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)     

Ovum transport in pregnant rats is little affected by RU486 and exogenous progesterone as compared to cycling rats

In cycling and pregnant rats, the eggs stay in the oviduct for approximately 66 and 90 h, respectively. The influence of progesterone in these timings was investigated. An excess or a simulated deficit of progesterone was induced with exogenous progesterone or the antiprogesterone RU486, respectively, beginning on the day of ovulation. The effect of these treatments on egg transport in cycling and pregnant rats was assessed in detail and complemented with determinations of estradiol and progesterone circulating levels and progesterone receptor levels in the oviduct. Accelerated transport of ova followed treatment with RU486 in cycling and pregnant rats but with different features. In cycling rats, acceleration began 24 h after the onset of treatment and was not associated with changes in estradiol levels; in pregnant rats, it started 72 h after treatment and was associated with a 5-fold increase in estradiol circulating levels. Thus, RU486 failed to accelerate ovum transport during the first three days of treatment in pregnant rats, in spite of the fact that no progesterone receptors were available in the oviduct as early as 24 h of treatment. Progesterone administration caused egg retention in the oviducts and a 50% reduction in circulating estradiol levels in cycling rats, whereas in pregnant rats progesterone excess did not change estradiol circulating levels and had no effect on the location of embryos on Days 4 and 5. These results demonstrate a different physiological importance of endogenous progesterone in slowing down oviductal ovum transport in cycling and pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta 2-adrenoceptor response in the rat uterus at the end of gestation and after induction of labor with RU 486

To study the relationship between the progesterone environment and beta-adrenoceptors in the myometrium, rats were treated with the antiprogestin RU 486 (10 mg per rat) at 08:30 h on day 21 of gestation. Under these conditions, more than 60% of animals delivered within 24 h after this treatment, while none of the control animals delivered within the same time period. beta-Adrenoceptors were identified using the radiolabeled antagonist (-)-[125I] iodocyanopindolol. The density (Bmax approximately 33-45 fmol/mg protein) and the affinity (KD approximately 0.105-0.106 nM) were not changed (during the late stages of gestation) in RU 486 treated rats compared with control rats. These results were correlated with the relaxation of longitudinal and circular strips of myometrium placed in high KC1 medium and exposed to beta-adrenoceptor agonists. The adrenoceptors implicated in the relaxation of myometrial strips were mainly of the beta 2-subtype. There was no difference in their affinity between control and RU 486 treated rats. Mean pA2 values were 8.46 for propranolol and 8.27 for ICI 118-551 against salbutamol. Altogether these results indicate in the rat that the blockade of the action of progesterone at its receptor site by RU 486 did not modify either the affinity or the sensitivity of beta 2-adrenoceptors in the myometrium, although it induced parturition.     

Inhibition of induction of 20 alpha-hydroxysteroid dehydrogenase activity in rat corpora lutea in vitro by the progesterone antagonist RU486.

To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20 alpha-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20 alpha-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 microM) the concentration of both progesterone and 20 alpha-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 microM RU486 a high ratio between 20 alpha-dihydro-progesterone and progesterone was found, while in the presence of 23 microM RU486 the concentrations of progesterone and 20 alpha-dihydro-progesterone were equal. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 microM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20 alpha-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.     

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.     

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.     

Differential effects of RU486 and indomethacin on follicle rupture during the ovulatory process in the rat

Ovulation (i.e., the release of mature oocytes from the ovary) requires spatially targeted follicle rupture at the apex. Both progesterone and prostaglandins play key roles in the ovulatory process. We have studied follicle rupture and ovulation in adult cycling rats treated with a progesterone receptor antagonist (RU486), an inhibitor of prostaglandin synthesis (indomethacin, IM), or both. All rats were treated with LHRH antagonist on the morning (0900 h) of proestrus to inhibit endogenous gonadotropins and with 10 microg of ovine LH (oLH) at 1700 h in proestrus to induce ovulation. Animals were treated from metestrus to proestrus with 2 mg/day of RU486 or vehicle (olive oil) and on the morning of proestrus (1200 h) with 1 mg of IM or vehicle (olive oil). Some rats treated with vehicle or RU486 were killed on the morning of proestrus to assess preovulatory follicle development. The remaining rats were killed on the morning of estrus to study follicle rupture and ovulation. In vehicle-treated rats, oLH induced ovulation in 98% of follicles. In IM-treated rats, spatial targeting of follicle rupture was disrupted. Most oocytes were released to the ovarian interstitium (50%) or to the periovarian space (39%), and a smaller percentage (11%) of oocytes remained trapped inside the luteinized follicle. RU486-treated rats showed, on the morning of estrus, unruptured luteinized follicles. Only occasionally (2.8%), the oocytes were released to the periovarian space. IM treatment induced follicle rupture in RU486-treated rats, and 25% of oocytes were released to the ovarian interstitium. However, the number of oocytes released to the periovarian space (i.e., ovulated) was not increased by IM treatment in rats lacking progesterone actions. Overall, these data indicate that RU486 and IM have opposite effects on follicle rupture and suggest that both progesterone and prostaglandins are necessary for the spatial targeting of follicle rupture at the apex.     

Pharmacokinetics and metabolism of RU 486

The effects of dose on the initial pharmacokinetics and metabolism of an antiprogesterone steroid RU 486 (mifepristone) were studied in healthy female volunteers after administration of RU 486 as a single dose of 50-800 mg. The concentrations of RU 486 and its monodemethylated, dimethylated and hydroxylated non-demethylated metabolites were measured specifically after Chromosorb-column chromatography by HPLC. Their relative binding affinities to the human uterine progesterone receptor were also determined. Micromolar concentrations of the parent compound in blood were reached within the first hour after oral administration. The pharmacokinetics of RU 486 followed two distinct patterns in a dose-dependent fashion. With a low dose of 50 mg the pharmacokinetics followed an open two-compartment model with a half-life of over 27 h. With the doses of 100-800 mg the initial redistribution phase of 6-10 h was followed by zero-order kinetics up to 24 h or more. Importantly, after ingestion of doses higher than 100 mg of RU 486 there were no significant differences in plasma concentrations of RU 486 within the first 48 h, with the exception of plasma RU 486 concentrations at 2 h. After single oral administration of 200 mg unchanged RU 486 was found 10 days later in two subjects. The elimination phase half-life with this dose, calculated between day 5 and 6, was 24 h. Micromolar concentrations of monodemethylated, didemethylated and non-demethylated hydroxylated metabolites were measured within 1 h after oral administration of RU 486. In contrast to plasma RU 486 concentrations, circulating plasma concentrations of metabolites increased in a dose-dependent fashion. With higher doses the metabolite concentrations were close to, or even in excess to the parent compound. The relative binding affinities of RU 486, monodemethylated, didemethylated and hydroxylated metabolites (progesterone = 100%) to the human progesterone receptor were 232, 50, 21, and 36, respectively. The existence of a high affinity-limited capacity serum binding protein would explain the long half-life and the observed diverging dose-dependent pharmacokinetics. The extravasation of RU 486 after the saturation of serum binding sites would explain the blunted serum peak concentrations of RU 486 with higher doses. The return of the drug back to circulation thereafter explains the zero-order kinetics. High concentrations of circulating metabolites capable of binding to the progesterone receptor suggest a significant contribution of these steroids in the overall antiprogestational action.     

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.     

Facilitation of Sexual Receptivity by Ventromedial Hypothalamic Implants of the Antiprogestin RU 486

RU 486 is known primarily as an antagonist to progestins and glucocorticoids. However, RU 486 has also been shown to have agonistic progestational properties in biochemical and behavioral studies. In the current study, RU 486 was implanted directly into tim ventromedial hypothalamus (VMH) to test for facilitative action on the receptive behavior of female ovariectomized Long-Evans rats primed with 5 μg of estradiol benzoate. Cannulae containing RU 486, progesterone (P), or empty cannulae were implanted 48 hr after estrogen priming. The lordosis quotient and the lordosis score were assessed 4 hr after the cannulae were lowered by a standardized test consisting of 10 mounts by a stimulus male. P and RU 486 significantly facilitated receptivity compared to blank implants in terms of lordosis quotient and lordosis score, with no significant difference between the hormone treatments. While only a single dose of each treatment was given in the current study, RU 486 facilitated lordosis when implanted to the VMH as well as progesterone in contrast to our previous results where the steroids were administered systemically.     

Luteolytic action of RU486: Modulation of luteal 3β-hydroxysteroid dehydrogenase and 20α-hydroxysteroid dehydrogenase activities in late pregnant rats

The effect of the synthetic antiprogestin RU486 on luteal function in late pregnant rats was studied by evaluating the activities of the enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). RU486 (2 mg/kg) administered to rats on day 18 of pregnancy at 10.00 h induced preterm delivery 26.4 ± 0.35 h (n = 8) after treatment. Luteal 3β-HSD activity increased 24 and 34 h after RU486 injection, but a significant and progressive decrease started at 48 h with the maximal reduction 72 h after RU486 treatment, when compared with controls. Serum progesterone concentration decreased at the time of 3β-HSD activity reduction. Interestingly, 20α-HSD activity started to increase 58 h after RU486 injection. The administration of the cyclooxygenase inhibitor, diclofenac (1.3 mg/kg), on days 17–19 of pregnancy to RU486-treated rats, delayed abortion and the duration of delivery, and prevented the decrease in 3β-HSD and the increase in 20α-HSD activities observed 58 h after antiprogesterone treatment. RU486 administered intrabursally (1 μg per ovary) on day 20 (14.00–15.00 h) increased 3β-HSD and decreased 20α-HSD luteal activities at 18.00 h on day 21 of pregnancy, without modifying serum progesterone concentration, when compared with normal pregnant rats. In conclusion, the luteolytic process after preterm delivery induced by RU486 administration in late pregnant rats is characterized by a decrease in luteal 3β-HSD activity and circulating progesterone, which may trigger the increase in luteal 20α-HSD activity. Prostaglandins seems to be involved in the increase of 20α-HSD activity and therefore, in the demise of corpora lutea.     

Facilitation of receptive behavior in estrogen-primed female rats by the anti-progestin, RU 486

The progestin receptor antagonist RU 38486 (henceforth referred to as RU 486) was tested for facilitative effects on female receptive behavior in ovariectomized Long-Evans rats primed with 2 micrograms estradiol benzoate (EB). RU 486 (0, 0.5, 1.6, or 5.0 mg) was administered 48 hr after estrogen priming. The lordosis quotient (LQ) and lordosis score (LS) were assessed 4 hr after RU 486 administration in a standardized test consisting of a 10-mount test by a stimulus male. A significant dose effect was found by both LQ and LS, with those subjects receiving 5 mg of RU 486 being significantly more receptive than vehicle control animals. Thus RU 486 acted as a weak progestin agonist under testing conditions typical for assessment of progestin facilitation of female sexual behavior in rats. Low levels of proceptive behavior (hops and darts) were seen in a minority of the tests, and did not vary systematically as a function of the dose of RU 486 administered. We also examined the effects of RU 486 given before progesterone (P) on receptivity in a blocking paradigm and confirmed previous reports that the antagonist significantly attenuates facilitation of sexual behavior when given in combination with P. A progestin receptor assay of the cytosols of the hypothalamus-preoptic area in estrogen-primed female rats treated with 5 mg RU 486 revealed a significantly greater depletion of available cytosolic P receptors than when rats were treated with a similarly facilitating dose of P (100 micrograms). The results suggest a possible dual mode of action for RU 486--a weak, receptor-mediated agonistic effect on sexual behavior when given alone to estrogen-primed rats, and a competitive blocking effect on receptivity when administered with P.     

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.     

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.     

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.     

Progesterone prevents the pregnancy-related decline in protein kinase A association with rat myometrial plasma membrane and A-kinase anchoring protein

The presence of cAMP-dependent protein kinase (PKA) in the plasma membrane compartment and its association with an A-kinase anchoring protein (AKAP150) is implicated in mediating cAMP regulatory events in the rat myometrium. The association of PKA with purified myometrial plasma membrane declined gradually between Day 16 and Day 21 of gestation, with a decrease of 53% +/- 11% of the catalytic subunit and of 61% +/- 7% of the regulatory subunit at Day 21 compared with Day 19. To determine the role of progesterone in this association, pregnancy was prolonged by administration of progesterone or shortened by administration of the antiprogestin RU486. Progesterone treatment maintained PKA association with plasma membrane at Day 21 at 123% +/- 23% (catalytic subunit) and 92% +/- 4% (regulatory subunit) of Day 19 levels. In contrast, protein phosphatase 1, protein phosphatase 2B, phospholipase Cbeta(3), and AKAP150 concentrations in the plasma membrane did not change over this interval or with progesterone treatment. Changes in PKA coimmunoprecipitated with membrane-associated AKAP150 paralleled those in total plasma membrane on Days 19 and 21 and on Day 21 following progesterone treatment. In contrast, plasma membrane PKA catalytic and regulatory subunits decreased by 20 h after RU486 injection on Day 15 of pregnancy to levels resembling those on Day 21. These data indicate that progesterone prevents the decline in PKA associated with myometrial plasma membrane and with AKAP150 in the pregnant rat. The decrease in membrane-bound PKA between Days 19 and 21 and after RU486 treatment precedes the onset of parturition in both experimental paradigms. The loss of plasma membrane PKA may be critical for the decrease in the inhibitory effect of cAMP on oxytocin-induced phosphatidylinositide turnover that occurs near the end of pregnancy and may contribute to enhanced myometrial contractile responsiveness near term.     

Fetal viability and fetal growth after prolonged uterine contractions induced by progesterone withdrawal in late pregnancy in rats.

The hypothesis that sustained uterine contractile activity is the direct cause of fetal death after progesterone withdrawal in late pregnancy in rats was investigated. Pregnant rats were subjected to progesterone withdrawal on day 15 of pregnancy by injecting 2 mg mifepristone (RU 486) kg-1 or by ovariectomy with oestradiol replacement (200 ng day-1). Uterine contractile activity (force and frequency) at 4 h, but not at 2 h, in rats injected with mifepristone was significantly higher than in rats injected with vehicle. The contractile activity in mifepristone-treated rats remained higher than in control rats, at 12, 24 and 48 h. Fetal viability 36 h after mifepristone injection, when uterine contractions had lasted for 32 h, was not significantly different from fetal viability in rats injected with vehicle, but at 42 h after mifepristone injection, fetal viability was significantly reduced. In ovariectomized rats, uterine contractile activity at 12, 24, 36 and 48 h, but not at 8 h, was significantly greater than in ovariectomized rats with progesterone replacement (4 mg day-1). Fetal viability at 42 h after the operation, when uterine contractions had lasted for 30 h, was not significantly reduced, but it was significantly reduced at 48 h. When ovariectomized rats had been left to develop uterine contractions for a period before progesterone was injected, deprivation of progesterone and prolonged uterine contractions for about 30 h did not reduce fetal viability or fetal growth determined on day 18, but it did so 3 days later, on day 21. Administration of 5 mg isoxuprine kg-1 twice a day, which suppressed uterine contractions, improved fetal viability in ovariectomized rats at the earlier stage, but not at the later stage. Nevertheless, isoxuprine did improve fetal growth at the later stage in these ovariectomized rats. It is concluded that increased uterine contractile activity sustained for 32 h or less does not reduce fetal viability, but longer periods of contraction may be the cause of fetal death.     

Evidence for separate binding sites for progesterone and RU486 in the chick oviduct

Binding characteristics of synthetic steroid, mifepristone (RU38486 - also referred to as RU486), were examined in cytosol prepared from the chick oviduct and the calf uterus, and were compared with those of progesterone and synthetic progestin R5020. Unlike [³H]progesterone binding, the [³H]RU486 binding in the oviduct cytosol did not saturate at 50 nM ligand concentration. The [³H]progesterone binding could not be eliminated in the presence of excess RU486, and [³H]RU486 binding was seen to be indisplaceable upon pretreatment of the chick oviduct cytosol with a 1000-fold excess progesterone. It is apparent that the chick oviduct cytosol is endowed with two separate sets of sites which interact with progesterone and RU486 independently. Furthermore, [³H]RU486 binding in the chick oviduct cytosol remained intact when incubated for 60 min at 37°C; it exhibited a single ionic form upon elution from DEAE-Sephacel and the [³H]RU486-associated radioactivity sedimented in the 4 S region both in salt-free and 0.3 M KCl-containing 5–20% sucrose gradients. In the calf uterus cytosol, both steroids exhibited comparable binding profiles. Our results provide evidence that chick oviduct possesses distinct binding sites that accept either progesterone or RU486, but not both, as is the case in the calf uterus.