conclude that ovarian progesterone, besides mediating the effects of the primary LH surge on the ovulatory process and luteinization, participates in the LH-dependent drop in the serum concentrations of inhibin in proestrous afternoon.     

RU486 blocks the secondary surge of follicle-stimulating hormone in the rat without blocking the drop in serum inhibin.

The stimulatory effect of progesterone on the release of the primary surges of serum LH and FSH is well characterized. Less is known about the relationship of progesterone to the secondary FSH surge. We used the antiprogesterone, RU486, to block the action of progesterone and studied the effects on the primary surges of LH and FSH, and especially on the secondary surge of FSH. Proestrous rats were treated with RU486 at 1,200 h. Rats were killed on proestrus and estrus, and serum levels of LH, FSH, and inhibin were measured. In all RU486-treated rats, the primary surges of LH and FSH were significantly attenuated, and the secondary FSH surge was completely abolished, despite a drop in inhibin levels following the primary surges. A high replacement dose of progesterone, delivered immediately after RU486 treatment, did not restore the primary surges of LH and FSH, or the secondary surge of FSH. These data suggest that other factors in addition to a drop in inhibin are responsible for producing the secondary FSH surge.     

Qualitative and quantitative data on experimental luteinization in the female rat

The dose-effect of 1.5-16 mcg luteinizing hormone (LH) per 100 gm body weight injected in rats at 1100-1200 on proestrus was compared with 30 mg meprobamate given to controls at the same time, on luteinization and ovulation seen in serial ovarian sections. The WII Wistar rats were killed. Luteinization with or without ovulation increased with dose (1.5, 3, 5, 8, and 16 mcg) of LH to a plateau (90%) above the 5 mcg dose, compared with 18% in controls. 2-5 animals in each dose group had preluteinized follicles, characterized by a dissociation of the cumulus oophorus from the granulosa. The absolute frequency of ovulation increased linearly with LH dose, but the frequency of ovulation among rats that luteinized was invariant. The coefficient of ovulation, calculated as the mean incidence of ovulation in relation to the total number of luteinized or preluteinized follicles in each rat, decreased from .769 in controls to .580 in the 3 mcg group, then rose to .916 in the 16 mcg group. Thus, in proestrous rats, low doses of LH induce corpora lutea with retained ova. The threshold dose of LH for luteinization and for ovulation is lower in proestrus than in diestrus II, but varies slightly in different strains of rats.     

Hypersecretion of follicle-stimulating hormone (FSH) on estrous afternoon in rats treated with RU486 in proestrus

Summary 1. Intact or ovariectomized (OVX) cyclic rats injected or not with RU486 (4 mg/0.2 ml oil) from proestrus onwards were bled at 0800 and 1800h on proestrus, estrus and metestrus. Additional RU486-treated rats were injected with: LHRH antagonist (LHRHa), estradiol benzoate (EB) or bovine follicular fluid (bFF) and sacrified at 1800 h in estrous afternoon. LH and FSH serum levels were determined by RIA.2. RU486-treated intact or OVX rats had decreased preovulatory surges of LH and FSH, abolished secondary secretion of FSH and hypersecretion of FSH in estrous afternoon. The latter was decreased by LHRHa and abolished by EB or bFF. In contrast, EB induced an hypersecretion of LH in RU486-treated rats at 1800h in estrus.3. It can be concluded that in the absence of the proestrous progesterone actions, the absence of the inhibitory effect of the ovary in estrus evoked a LHRH independent secretion of FSH.     

Integration of the effects of estradiol and progesterone in the modulation of gonadotropin secretion

Estradiol secreted by the maturing follicle is the primary trigger for the surge of gonadotropins leading to ovulation. Progesterone has stimulatory or inhibitory actions on this estrogen-induced gonadotropin surge depending upon the time and dose of administration. The administration of progesterone to immature ovariectomized rats primed with a low dose of estradiol induced a well-defined LH surge and prolonged FSH release, a pattern similar to the proestrus surge of gonadotropins. A physiological role of progesterone is indicated in the normal ovulatory process because a single injection of the progesterone antagonist RU 486 on the day of proestrus in the adult cycling rat and on the day of the gonadotropin surge in the pregnant mare's serum gonadotropin stimulated immature rat resulted in an attenuated gonadotropin surge and reduced the number of ova per ovulating rat. Progesterone administration brought about a rapid LHRH release and an decrease in nuclear accumulation of estrogen receptors in the anterior pituitary but not the hypothalamus. The progesterone effect was demonstrated in vitro in the uterus and anterior pituitary and appears to be confined to occupied estradiol nuclear receptors. In in vivo experiments the progesterone effect on estradiol nuclear receptors appeared to be of approximately 2-h duration, which coincided with the time period of progesterone nuclear receptor accumulation after a single injection of progesterone. During the period of progesterone effects on nuclear estrogen receptors, the ability of estrogens to induce progesterone receptors was impaired. Based on the above results, a model is proposed for the stimulatory and inhibitory effects of progesterone on gonadotropin secretion.     

Progesterone promotes oocyte maturation, but not ovulation, in nonhuman primate follicles without a gonadotropin surge

During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% +/- 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% +/- 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% +/- 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 +/- 9) and DHT- (36 +/- 15) but not vehicle- (4 +/- 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.     

Anti-ovulatory effects of RU486 and trilostane involve impaired cyclooxygenase-2 expression and mitotic activity of follicular granulosa cells in rats

To explore the mechanism for anti-ovulatory effects of blockade of preovulatory synthesis and action of progesterone, we focused on cyclooxygenase (COX)-2 induction and mitotic activity of granulosa cells in gonadotropins-treated rats. Treatment with RU486 (a progesterone receptor antagonist) or trilostane (a 3β-hydroxysteroid dehydrogenase inhibitor) just prior to or 4h after human chorinonic gonadotropin (hCG) (hCG4h) decreased ovulation rates and circulating progesterone level. Human CG induction of immunoreactive COX-2 in the granulosa layer of mature Graafian follicles at hCG8h was reduced by RU486 treatment at hCG0h and trilostane treatment at hCG4h. RU486 treatment further attenuated ovarian prostaglandin E(2) (PGE(2)) level significantly. Cell proliferative activity in mural granulosa layer of the inhibitors-treated follicles was significantly lower than in intact group. Obtained results show that inhibition of synthesis and action of progesterone caused attenuated COX-2/PGE(2) system and dysregulated mitotic response of granulosa cells, resulting in decreased ovulation.