Uterine production of prostaglandins F2 alpha and 6-keto-F1 alpha by ovariectomized pregnant rats receiving antiprogesterone steroid or in which progesterone has been withdrawn.

Ovariectomized early pregnant rats given continuous steroid replacement therapy have been treated with antiprogesterone steroid, ZK98299 or RU38486. At 24 h following treatment, uterine explants in culture were found to produce significantly greater amounts of PGF2 alpha, but not of 6-keto-PGF1 alpha, when compared to controls. ZK98299 and RU38486 gave almost identical levels of uterine PG production. The 6-keto-PGF1 alpha/PGF2 alpha production ratio for uteri of treated rats was decreased by 45% relative to controls. Similar changes in uterine PGF2 alpha production and 6-keto-PGF1 alpha/PGF2 alpha ratio have been shown for ovariectomized early pregnant rats in which progesterone has been withdrawn when compared to control animals. It has been suggested that inhibiting or withdrawing progesterone in rat uteri exposed to estradiol and progesterone may lead to a stimulation of endoperoxide F-reductase and/or E2 9-ketoreductase activities. The presence of luminal fluid in the uteri was observed for animals treated with antiprogesterone steroid or in which progesterone had been withdrawn. This was associated with a decrease in % dry weight for the uteri of these animals.     

Prostaglandin receptors EP and FP are regulated by estradiol and progesterone in the uterus of ovariectomized rats

Background  

Prostaglandins are important for female reproduction. Prostaglandin-E2 acts via four different receptor subtypes, EP1, EP2, EP3 and EP4 whereas prostaglandin-F2alpha acts through FP. The functions of prostaglandins depend on the expression of their receptors in different uterine cell types. Our aim was to investigate the expression of EPs and FP in rat uterus and to identify the regulation by estradiol, progesterone and estrogen receptor (ER) selective agonists.     

Prostaglandin production by isolated cells of rat uterus incubated with or without progesterone

The production of prostaglandins F2 alpha and 6-keto F1 alpha in vitro by the luminal epithelial and the residual (mainly stomal) cells isolated from the uteri of proestrous rats have been measured. The basic procedure involved culturing the cells overnight, washing, and then incubating for 2 h when the amounts of prostaglandins released into the medium were determined by radioimmunoassay. No significant change occurred when the epithelial cells were incubated in the combined presence of arachidonic acid and progesterone for the short-term culture compared to being incubated with arachidonic acid alone. However, when progesterone was incorporated into the medium for the overnight culture, a significant increase occurred in 6-keto PGF1 alpha production by the epithelial cells, without any change in PGF2 alpha, compared to when the cells were incubated overnight in the absence of progesterone. It is suggested that overnight treatment of the epithelial cells with progesterone increases the amount or activity of prostacyclin synthetase, responsible for converting PGH2 endoperoxide to PGI2. Very low levels of prostaglandin production were found for the residual cells.     

The effect of frozen ovarian autografts on compensatory ovulation and steroid production in unilaterally ovariectomized rats.

In the present study rats were unilaterally ovariectomized (ULO) and the surgically removed ovary was frozen for 13 days. After allowing the remaining ovary to compensate with respect to number of ova shed, the frozen graft was thawed and transplanted subcutaneously to determine the effect on ovulation number, cycle length, uterine weight, ovarian weight and plasma levels of estradiol-17beta (E2) and progesterone. Rats ULO at 45 days of age, which received an autograft 13 days later, had a decrease in the number of eggs shed as compared to control ULO rats (6.4 +/- 0.8 vs. 11.1 +/- 0.9 eggs, respectively) and a decrease in plasma E2 (14.5 +/- 1.7 VS. 21.0 +/- 1.5 PG/ML, respectively). No differences were observed in progesterone concentration, uterine weight, ovarian weight or cycle length. In contrast, rats ULO at 31 days of age, which received an autograft 13 days later, showed no differences in comparison to control ULO rats. Castrates which received ovarian autografts developed cycling vaginal smears and had increased E2 (31.9 +/- 4.3 pg/ml) and decreased progesterone (18.3 +/- 1.9 ng/ml) levels. Since ULO animals with autografts shed fewer ova, the present study demonstrates that the amount of ovarian tissue influences ovulation number either by utilization of gonadotropins or by an, as yet, undefined mechanism.     

Estrogen receptor mRNA in uteri of normal estrous cycling and ovariectomized rats by in situ hybridization.

Biological effects of estrogen are mediated via its binding to the estrogen receptor (ER), the contents of its protein and mRNA varying during the estrous cycle. In the present study, the ERalpha mRNA expression in different cell components of the uterus was investigated in normal estrous cycling rats using nonisotopic in situ hybridization. Additionally, ovariectomized (OVX) rats treated with 17beta-estradiol (E2: 5 microg/kg, sc injection daily) were also investigated to clarify the effects of exogenous E2. At proestrus and diestrus, and especially the former, the luminal and glandular epithelial (LE and GE) cells were strongly positive, along with stromal cells beneath the luminal epithelium. At estrus, the expression was slightly diminished in LE cells, but almost completely lacking in GE cells. At metestrus, positive signals appeared again in GE cells. In the myometrium, ER mRNA was demonstrated to be constantly positive in all estrous cycle stages. OVX rat uteri underwent marked atrophy, but ER mRNA still remained in all cell types. After 2 consecutive days of E2 treatment, markedly increased intensity was observed, especially in LE and GE cells. The uteri of OVX rats treated with E2 for 14 days, however, showed slightly diminished expression, whereas the serum concentration of E2 was comparable to that in rats after 2 days. These results provide evidence that cell-type specific patterns of ER mRNA expression characterize the uteri of both normal estrous cycling rats and OVX rats after estrogen treatment.     

Factors subserving the enhancing effect of progesterone on the output of prostaglandin F2 alpha in uteri from ovariectomized rats

The effects of progesterone (P4) and of calcium-ionophore A-23187, on the release of prostaglandins (PGs) E2 and F2 alpha, in uteri isolated from ovariectomized rats and the influences of mepacrine and nifedipine, were explored. The metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF 1 alpha, PGE 2 and PGF2 alpha) in uterine segments from spayed rats, injected or not with P4, was also studied. In all cases ovariectomy was performed 20-25 days prior to sacrifice. One group of spayed rats were injected with 4.0 mg of P4 during two days and sacrificed 24 h after the last injection. The remaining spayed animals were considered as controls. Tissue samples from both groups were incubated for one hour in the absence or in the presence of either A-23187 (1.0 microgram/ml), mepacrine (10(-3) M) or nifedipine (10(-6) M), or a combination of A-23187 plus mepacrine. At the end of the incubating period PGs in the suspending solution were extracted, separated, identified (TLC) and quantitated. The metabolism of 14C-AA into different prostanoids was explored in uterine segments from spayed rats, injected or not with P4 prior to sacrifice. Tissue prepared from P4-injected rats as well as those from rats not receiving P4 but incubated with ionophore A-23187, generated and released significantly more PGF2 alpha into the incubating solution than basal controls, but failed to exhibit changes in the basal output of PGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment.

The transsulfuration pathways allow the interconversion of homocysteine and cysteine with the intermediary formation of cystathionine. The various organisms studied up to now incorporate reduced sulfur into a three- or a four-carbon chain and use differently the transsulfuration pathways to synthesize sulfur amino acids. In enteric bacteria, the synthesis of cysteine is the first step of organic sulfur metabolism and homocysteine is derived from cysteine. Fungi are capable of incorporating reduced sulfur into a four-carbon chain, and they possess two operating transsulfuration pathways. By contrast, synthesis of cysteine from homocysteine is the only existing transsulfuration pathway in mammals. In Saccharomyces cerevisiae, genetic, phenotypic, and enzymatic study of mutants has allowed us to demonstrate that homocysteine is the first sulfur amino acid to be synthesized and cysteine is derived only from homocysteine (H. Cherest and Y. Surdin-Kerjan, Genetics 130:51-58, 1992). We report here the cloning of genes STR4 and STR1, encoding cystathionine beta-synthase and cystathionine gamma-lyase, respectively. The only phenotypic consequence of the inactivation of STR1 or STR4 is cysteine auxotrophy. The sequencing of gene STR4 has allowed us to compare all of the known sequences of transsulfuration enzymes and enzymes catalyzing the incorporation of reduced sulfur in carbon chains. These comparisons reveal a partition into two families based on sequence motifs. This partition mainly correlates with similarities in the catalytic mechanisms of these enzymes.     

Studies on aspartase. V. Denaturant-mediated reactivation of aspartase,which has been otherwise irreversibly inactivated by various causes

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, inactivated by heat-treatment at 55°C or above in the presence of 10 mM 2-mercaptoethanol does not recover the activity at all upon simple chilling and remains to be seriously denatured resulting in the formation of insoluble aggregates. When the heat-denatured enzyme is further treated with 6 M guanidine-HCl followed by 51-fold dilution at 25°C adn pH 6.8, the enzyme activity is gradually restored and reaches almost 40% that of the native enzyme in 20 min. The secondary and the quaternary structures of the reactivated enzyme exhibit a close similarity to those of the native enzyme, as revealed by circular dichroism and electron micrograph, respectively. A similar reactivation is attained, when proton-inactivated aspartase at acidic pH is treated with 6 M guanidine-HCl followed by dilution.As previously reported, aspartase requires the sulfhydryl group for its activity (Mizuta, K. and Tokushige, M. (1975) Biochim. Biophys. Acta 403, 221–231) and is readily inactivated by sulfhydryl reagents. Although the inactivated enzyme can usually be reactivated with sulfhydryl compounds, the reactivation becomes impossible, when a large excess of the reagent is applied. Chemically-inactivated aspartase with a large excess of 5,5′-dithiobis(2-nitrobenzoic acid) is, however, reactivated by way of the reduction of fully exposed polypeptide side chains with dithiothreitol in the presence of 6 M guanidine-HCl.     

Effect of genistein on steroid hormone production in the pregnant rhesus monkey.

Genistein is a phytoestrogen found in soy beans. Phytoestrogens have been reported to cause reproductive problems in sheep and rats. This research was conducted to determine the effects of genistein fed to rhesus monkeys during pregnancy, with specific interest on fetal growth and steroidogenesis in the maternal-fetoplacental unit. Two groups of five monkeys each were selected in early stages of pregnancy. One group was administered genistein in a fruit treat each weekday until Cesarean section 10 days prior to term. The second, control group, received fruit treats without genistein. Maternal blood samples were collected on Tuesday and Friday of each week. At delivery, samples were collected from the maternal peripheral circulation, uterine veins, uterine-ovarian veins, and the fetal heart. Comparisons between control and genistein-treated monkeys revealed no differences in the maternal weight gained during pregnancy, or in fetal weights or placental weights at delivery. Serum was assayed by radioimmunoassay (RIA) for estradiol, progesterone, dehydroepiandrosterone sulfate (DHEA-S), and estrone. No significant differences (P > 0.05) were noted in progesterone or DHEA-S levels at delivery or during the pregnancy; however, estradiol levels were higher (P < 0.05) in the four areas studied at delivery and in the maternal blood with advancing gestation. Additionally, estrone levels tended to increase more rapidly (P = 0. 057) in the maternal blood of monkeys receiving genistein than in untreated controls, suggesting that genistein may stimulate the deconjugation of estrone in the gut, thus allowing its reabsorption into the peripheral circulation and conversion to estradiol.     

Inhibition of progesterone synthesis in placenta by administration of dexamethasone to pregnant rats

Glucocorticoid receptors have been detected in placenta from several species, including the rat, although the biological function of corticoids is unknown in placenta from the latter species. The present experiments examined the effect of glucocorticoid treatment on placental progesterone biosynthesis from endogenous precursors by incubated basal zone trophoblast and labyrinthine zone of placentas from adrenalectomized-ovariectomized rats at the end of pregnancy. It was found that a higher proportion of synthesized progesterone was retained in the tissue than that released into the incubation medium. Treatment of rats on the 17th-18th day of pregnancy with 10 micrograms/ml of dexamethasone in the drinking saline for 3 days, produced a significant inhibition of progesterone detected in tissue and medium of incubated placental zones. In vitro addition of dexamethasone (10(-4) M) was also effective in reducing progesterone in the placental zone studied (LZ). Serum progesterone of intact rats was in the range of rats near parturition (approx 25 ng/ml) and dropped to almost undetectable levels in rats with adrenalectomy and ovariectomy, with or without dexamethasone treatment, suggesting that in late pregnancy the rat placenta does not contribute significantly to circulating levels of progesterone. This glucocorticoid effect could not be extended to estrogens, as we, in accord with the work of other groups, failed to detect estrogen synthesis in rat placenta. It is suggested that a function for glucocorticoid receptors in rat placenta may be the inhibition of local progesterone production.     

Prostaglandin E2 enhancement of interferon-gamma production by antigen-stimulated type 1 helper T cells.

Prostaglandin E2 (PGE2) is a potent mediator generated in immune tissues by cyclooxygenation of arachidonic acid. PGE2 affects T cell functions through four homologous G protein-coupled receptors termed EP1R, EP2R, EP3R, and EP4R that differ in tissue distribution and signaling. Antigen-evoked secretion of interferon-gamma (IFN-gamma) by sperm whale myoglobin-specific Th1 cells of DBA/2 mouse I-Ed-restricted clones, that express EP3Rs and EP4Rs, was enhanced a maximum of 3-fold by 10(-10) to 10(-8) M PGE2 and 2.5-fold each for the EP1R/EP3R-directed agonist sulprostone (10(-8) and 10(-7) M) and for the EP4R/EP3R/EP2R agonist misoprostol (10(-9) M). Neither PGE2 nor the synthetic analogs affected secretion of IFN-gamma by PMA plus ionomycin-stimulated clones of Th1 cells. Antigen-evoked secretion of IFN-gamma by influenza hemagglutinin-specific mouse lymph node Th1 cells, that also express EP3Rs and EP4Rs, was increased a maximum of 12-fold by 10(-9) to 10(-8) M PGE2, 14-fold by 10(-9) M sulprostone, and 10-fold by 10(-9) M misoprostol. Production of IFN-gamma by either type of Th1 cell was not affected significantly by 10(-6) M PGE2 alone. The generation of IFN-gamma by antigen-stimulated Th1 cells thus is significantly enhanced by physiologically relevant concentrations of PGE2.     

Effect of estradiol and progesterone on UDPgalactose pyrophosphatase activity in the endometrium of ovariectomized rats.

Rat endometrium was found to contain a UDPgalactose pyrophosphatase for the hydrolysis of UDPgalactose into galactose 1-phosphate and UMP. The adminstration of 17beta-estradiol to ovariectomized rats resulted in a significant decrease in the activity of the enzyme in endometrium while have little effect on that in myometrium. The response was linear with the dose of estradiol and as little as 0.07 mug per 100 g body weight produced maximum inhibition of the enzyme. Progesterone on its own had little effect on the enzyme activity but in combination with estradiol, it effectively prevented the inhibitory effect of estradiol. This inhibitory effect of estradiol on the activity of UDPgalactose pyrophosphatase may function in the regulation of glycoprotein biosynthesis in endometrium.     

Effect of long-term treatment with steroid hormones or tamoxifen on the progesterone receptor and androgen receptor in the endometrium of ovariectomized cynomolgus macaques

The progesterone receptor (PR) and androgen receptor (AR) belong to the nuclear receptor superfamily. Two isoforms of PR (A and B) have been identified with different functions. The expression of AR, each isoform of PR and their involvement in long-term effects on the endometrium after hormonal replacement therapy (HRT) or tamoxifen (TAM) treatment is not known. The aims of this study were to determine PR(A+B), PRB and AR distribution by immunohistochemistry in the macaque (Macaca fascicularis) endometrium. Ovariectomized (OVX) animals were orally treated continuously for 35 months with either conjugated equine estrogens (CEE); medroxyprogesterone acetate (MPA); the combination of CEE/MPA; or TAM. Treatment with CEE/MPA tended to down-regulate PR in the superficial glands, but increased it in the stroma. TAM treatment increased both the PR and PRB levels in the stroma. Overall, less than 20% of the cells were positive for the PRB isoform and less variation was observed after steroid treatment. AR was found in the stroma, mainly distributed in the basal layer of the endometrium in the OVX and steroid treated groups, but was absent in the TAM treated group. No AR was found in the glandular epithelium. The present data show that long-term hormone treatment affects the PR level, and also the ratio between PRA and PRB in the endometrium.     

Effect of Andrographis paniculata extract on progesterone in blood plasma of pregnant rats.

The effect of the powdered extract of Andrographis paniculata leaves (APE), an active principle of Kan Jang tablets [standardized for content of andrographolide (4.6%) and 14-deoxo-andrographolide (2.3%) content (total andrographolids--6.9%)] on blood progesterone content in rats was studied. Peroral administration of APE during the first 19 days of pregnancy in doses of 200, 600, and 2000 mg/kg (i.e. doses 30, 90, and 300 fold higher than its daily therapeutic dose in humans) does not exhibit any effect on the elevated level of progesterone in the blood plasma of rats. Let us assume then that in therapeutic dose, Andrographis paniculata extract cannot induce progesterone-mediated termination of pregnancy.     

Relationship between occupied form of nuclear estrogen receptor and cytosolic progesterone receptor or DNA synthesis in uteri of estradiol implanted rats

The effect of 17 beta-estradiol (E2) implantation on the cytosolic progesterone receptor (RcP), DNA and occupied form of nuclear estrogen receptor (o- Rn) content in the uterus of ovariectomized adult rats, is described. Animals were implanted with oil or E2-oil solution in Silastic capsules. The latter group animals were divided into two subgroups: in subgroup (a), capsules remained in situ until decapitation time. In subgroup (b) they were removed 48 h after implantation. The E2 implantation caused a significant increase in uterine weight, RcP and o-Rn content 48 h later. However, the DNA content increased significantly only after 72 h, but there was no significant difference in the t-Rn concentration in relation to the non-estrogenized animals. In subgroup (a) animals, these values remained unchanged until 96 h. In subgroup (b), the removal of E2 implants 48 h later caused an almost complete return to the values before the E2 implantation in terms of uterine weight, RcP and o-Rn content. However the DNA concentration remained higher and the t-Rn level was lower than those values that were obtained for the non- estrogenized rats. These results suggest that the RcP and DNA synthesis induced by E2 would be connected to the level of o-Rn, although a closer dependency over time seems to exist between the o-Rn and RcP levels than between the o-Rn and DNA concentrations.