Regulation of myometrial beta 2-adrenergic receptors by progesterone and estradiol-17 beta in late pregnant rats

Rat myometrium exhibited a marked decrease in the concentration of beta 2-adrenergic receptors immediately before parturition, i.e., in the last 6 h of pregnancy. This phenomenon continued until the withdrawal of myometrial progesterone (-94% from Day 18 of pregnancy to term) and coincided with the sharp increase (+200%) of the myometrial concentration of estradiol. A linear positive correlation was found (r2 = 0.645) between the concentration of beta 2-adrenergic receptors and the log ratio of myometrial concentration of progesterone/myometrial concentration of estradiol (P/E2), suggesting a modulation of beta 2-adrenergic receptors by steroids. In rats with estrogen-dominated uteri (intact of ovariectomized late pregnant rats injected with estradiol), there was no change either in concentration or affinity of beta 2-adrenergic receptors relative to untreated control pregnant rats. In contrast, rats with progesterone-dominated uteri (intact or ovariectomized late pregnant rats treated with progesterone or ovariectomized rats) have an increased number of beta 2-adrenergic receptors, with a decreased affinity of these receptors compared to untreated control pregnant rats or to estrogen-treated rats. These results suggest that progesterone regulates the number of beta 2-adrenergic receptors in myometrium of late pregnant rats. The mechanisms by which progesterone exerts this regulation remains to be elucidated.     

Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture

Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2-12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p greater than 0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p less than 0.05) with a 24-h pulse of dibutyryl adenosine 3', 5'-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal: Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3-12) resulted in continuously stimulated (p less than 0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p less than 0.05) percentage of cells staining positive for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase (3 beta HSD) activity were recovered on Day 12 in cultures incubated (Days 3-12) with dbcAMP. Incubation of cultures continuously with prostaglandin F2 alpha (PGF2 alpha) produced dose-dependent inhibition (p less than 0.05) of progesterone secretion. Reduced numbers of 3 beta HSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF2 alpha) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.     

Further characterization of the electromyographic activity of the myometrium and mesometrium in nonpregnant sheep under estrogen supplementation.

The effects of estrogen administration on uterine contractility varies with animal species. In nonpregnant ovariectomized sheep, estrogen administration has been reported either to inhibit, inhibit then stimulate, or only stimulate uterine contractility. The aim of the present study was to determine the effects of prolonged estrogen administration in the electromyographic (EMG) activity recorded from the myometrium and mesometrium in nonpregnant ovariectomized sheep after estrous synchronization by inserting vaginal progesterone sponges 14 days before surgery. Surgery was performed on four ewes under halothane anesthesia. Bilateral oophorectomy was performed, and stainless steel EMG electrodes were sewn to the mesometrium and myometrium in both left and right horns of the uterus. Blood samples were taken at 1000 h from the uterine vein for 13,14-dihydro-15-keto-prostaglandin F2 alpha determination, and from the femoral artery for estradiol determination. Starting on Day 7 after surgery, estradiol 17 beta (50 micrograms/24 h) was infused continuously into the jugular vein. Estrogen administration had a different effect on the EMG activity recorded from myometrium and mesometrium. The myometrial response to estrogen was an increase in the frequency of short EMG events from 19.0 +/- 8.7 to 57.0 +/- 5.0 (p less than 0.05) for events less than 60 sec, and from 2.70 +/- 0.83 to 10.30 +/- 1.36 (p less than 0.05) for events lasting greater than 60 sec but less than less than 180 sec. In contrast, there was no stimulatory effect of estrogen on mesometrial EMG for both types of short events less than 60 sec, and greater than 60 but less than less than 180 sec.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin-induced stimulation of progesterone production by cow luteal cells in vitro

The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.     

Oxytocin and bovine parturition: a steep rise in endometrial oxytocin receptors precedes onset of labor.

Oxytocin receptors were measured in myometrium and intercaruncular endometrium of cows during pregnancy and parturition. Concentrations of estradiol-17 beta, estrone, and progesterone in peripheral blood were also measured. Receptor concentrations in the endometrium rose almost 200-fold from Day 20 to term (p < 0.0001, ANOVA), from 40 +/- 11 to 7300 +/- 1430 fmol/mg protein. Myometrial receptor concentrations increased 10-fold from 180 +/- 36 fmol/mg on Day 20 to 1850 +/- 360 fmol/mg protein at term (p < 0.0001, ANOVA). During labor, endometrial receptors (6600 +/- 1300 fmol/mg) remained at prelabor values, whereas myometrial receptor concentrations had decreased to 1190 +/- 316 fmol/mg (not significant) and declined further postpartum. Plasma concentrations of progesterone declined from 4-5 ng/ml to about 2 ng/ml between Days 250 and 282 and dropped to < 0.2 ng/ml shortly before delivery. Plasma concentrations of estrone and estradiol-17 beta were below 10-20 pg/ml until Day 230. Estrone concentrations were significantly (p < 0.05) increased by Day 250 and estradiol-17 beta by Day 270, and then both rose rapidly. During labor, plasma estrone was 1135 +/- 245 pg/ml and plasma estradiol-17 beta was 226 +/- 131 pg/ml. The molar ratio of estrone and estradiol-17 beta to progesterone rose from less than 0.01 to 4.4 during labor, and was correlated with oxytocin receptor concentrations in endometrium (r = 0.5160, p < 0.001), but not those in myometrium (r = 0.0122). The regulation of oxytocin receptors by ovarian hormones in the two tissues may therefore differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of gonadotropin treatment interval on follicular maturation, in vitro fertilization, circulating steroid concentrations, and subsequent luteal function in the domestic cat.

The impact of the eCG-hCG interval on in vitro fertilization (IVF), endogenous hormonal patterns, and luteal integrity was studied in the domestic cat. Adult cats with inactive ovaries were given eCG (i.m.) and then hCG (i.m.) 80, 84, 88, 92, or 96 h later. Oocytes were aspirated 25-27 h after hCG and co-cultured with swim-up-processed cat spermatozoa. Blood samples were collected daily from 2 days before eCG treatment (Day -2) through Day 14, and sera were analyzed for estradiol-17 beta and progesterone. The mean number of oocytes recovered from the 80-92-h groups (range, 17.2 +/- 2.1 to 21.1 +/- 3.0) did not differ (p greater than 0.05); however, oocyte number was reduced (p less than 0.05) in the 96-h group (10.3 +/- 2.1). The proportion of all oocytes classified as mature was greater (p less than 0.05) when hCG was given 80, 84, or 88 h compared to 92 or 96 h after eCG. Delaying hCG treatment until 96 h caused more than 25% of all oocytes to degenerate, which was a greater rate (p less than 0.05) than in all other groups. The IVF rate at 80 (57.1%), 84 (56.5%), 88 (65.0%), and 92 (52.5%) h was greater (p less than 0.05) than that observed at 96 h (33.6%). Circulating estradiol-17 beta concentrations began to rise above nadir within 24 h of eCG injection in all interval groups. On the basis of areas under the curve, cats in the 80- and 84-h treatments produced more (p less than 0.05) estradiol-17 beta than other groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cytosol and nuclear progesterone receptors in rabbit uterus by estrogen, antiestrogen and progesterone administration.

A synthetic progestin, 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione (ORG 2058), was utilized to measure progesterone receptors from the rabbit uterus. This steroid has a high affinity for both cytosol and nuclear receptors, with KD values of 1.2 nM (at 0--4 degrees C) and 2.3 nM (at 15 degrees C), respectively. Administration of estradiol-17 beta or a non-steroidal antiestrogen, tamoxifen, for 5 days to estrous rabbits led to a progressive rise in the cytosol receptor levels: from 34,000 to 120,000 (estradiol-17 beta) and 80,000 (tamoxifen) receptors/cell, without any major influence on the nuclear receptor content. A single intravenous injection of progesterone (5 mg/kg) elicited a 3-fold increase in the mean nuclear receptor content at 30 min after injection (from 18,000 to 48,000 receptors/nucleus). Nuclear receptor accumulation was short-lived and returned to control levels within 4 h after treatment. A second dose of progesterone given 24 h later doubled the nuclear receptor level (from 18,000 to 35,000 receptors/nucleus). The concomitant decline in the cytosol receptor content was twice that accounted for by the nuclear receptor accumulation (70,000 vs. 30,000, and 40,000 vs. 17,000 receptors/cell, after the first and second progesterone injection, respectively). Following progesterone administration, the cytosol receptor level reached a nadir by 30 min, exhibited minimal replenishment within the ensuing 24 h, and remained at approx. 50% of the pretreatment values. After a single dose or two consecutive doses of progesterone, total uterine progesterone receptor content declined to about 60% of the level prior to each dose, a nadir being reached at 2 h after treatment.     

Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappaB activation of cyclooxygenase 2 expression

Spontaneous labor in women and in other mammals is likely mediated by a concerted series of biochemical events that negatively impact the ability of the progesterone receptor (PR) to regulate target genes that maintain myometrial quiescence. In the present study, we tested the hypothesis that progesterone/PR inhibits uterine contractility by blocking nuclear factor kappaB (NF-kappaB) activation and induction of cyclooxygenase-2 (COX-2), a contractile gene that is up-regulated in labor. To uncover mechanisms for regulation of uterine COX-2, immortalized human fundal myometrial cells were treated with IL-1beta +/- progesterone. IL-1beta alone caused a marked up-regulation of COX-2 mRNA, whereas treatment with progesterone suppressed this induction. This was also observed in human breast cancer (T47D) cells. In both cell lines, this inhibitory effect of progesterone was blocked by RU486. Using chromatin immunoprecipitation, we observed that IL-1beta stimulated recruitment of NF-kappaB p65 to both proximal and distal NF-kappaB elements of the COX-2 promoter; these effects were diminished by coincubation with progesterone. The ability of progesterone to inhibit COX-2 expression in myometrial cells was associated with rapid induction of mRNA and protein levels of inhibitor of kappaBalpha, a protein that blocks NF-kappaB transactivation. Furthermore, small interfering RNA-mediated ablation of both PR-A and PR-B isoforms in T47D cells greatly enhanced NF-kappaB activation and COX-2 expression. These effects were observed in the absence of exogenous progesterone, suggesting a ligand-independent action of PR. Based on these findings, we propose that PR may inhibit NF-kappaB activation of COX-2 gene expression and uterine contractility via ligand-dependent and ligand-independent mechanisms.     

Regulation of alpha- and beta-adrenergic receptor subclasses by gonadal steroids in human myometrium

Both alpha- and beta-adrenergic receptors have been identified in the human myometrium by radioligand binding. Both adrenergic receptor subclasses have been shown to mediate the contractile response of the uterus upon catecholamine stimulation: alpha-adrenergic receptors cause uterine contraction while beta-adrenergic receptors induce relaxation. We have identified alpha 1- and alpha 2-adrenergic receptors in myometrial membranes using the newly developed radiolabelled specific antagonists [3H]-prazosin and [3H]-rauwolscine. This enabled us to characterize both receptor subclasses individually. Beta adrenergic receptors were identified using the radiolabelled antagonist (-)-[3H]-dihydroalprenolol. Binding of radioligands to the myometrial membrane receptors was rapid, readily reversible, of high affinity and stereoselective. The total number of alpha 1-, alpha 2- and beta-receptors was determined by Scatchard analysis of radioligand saturation binding and the beta/beta 2-receptor ratio was determined by computer analysis of the beta 2-selective antagonist ICI 118 551) (-)-[3H]-dihydroalprenolol competition binding curves. This enabled us to study the regulation of both alpha- and beta-receptor subclasses under various physiological and pharmacological conditions in the human, i.e., during different phases of the menstrual cycle, in postmenopausal women and during depo-progestin (Medroxyprogesterone acetate) therapy. Only the alpha 2- and beta 1-adrenergic receptor concentrations were found to be subjected to gonadal steroid regulation. The number of alpha 2- and beta 1-adrenergic receptors increased concomitantly with circulating plasma oestradiol levels. This effect was counteracted by progesterone. The number of alpha 1- and beta 2-adrenergic receptors was unaffected by the gonadal steroid environment. These results are an example of the heteroregulation of membrane receptors by oestrogens and progesterone and cast new light on the regulatory mechanisms involved in uterine contractility in the human.     

Endocrine, luteal and follicular responses after the use of the short-term protocol to synchronize ovulation in goats

The effect of the so-called Short-Term Protocol (5-day progesterone treatment+PGF(2)alpha) on ovarian activity and LH surge was studied in goats. The goats received 250IU eCG at the time of device withdrawal (eCG group; n=7), or 200microg of EB (estradiol benzoate) 24h after device withdrawal (EB group; n=8), or received neither eCG nor EB (control group; n=8). The Short-Term Protocol induced greater (4.1+/-1.1ng/ml) progesterone serum concentrations at 24h after start of the treatment, that declined to 0.2+/-0.1ng/ml at 12h after device withdrawal. In all of the groups, the maximum concentration of estradiol-17beta was reached at about 36h after device withdrawal. Maximum concentration was greater in the EB group (76.9+/-24.6pmol/l) than in the control group (41.8+/-9.0pmol/l; P<0.01), with the eCG group showing intermediate concentration (70.3+/-32.5pmol/l; P=NS). The LH peak occurred earlier in the eCG group (38.4+/-2.0h after device withdrawal) and in the EB group (41.0+/-4.1h), than in the control group (46.3+/-5.1h; P<0.05). Ovulation occurred earlier in the eCG group (5/7) and in the EB group (8/8) (58.8+/-2.7h and 63.0+/-5.6h, respectively), than in the control group (7/8) (70.2+/-8.3h; P<0.05). In summary, the Short-Term Protocol induced similar concentrations of progesterone among treated goats. In addition, eCG or EB resulted in a similar increase in estradiol-17beta and a similar LH surge, which induced ovulation in most females (86.7%) in a consistent interval (about 60h) after the end of progesterone exposure.     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of plasminogen activitor inhibitor-1 in human adipose tissue: interaction between cytokines, cortisol and estrogen.

To investigate further the role of plasminogen activator inhibitor-1 (PAI-1) in human adipose tissue, the regulation of cytokines, cortisol (dexamethasone) as well as estrogen on PAI-1 were determined in human adipose tissue fragments. PAI-1 activity was increased in human adipose tissue fragments incubated for 48 h with interleukin-1beta (IL-1beta) (2.6-fold, p < 0.01) and tumor necrosis factor-alpha (2.3-fold, p < 0.01). Incubation with interleukin-6 revealed a non-significant decrease in PAI-1 activity. Parallel findings were obtained when studying the PAI-1 mRNA expression. Dexamethesone increased PAI-1 activity after incubation for 8 h (p < 0.05) and enhanced the stimulation of IL-1beta after 8 h incubation. However, after 24 and 48 h, dexamethasone significantly reduced the IL-1beta induced increase in PAI-1 activity by 24-52% (p < 0.05), accordingly, PAI-1 mRNA expression was reduced 60%. Finally, the induction of PAI-1 activity and PAI-1 mRNA expression by IL-1beta was attenuated by estrogen (17.8+/-4.9%, p < 0.05 and 20.9+/-5.8%, p < 0.05, respectively). These results indicate that multiple cytokines, estrogen and dexamethasone may be involved in the regulation of PAI-1 biosynthesis in human adipose tissue, and suggest that there are interactions between cytokines and these steroid hormones. The interplay between these hormones may be of importance for the levels of PAI-1 observed in obesity and associated states.