Luteal expression of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase, and 20alpha-hydroxysteroid dehydrogenase genes in late pregnant rats: effect of luteinizing hormone and RU486

A decrease in serum progesterone at the end of pregnancy is essential for the induction of parturition in rats. We have previously demonstrated that LH participates in this process through: 1) inhibiting 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and 2) stimulating progesterone catabolism by inducing 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity. The objective of this investigation was to determine the effect of LH and progesterone on the luteal expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450(scc)), 3beta-HSD, and 20alpha-HSD genes. Gene expression was analyzed by Northern blot analysis 24 and 48 h after administration of LH or vehicle on Day 19 of pregnancy. StAR and 3beta-HSD mRNA levels were lower in LH-treated rats than in rats administered with vehicle at both time points studied. P450(scc) mRNA levels were unaffected by LH. The 20alpha-HSD mRNA levels were not different between LH and control rats 24 h after treatment; however, greater expression of 20alpha-HSD, with respect to controls, was observed in LH-treated rats 48 h after treatment. Luteal progesterone content dropped in LH-treated rats at both time points studied, whereas serum progesterone decreased after 48 h only. In a second set of experiments, the anti-progesterone RU486 was injected intrabursally on Day 20 of pregnancy. RU486 had no effect on 3beta-HSD or P450(scc) expression but increased 20alpha-HSD mRNA levels after 8 h treatment. In conclusion, the luteolytic effect of LH is mediated by a drop in StAR and 3beta-HSD expression without effect on P450(scc) expression. We also provide the first in vivo evidence indicating that a decrease in luteal progesterone content may be an essential step toward the induction of 20alpha-HSD expression at the end of pregnancy in rats.     

Participation of ovarian 20 alpha-hydroxysteroid dehydrogenase in luteotrophic and luteolytic processes during rat pseudopregnancy

In normal rats, before Day 12 of pseudopregnancy, minimal levels of 20 alpha-HSD activity were detected in functional CL whereas those in the residue were 3-5 times higher. When ovulation was blocked for more than 2 weeks by placing rats in a continuously lit environment before the induction of pseudopregnancy, only minimal levels of 20 alpha-HSD activity were detectable in the functional CL and residue before Day 12. In normal pseudopregnant rats, there was a linear increase in 20 alpha-HSD activity from Day 12 to 15 in the functional CL and residue, but the rate of elevation was much higher in functional CL. This tendency was much more clear-cut in rats in the continuous lighting. In immature rats in which pseudopregnancy was induced by PMSG and hCG treatment, 20 alpha-HSD activity peaked twice. The first small peak was attributed to the early regression of some of the large number of corpora lutea, and the changes in 20 alpha-HSD activity in most of the corpora lutea paralleled those in rats in continuous lighting. Bromocriptine abolished the prolactin surges, and in normal pseudopregnant rats an increase in 20 alpha-HSD activity in functional CL started from 12 h and the rate of the increase was accelerated from 36 h afterwards, while a relatively small increase was observed in the residue at 18 h and later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of RU 486 on luteal function in the early pregnant rat

A dose of 30 mg RU 486/kg, an antiprogesterone, was administered to pregnant rats on Day 2 (Group 1) or Day 4 (Group 2) of pregnancy. RU 486 significantly changed serum progesterone and oestradiol concentrations and luteal 3 beta-HSD and 20 alpha-HSD activities in Group 1, and implantation was significantly inhibited. The luteal 3 beta-HSD activity in Group 2 rats on Day 6 was significantly (P less than 0.01) lower than the control value (7.5 +/- 0.6 and 10.1 +/- 0.6 mU/mg protein respectively). This decline in the 3 beta-HSD activity was followed by a marked decrease in the serum progesterone concentration, resulting in a significant decrease of the progesterone/oestradiol ratio and implantation was completely inhibited. The 20 alpha-HSD activity, which could not be detected on Day 6 in the control rats, was twice as great in Group 2 than in Group 1 rats (17.5 +/- 1.2 and 7.4 +/- 3.1 mU/mg protein respectively). Ultrastructural examination of corpora lutea of Group 2 rats confirmed luteolysis. These results suggest that RU 486 has a luteolytic effect and its anti-implantation effect is concomitant with luteolysis of the corpora lutea of pregnancy.     

Progesterone receptor is not required for progesterone action in the rat corpus luteum of pregnancy

In this study, we investigated whether progesterone exerts a local action regulating the function of the corpus luteum of pregnancy in rats. The luteal activities of the enzymes 3beta-hydroxysteroid dehydrogenase (3beta-HSD), involved in progesterone biosynthesis, and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), that catabolizes progesterone and reduces progesterone secretion by the corpus luteum, were evaluated after intrabursal ovarian administration of progesterone in pregnant rats that had received a luteolytic dose of prostaglandin F2alpha (PGF2alpha). Luteal 3beta-HSD activity decreased and 20alpha-HSD activity increased after PGF2alpha treatment (100 microg x 2 intraperitoneally on Day 19 of pregnancy at 12:00 p.m. and 4:00 p.m.) when compared with controls sacrificed at 8:00 p.m. on Day 20 of pregnancy. This effect of PGF2alpha on the luteal 3beta-HSD and 20alpha-HSD activities was abolished in animals that also received an intraovarian dose of progesterone (3 microg/ovary on Day 19 of pregnancy at 8:00-9:00 a.m.). In a second functional study, luteal cells obtained from 19-day pregnant rats responded to the synthetic progestin promegestone (R5020) in a dose-dependent manner, with an increase in the progesterone output. In addition, the glucocorticoid agent hydrocortisone did not affect progesterone accumulation in the same luteal cell culture. We also examined by immunocytochemistry the expression of progesterone receptors (PR) in the corpora lutea during pregnancy and demonstrated the absence of PR in this endocrine gland in all the days of pregnancy studied. In the same pregnant rats, positive staining for PR was observed in cells within the uteroplacental unit, such as cells of the decidua basalis and trophoblast giant cells of the junctional zone. In addition, positive PR staining was observed in the ovarian granulosa and theca cells of growing follicles, but not in corpora lutea of ovaries obtained from cycling rats at proestrus. In summary, this report provides further evidence of a local action of progesterone regulating luteal function in the rat despite the absence of a classic PR.     

Dependence on prolactin of the luteolytic effect of prostaglandin F2alpha in rat luteal cell cultures

Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20alpha-dihydroprogesterone, a product of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20alpha-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450(scc)) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2alpha (PGF2alpha) was added 7-8 days after seeding. In prolactin-treated cells, PGF2alpha reduced steroidogenesis after 4-45 h, and at 45 h total progestins and P450(scc) mRNA were reduced by 45%. At 8-45 h PGF2alpha reduced the percent progesterone out of total progestins, and at 45 h 20alpha-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2alpha had little effect on total progestins or 20alpha-HSD mRNA but doubled P450(scc) mRNA. Phospholipase C activity was stimulated by PGF2alpha regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2alpha; the finding that without prolactin PGF2alpha has an alternative set of actions could help in identifying the signaling pathways of PGF2alpha responsible for its luteolytic effects.     

Termination of pregnancy after accelerated lactation in the rat. III. Effects of hormonal treatments.

The relationship was investigated of the hormones associated with pregnancy and lactation and the termination of pregnancy that occurs when rats mated at the post-partum oestrus are allowed to suckle a large litter after implantation. The primary cause for pregnancy termination was found to be an insufficient level of progesterone with the possible need for a primary or synergistic dose of oestrogen. The progesterone deficiency was not due to the high levels of prolactin present in nursing rats, since pregnancy termination could not be produced by prolactin administration in the absence of accelerated lactation, and could not be prevented by blocking prolactin secretion after accelerated lactation. Administration of LH to rats after accelerated lactation did prevent termination of pregnancy.     

Microsomal 20 alpha-hydroxysteroid dehydrogenase activity for progesterone in human placenta

Placental 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity was studied in order to evaluate the mechanism of continuation of pregnancy and initiation of labor. The placentas obtained at various gestational weeks were homogenized and fractionated into "nuclear", "mitochondrial", "microsomal" and "supernatant" fractions. Each fraction was incubated with 14C-progesterone and a hydrogen donor. Enzymatic activity was measured by the conversion of progesterone to 20 alpha-dihydroprogesterone. The highest activity of 20 alpha-HSD for progesterone was found to be localized in "microsomal" fraction. The Km constant of 20 alpha-HSD was 4.5 X 10(-6)M for progesterone in "microsomal" fraction. It was found that placental microsomal 20 alpha-HSD required NADPH as well as NADH. 20 alpha-HSD activity for progesterone increased as gestational weeks advanced. The addition of DHA-sulfate and DHA inhibited 20 alpha-HSD activity for progesterone significantly, suggesting that the steroid produced by the feto-placental unit may be involved in the metabolism of progesterone in human placenta.     

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.     

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.     

In vitro secretion of progestins by rat luteal cells and their 20 alpha-hydroxysteroid dehydrogenase activity

Functionally active or regressing corpora lutea were harvested from pseudopregnant (psp) rats between days 5-8 of psp or day 15 of psp, respectively. They were enzymatically dispersed and cultured for 24 h to assess progestins in the medium and 20 alpha-hydroxysteroid dehydrogenase [20 alpha-HSD, catalyzing the conversion of progesterone to 20 alpha-dihydroprogesterone (20 alpha-OH-P)] activity in the cell. Though the active luteal cells retained low 20 alpha-HSD activity, they secreted 6-7 times more 20 alpha-OH-P than progesterone as the regressing luteal cells did. There was no significant difference between the total amounts of progestins in the 2 groups. When increasing doses of pregnenolone were added to the media, progesterone secretion from the active luteal cells was promoted and the progesterone to 20 alpha-OH-P ratio became comparable to the circulating progestins ratio during the mid-luteal phase. In contrast, from the regressing luteal cells only 20 alpha-OH-P secretion was promoted. These results indicate that an insufficient precursor supply results in the catabolism of a large part of synthesized progesterone before its release from luteal cells and suggest the presence of a high affinity but low capacity 20 alpha-HSD in active corpora lutea.     

Characteristics and inhibition by flavonoids of 20alpha-hydroxysteroid dehydrogenase activity in mouse tissues

Progesterone was stereoselectively reduced to a metabolite 20alpha-hydroxy-4-pregnen-3-one in the cytosolic fraction from the liver of male mice, indicating that the reduction of progesterone is catalyzed by 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). The cytosolic 20alpha-HSD activity was observed not only in the liver, but also in the kidney and lung. In liver cytosol, both NADPH and NADH were effective as cofactors for 20alpha-HSD activity, although NADPH was better than NADH for the enzyme activity. On the other hand, 20alpha-HSD activity in kidney cytosol required only NADPH as a cofactor. No significant sex-related difference of 20alpha-HSD activity was observed in liver and kidney cytosols. Flavonoids have been reported to inhibit the biosynthesis and metabolism of steroids. However, little is known about inhibitory effects of flavonoids on 20alpha-HSD activity. Thus, the effects of 16 flavonoids on 20alpha-HSD activity were examined, using liver cytosol of male mice. Among flavonoids tested, fisetin, apigenin, naringenin, luteolin, quercetin and kaempferol exhibited high inhibitory potencies for the 20alpha-HSD activity. We propose the possibility that these flavonoids augment progesterone signaling by inhibiting potently 20alpha-HSD activity in non-reproductive tissues.     

The effect of indomethacin on uterine contractility and luteal regression in pregnant rats at term.

Treatment of pregnant rats with 1 mg indomethacin/kg twice daily i.m. beginning on Day 20 delayed the onset of parturition by about 21 hr and prolonged the duration of spontaneous parturition by 4 hr. Plasma progesterone and oestradiol levels were determined in daily samples of peripheral blood, and uterine contractions were recorded before and during parturition by means of small, chronically implanted intrauterine balloons which were connected to pressure transducers via fluid-filled catheters. Indomethacin treatment did not inhibit or suppress spontaneous or oxytocin-induced contractions, which were of the same intensity in indomethacin-treated as in control rats. Parturition was induced with oxytocin in the same proportion of treated and control rats, but its induction was not successful in treated rats until 1 day later than in control rats, but its induction was not successful in treated rats until 1 day later than in controls. The onset of parturition was always related to the plasma progesterone level, which declined at a slower rate in indomethacin-treated than in control rats, reaching baseline values approximately 1 day later in the treated animals. The appearance of 20alpha-hydroxysteroid dehydrogenase in the CL of pregnant rats normally occurs on Day 21 of gestation, but activity was not observed until about 1 (0-3) day later in the indomethacin-treated rats, indicating that luteolysis was retarded. Prostaglandin F-2alpha infusions given on Day 21 reversed the effects of indomethacin treatment on plasma progesterone, luteal 20alpha-hydroxysteroid dehydrogenase activity and the timing and duration of parturition, and reduced the high perinatal mortality associated with indomethacin treatment, suggesting that the effects of indomethacin were related to its inhibitory action on prostaglandin synthetase activity. It is concluded that, in rats, indomethacin exerts its effects on parturition through inhibition of luteal regression which was significantly retarded but not prevented, and that indomethacin does not have a direct effect on myometrial contractility.     

Changes in plasma progesterone and prolactin concentrations during the annual cycle and the role of prolactin in the maintenance of lactation and luteal development in the Antarctic fur seal (Arctocephalus gazella)

Progesterone in Antarctic fur seals was undetectable from 1-2 days before parturition to 4-6 days after parturition. There was a rapid increase in progesterone to 20 ng/ml between 6 and 10 days post partum and this increase coincided with peak concentrations of oestradiol-17 beta at the time normally associated with oestrus and mating in this species. Newly formed corpora lutea were present in the ovaries by Day 9 post partum even though the seals had been isolated in an enclosure and not mated. Thereafter, progesterone remained detectable, but at a low concentration (5 ng/ml) throughout embryonic diapause. A similar pattern was observed in unmated females which suggests they enter a period of pseudopregnancy. Progesterone increased to 35 ng/ml between late February and mid-March, indicating activation of the corpus luteum at the end of diapause, and then declined slowly through the remainder of gestation. Plasma prolactin, measured against a human prolactin standard, was elevated from 1-2 days before parturition and peaked at 0-3 days post partum. It then declined slowly throughout the post-partum period and remained at a low level throughout embryonic diapause. Prolactin concentration declined to undetectable at the end of diapause and before the end of lactation. Reduction of prolactin secretion by injections of bromocriptine from Days 3 to 5 post-partum terminated lactation. Mothers, which normally leave their pups to feed at sea on about Day 7 post partum, did not continue to lactate beyond Day 7 although this did not appear to be associated with reduced prolactin secretion. Bromocriptine treatment appeared to prevent the post-ovulatory surge of progesterone although there was no long-term effect of bromocriptine on progesterone secretion during the early stages of embryonic diapause/pseudopregnancy. This study has shown that prolactin is an important hormone for maintaining early lactation in the fur seal and it probably also has a role in the control of ovulation and luteal development. Prolactin does not appear to be implicated in the control of lactation cycles in fur seals. Changes in plasma progesterone during the annual cycle show that the pattern in fur seals resembles that of some carnivores with embryonic diapause.     

Hormonal regulation of male-specific 20beta-hydroxysteroid dehydrogenase with carbonyl reductase-like activity present in kidney microsomes of rats.

Progesterone, 17alpha-hydroxyprogesterone, cortisone and cortisol, which are C(21)-steroids with a ketone group at the 20-position, potently inhibited the activity of enzyme acetohexamide reductase (AHR) responsible for the reductive metabolism of acetohexamide in kidney microsomes of male rats. Furthermore, progesterone was a competitive inhibitor of AHR. In the case of progesterone usage as the substrate, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) activity was much higher than 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity in kidney microsomes of male rats. These results indicate that AHR present in kidney microsomes of male rats, functions as 20beta-HSD with carbonyl reductase-like activity. In male rats, both testectomy and hypophysectomy decreased the renal microsomal 20beta-HSD activity, but the decreased enzyme activities were increased by the treatment with testosterone propionate (TP). We propose the possibility that TP treatment regulates the renal microsomal 20beta-HSD activity by acting directly on the kidney of male rats. This is supported from the fact that when TP was given to ovariectomized and hypophysectomized female rats, the male-specific 20beta-HSD activity was detected in their kidney microsomes.     

Progestin regulation of progesterone biosynthetic enzymes in cultured rat granulosa cells

Progestins have recently been shown to augment gonadotropin-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) biosynthesis in cultured rat granulosa cells. The mechanism by which progestins autoregulate ovarian progestin biosynthesis was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of the enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). Granulosa cells obtained from immature hypophysectomized, estrogen-treated rats were cultured with FSH and/or progestins. Pregnenolone production was measured in the presence of cyanoketone (10(-6) M) to inhibit 3 beta-HSD activity. Enzymatic activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. FSH stimulated pregnenolone production, while treatment with progesterone or R5020 alone was ineffective. Concomitant treatment with the progestins further enhanced FSH-stimulated pregnenolone production in a dose-dependent manner with minimal effective doses of 10(-8) and 10(-7) M for R5020 and progesterone, respectively. In FSH-primed cells, LH increased pregnenolone accumulation, and concomitant treatment with R5020 also enhanced the LH action. Furthermore, the gonadotropins stimulated the activity of 3 beta-HSD, and this effect was further enhanced by concomitant treatment with either R5020 or progesterone in a dose-dependent manner. In addition, the 20 alpha-HSD activities were enhanced by progestins in cells treated with FSH but not with LH. Thus, both natural and synthetic progestins stimulate the gonadotropin-induced progesterone production in cultured granulosa cells via enhancing the 3 beta-HSD enzyme as well as pregnenolone biosynthesis.     

Sex-related expression of 20alpha-hydroxysteroid dehydrogenase mRNA in the adult mouse.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone into its inactive form, 20alpha-hydroxyprogesterone. To gain information about the exact sites of 20alpha-HSD mRNA expression, we performed in situ hybridization using a (35)S-labeled cRNA probe in tissues of adult mice of both sexes. 20alpha-HSD mRNA was expressed in both male and female gonads. In the ovary, high expression was found in luteal cells of corpora lutea, while much lower expression could be detected in granulosa cells of growing follicles. In the testis, a specific hybridization signal was detected only in Leydig cells. In the female reproductive tract, 20alpha-HSD mRNA was found in the epithelial cells of the uterine cervix. In the adrenal cortex, only the zona reticularis exhibited specific radiolabeling, the expression being very high in the female and very low in the male. In the skin, specific labeling was restricted to sebaceous glands, the hybridization signal being much higher in the female than in the male. In the liver, 20alpha-HSD mRNA was found in hepatocytes, with a higher degree of expression in the female. In the kidney, specific labeling was observed in the epithelial cells of distal convoluted tubules, the signal being also much more striking in the female than in the male. In non-reproductive tissues, it clearly appears that the expression of 20alpha-HSD mRNA is higher in the female than in the male, suggesting that 20alpha-HSD may play an important role in reducing the intracellular concentration of progesterone originating from the circulation at a much higher level in the female.