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.     

Insulin regulation of steroidogenic activity in rat culture luteal cells

The effect of insulin on the function of rat luteal cells in monolayer culture was examined. Cells were obtained from PMSG-hCG primed immature rats and further cultured in serum free medium with or without insulin. The hormone produced an increase of progesterone production and maximal stimulation was achieved at 0.2 nM of insulin (100% stimulation). This effect was enhanced by addition of methyl-isobutyl-xantine (MIX 0.1 mM) to the culture medium. However, the stimulation produced by LH was not augmented by the presence of insulin. The conversion of progesterone into 20 alpha-hydroxy-progesterone was also enhanced after insulin treatment. Luteal cells were also cultured in the presence of 25-hydroxy-cholesterol (10 micrograms/ml). In these conditions insulin produced a 2-fold increase in progesterone production. Aromatase activity was assessed by adding androstenedione (0.25 microM) as substrate. Insulin produced a 14-fold stimulation of estradiol production after 24 h of culture. Insulin action was tested in short time incubations of luteal cells in a glucose free medium, in these experiments the hormone was able to induce a significant increase in progesterone and 20 alpha-hydroxy-progesterone production. These data suggest that luteal cell function is regulated by insulin and that this hormone has a direct effect on the steroidogenic process.     

Weak estrogenic activity of phenol red in the pituitary gonadotroph: re-evaluation of estrogen and antiestrogen effects

Phenol Red (Phr) which is widely used as a pH indicator in cell culture media has recently been described to possess estrogenic activity in different cell types. In the present study we investigated if the dye shows such activity on LH secretion of cultivated rat pituitary cells and controlled the established effects of estradiol (E2) and keoxifene (K) in this model in the absence of Phenol Red. 24 h treatment of pituitary cell cultures with Phr led to enhancement of GnRH-stimulated LH secretion whereas 4 h treatment reduced LH secretion. When the cells received E2 instead of Phr for the indicated incubation periods we observed nearly identical results i.e. a short-term inhibitory and a long-term stimulatory effect on LH secretion. 24 h treatment of pituitary cell cultures with increasing concentrations of Phr led to a stimulatory effect on GnRH-stimulated LH secretion an effect that occurred at 10 microM got maximal at 100 microM and was lost at higher concentrations resulting in a bell-shaped dose-response curve. The inhibitory action of Phr was present at concentrations greater than or equal to 10 microM. Both effects could be blocked by the antiestrogen K indicating their specificity. K has recently been described to induce an antigonadotrophic effect in this model. Although high concentrations of the antiestrogen were still able to inhibit LH secretion this effect was not present at lower concentrations when Phr-free culture medium was used in the experiments. Thus Phr showed weak estrogenic activity in the gonadotroph. The established actions of E2 and K on LH secretion were qualitatively reproducible when Phr was excluded from the culture medium.     

The effect of progesterone and human interferon alpha-2 on the release of PGF2 alpha and PGE from epithelial cells of human proliferative endometrium.

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of menstrual cycle (n = 8). Progesterone at a concentration of 1 microM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 microM suppressed both the release of PGF2 alpha and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 microM arachidonic acid (AA) abolished this effect of progesterone on both PGF2 alpha and PGE release. Interferon alpha-2 did not suppress the basal release of PGF2 alpha nor PGE. In the presence of progesterone, interferon alpha-2 attenuated the progesterone mediated suppression of PGF2 alpha but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon alpha-2 does not exert this action on human endometrium.     

The suppressive effect of delta-1-tetrahydrocannabinol on the steroidogenic activity of rat granulosa cells in culture

In previous study the major psychoactive ingredient of marihuana (delta 1-THC) has been shown to inhibit ovarian prostaglandin synthesis when administered to normally cycling rats in the early afternoon of proestrus. These results suggested a direct suppressive effect of the drug on the ovary. The purpose of this study was to evaluate the effect of delta 1-THC on steroidogenesis in granulosa cells (GC) in vitro. Incubation of GC with delta 1-THC (10-50 microM) effectively inhibited LH-stimulatable progestin production. This suppressive effect was not abolished by washing the cells after 24 h of culture in the presence of delta 1-THC, indicating the irreversible nature of the blocking effect of delta 1-THC. By contrast, estradiol production following incubation of GC with testosterone (1 microgram/ml) was not inhibited by similar concentrations of delta 1-THC, thus suggesting that delta 1-THC does not inhibit aromatase activity in GC. In addition, delta 1-THC was shown to inhibit cAMP production as well as 125I-hCG binding capacity to GC. Administration of 8-Br-cAMP did not abolish the delta 1-THC-induced block, suggesting that the drug probably acts distal to the cAMP site of action.     

In vitro effects of cannabinoids on follicular function in the rat

delta 1-Tetrahydrocannabinol (delta 1-TCH), the major psychoactive constituent of marihuana, was found to suppress the preovulatory surge of gonadotropins and thereby to prevent ovulation in rats, rabbits and rhesus monkeys. These studies suggested that the drug acts primarily on the hypothalamus to suppress luteinizing hormone releasing hormone (LHRH) secretion. The aim of the present study was to examine the direct effect of delta 1-THC, the psychoactive constituent of marihuana and cannabidiol (CBD), one of its nonpsychoactive constituents, on preovulatory rat follicles in vitro. Both cannabinoids inhibited follicular steroidogenesis in a dose-dependent manner. Basal accumulation of progesterone (P), testosterone (T) and estradiol-17 beta (E2) was reduced up to 60% by the highest doses examined (100-200 microM). The luteinizing hormone (LH)-stimulated increase in P and T was inhibited by 75-88% by the highest doses of both cannabinoids (50-200 microM), while E2, accumulation was inhibited by only 40%. It appears that the inhibitory action of cannabinoids is exerted beyond LH binding and activation of adenylate cyclase and prior to pregnenolone formation in the gonadal steroidogenic pathway. In addition to this anti-steroidogenic effect, both cannabinoids induced resumption of meiosis in follicle-enclosed oocytes cultured in hormone-free medium; 200 microM delta 1-THC resulted in 80% maturation and CBD in 75%. It seems that the action of cannabinoids on rat follicles in vitro is unrelated to their psychotropic activity.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct opioid regulation of pituitary release of bovine luteinizing hormone

Although endogenous opioid peptides (EOP) are thought to alter pituitary release of luteinizing hormone (LH) by modifying the release of gonadotropin-releasing hormone (GnRH) from the brain, EOP may also directly affect the release of LH from pituitary cells. This hypothesis was tested using dispersed cells from the bovine anterior pituitary gland. Pituitaries were enzymatically dissociated, preincubated for 18 h and then cultured for either 2 or 24 h with GnRH, naloxone, methionine-enkephalin (Met-enk) or their combinations. Basal release of LH into media was 18.2 and 38.4 ng/100,000 cells after culture for 2 or 24 h, respectively. When cultured for 2 or 24 h with 10 nM GnRH, LH release was 296% and 131% of the basal release for each culture period. Cellular viability (75% vs 68%) and total (cells + medium) LH (128 vs 134 ng/100,000 cells) did not differ (P greater than .05) between cells cultured for 2 or 24 h. Naloxone (1 microM) increased (P less than .01) basal release of LH by 57% after 2 h of culture but not after 24 h of culture. Naloxone did not augment the amount of LH released in response to 10 nM GnRH. Addition of Met-enk (1 nM to 1 microM) suppressed (P less than .05) basal release of LH (23% to 62%) after 2 h of culture. Similar suppressive effects (8% to 49%) occurred in a dose-dependent manner (0.1 nM to 1 microM) after 24 h of culture. Met-enk (1 and 100 nM) antagonized (P less than .05) the stimulatory effect of naloxone and reduced (P less than .05) the amount of LH released in response to GnRH after 2 h of culture. In summary, the stimulatory effect of naloxone on the basal release of LH suggests that EOP may directly regulate pituitary cell function; the inhibitory effect of physiological concentrations of Met-enk on the basal in vitro release of LH suggests that EOP may directly affect the release of LH in vivo; the antagonism between the stimulatory effect of naloxone and the inhibitory effect of Met-enk is consistent with effects exerted through opioid receptors; and the stimulatory effect of GnRH may be partially reduced by Met-enk. These results are consistent with the hypothesis that opioids may directly modulate the release of LH at the pituitary level.     

The effect of Λ9-tetrahydrocannabinol on the positive and negative feedback control of luteinizing hormone release

The principal psychoactive component of marihuana, Λ⁹-Tetrahydrocannabinol (THC), decreases serum LH levels when given to ovariectomized rats at a dose of 10 mg/kg. This same dose of THC blocks both the estrogen and progesterone mediated positive feed-back release of LH. The steroid-induced LH surge, when blocked by THC, reappears 24 h later. The positive feedback release of LH should serve as a suitable model to study the mechanism of action by which THC blocks the ovulatory LH surge.     

The interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated from pigs in early and midluteal phase.

The first objective of this research was to define the capacity of corpora lutea of pig to secrete estradiol in the presence of an androgen substrate which was testosterone. The second objective was to define the synergism between gonadotropic hormones such as LH, FSH, and PRL and testosterone as measured by estradiol and progesterone secretion by two types of porcine luteal cells. Luteal cells were collected from newly forming corpora lutea (0-3 days after ovulation) and from mature corpora lutea (8-10 days after ovulation). After dispersion, luteal cells were suspended in medium M199 supplemented with 10% of calf serum and grown as monolayers at 37 degrees C. Control cultures were grown in medium alone while other cultures were supplemented with either testosterone alone at a concentration of 1 x 10(-7) M or with 10, 100, 500 ng LH plus testosterone, 10, 100, 500 ng FSH plus testosterone or 10, 100, 500 ng PRL plus testosterone. After 2 days of cultivation all cultures were terminated and media were frozen at 20 degrees C for further steroid analysis. Testosterone added to the culture medium in the absence of gonadotropins was without effect on estradiol and progesterone secretion by luteal cells collected in the corpora lutea of the early luteal phase. On the other hand testosterone added to the medium significantly increased progesterone and estradiol secretion by cultured luteal cells collected in the midluteal phase of the cycle. No additive stimulatory action of gonadotropins and testosterone on progesterone secretion was observed in cultures of luteal cells from the early luteal phase but this was not the case in cultures of luteal cells from the midluteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cyclodextrin-encapsulated beta-carotene on progesterone production by bovine luteal cells

Experiments were conducted to examine the effect of cyclodextrin-encapsulated beta-carotene on basal or cholesterol (cyclodextrin-encapsulated), LH and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production by bovine corpus luteum cells isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with serum-free DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. Medium was replaced after 24h and thereafter every 48 h. Beta-carotene was added to cultures in a carrier molecule, dimethyl-beta-cyclodextrin, to facilitate dissolution. All treatments were started on day 3 of culture. Treatment of cells with 1 or 2 micromol/l beta-carotene resulted in sharp inhibition of progesterone production. On the contrary, treatment of cells with 0.1 micromol/l beta-carotene resulted in significant stimulation (P<0.05) of both basal and cholesterol-stimulated progesterone secretion. The effect of beta-carotene on LH or dbcAMP-stimulated progesterone production was also examined. Treatment of cells with LH or dbcAMP always resulted in stimulation of progesterone secretion (P<0.001). However, cells treated with LH plus beta-carotene or dbcAMP plus beta-carotene both produced significantly (P<0.01) less progesterone relative to those cells treated with LH or dbcAMP alone on days 7, 9 and 11 of culture. These results indicate that beta-carotene can enhance luteal steroidogenesis when present at low concentrations but is inhibitory at higher concentrations and that encapsulation of beta-carotene in cyclodextrin is an effective method of supplying it to cells in culture.     

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenol's stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.     

Effect of histone H2A on progesterone production by bovine luteal cells

Histone H2A competitively inhibits binding of GnRH to high affinity rat ovarian receptor sites and blocks gonadotropin-stimulated steroid and cAMP accumulation during culture of rat granulosal or luteal cells. The objective of our study was to examine the progesterone suppressive effects of histone H2A on bovine luteal cells. In the first study, luteal cells were treated at Time = 0 h with a partially purified preparation of bovine ovarian histone H2A (3 ng GnRH equivalents, 800 micrograms protein), equivalent amounts of GnRH (3 ng), or BSA (800 micrograms) and incubated for a total of 4 h. At Time = 2 h, cells were treated with 5 ng bovine LH (bLH) or with medium. Histone H2A completely blocked both basal and LH-induced accumulation of progesterone compared with untreated cultures or cultures treated with bLH. Neither BSA nor GnRH suppressed LH-induced progesterone accumulation. In the second study, histone H2A was added to cultures at Time = 0 h and bovine luteal cells were cultured for 8 h. After 2 h of treatment, histone H2A (3 ng GnRH equivalents) was removed from selected cultures and replaced with fresh medium. Four hours later cultures were treated with 5 ng bLH or medium. LH treatment of cultures from which histone H2A had been removed resulted in an increase in accumulation of progesterone compared with control cultures treated throughout the treatment period with histone H2A. The third study examined the effect of 9-181 pg GnRH equivalents (1.7-34 micrograms protein) of a highly purified preparation of bovine ovarian histone H2A on basal and LH-induced progesterone production during 2 or 3 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Stimulatory and inhibitory effects of progesterone on FSH secretion by the anterior pituitary.

The purpose of this study was to investigate whether progesterone exerted progesterone receptor mediated direct effects on the anterior pituitary in the secretion of FSH and whether such effects were mediated through the 5 alpha-reduction of progesterone. Treatment of anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the ED50 for gonadotropin releasing hormone (GnRH)-stimulated FSH release from 0.58 to 0.36 ng/ml and the ED50 for GnRH-induced LH release from 0.54 to 0.19 ng/ml. When dispersed pituitary cells were treated with 0.5 nM estradiol and exposed to various doses of progesterone for 1 to 6 h, the most consistent rise in basal and GnRH-stimulated FSH release was observed with the 50 nM dose of progesterone with a 3-h exposure period. All three doses of progesterone elevated basal LH and GnRH-stimulated LH was increased by the 50 and 100 nM doses of progesterone during the 3-h period of treatment. Using the 50 nM dose of progesterone, basal and GnRH-stimulated LH was increased after 2, 3 and 6 h of progesterone treatment. When the period of exposure of progesterone was extended to 12, 36 or 48 h, there was a significant inhibition of GnRH-stimulated FSH release. GnRH-stimulated LH release was inhibited at 36 and 48 but not 12 h after progesterone treatment. These studies showed that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. These results are similar to those observed in vivo after progesterone treatment. Furthermore estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone. The progesterone antagonist RU486 prevented the progesterone-induced rise in GnRH-stimulated FSH release. Furthermore the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide also prevented the progesterone-induced rise in GnRH-stimulated FSH release in estrogen-treated dispersed pituitary cells. These results indicate that the anterior pituitary is a major site of action of progesterone in the release of FSH and that 5 alpha-reduction of progesterone plays an important role in FSH release.     

Hormonal regulation of estrogen and progestin receptors in decidual cells

Total estrogen receptor (Re) and total progestin receptor (Rp) were measured in the cytosol and nuclear fractions from hamster deciduomal tissue and decidual cell cultures. Correlation of serum steroid (estradiol and progesterone) and deciduomal receptor profiles revealed a significant loss of Re during the first four days of decidualization that was not attributable to changes in serum steroid levels. A decidual cell-tissue culture system was used to study the receptor's recovery response to progesterone withdrawal. Decidual cells were plated and grown in Ham's F12/Dulbecco's modified Eagle's medium with 5% horse serum supplemented with insulin, transferrin, selenium and progesterone (10 ng/ml). Within 48 h of culture large, multinucleate decidual cells were observed by phase microscopy. At 72 h of culture in medium containing progesterone, only Rp was detectable in decidual cells. Re was not detectable (less than 200 fmol/mg DNA) in either cytosol or nuclei from cells maintained in the presence of progesterone. However, when progesterone was deleted from the medium, cytosol Re recovered progressively from 8 h to 16 h of culture. Progesterone withdrawal also caused parallel increases in cytosol and nuclear Rp, and estradiol treatment (2 ng/ml) in combination with progesterone withdrawal further enhanced Rp levels in decidual cell cultures. These results with cultured decidual cells demonstrate that progesterone down-regulates Re and Rp, Re recovers rapidly upon progesterone withdrawal, and the Re system is competent to respond to estrogen action in terms of Rp induction. We used the density-shift method to determine that progestin increases the turnover of nuclear Re in hamster decidual cells within 3 h. Hamster decidual cells were isolated from the endometrium and cultured in progesterone-free medium containing normal amino acids (1H, 12C, 14N) for 2 days. Confluent monolayers of cells were exposed to 1 nM estradiol (E2) for 1 h to maximize the amount of occupied Re in the nuclear fraction. Then, at time 0, cells were transferred to medium supplemented with dense (2H, 13C, 15N) amino acids and either 1 nM E2 or E2 plus 100 nM progesterone. After Re was labeled with dense amino acids for 1, 3, 6 and 9 h, nuclear Re was extracted with 10 mM pyridoxal -5' phosphate and labeled with 125I-iodoestradiol (5 nM). Two radioactive peaks representing preexisting and newly synthesized Re were separated by sucrose density-gradient centrifugation. The halflife of nuclear Re in decidual cells was 3.7 h when cells were treated with E2 alone.(ABSTRACT TRUNCATED AT 400 WORDS)