The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17β

We reported that spermidine and spermine pools in the uterus both doubled within 24h after oestradiol administration to castrated rats (Russell & Taylor, 1971). Now we have studied the enzymic synthesis of spermine (by spermidine-dependent S-adenosyl-l-methionine decarboxylase) and find that the activity of the enzyme(s) involved is elevated soon after hormone administration. Enzyme activity is increased within 4h and is five times that of controls within 24h. Cycloheximide or actinomycin D administered at the time of oestradiol injection completely blocked the increase in enzyme activity. The enzyme involved in spermine synthesis, S-adenosyl-l-methionine decarboxylase, with S-adenosyl-l-methionine and spermidine as required substrates, was partially purified on Sephadex and DEAE-cellulose columns. The decarboxylation of S-adenosyl-l-methionine could not be separated from the transfer of a propylamine moiety from the decarboxylated S-adenosyl-l-methionine to spermidine to form spermine. We were unable also to separate this system from the enzyme that formed spermidine when S-adenosyl-l-methionine and putrescine are used as substrates. Spermidine-stimulated S-adenosyl-l-methionine decarboxylase has an apparent half-life of 60min, identical with the half-life reported for putrescine-stimulated S-adenosyl-l-methionine decarboxylase. These results strongly suggest that the same enzyme(s) operate in the synthesis of both spermidine and spermine.     

Nuclear binding of oestradiol-17β and induction of protein synthesis in the rat uterus during postnatal development

1. The uterine response to a single injection of oestradiol-17beta during postnatal development of the rat was studied with respect to (i) nuclear binding of oestradiol-17beta; (ii) induction of the synthesis of a specific cytoplasmic protein (;induced protein' of Gorski); (iii) rate of incorporation of (3)H-labelled amino acids into total protein and into nuclear acid-soluble and acid-insoluble protein; and (iv) rate of [(3)H]thymidine incorporation into DNA. 2. Specific nuclear binding of oestradiol-17beta could be demonstrated even at birth. Administration of oestradiol-17beta in vivo caused a significant increase in the number of nuclear binding sites in rats aged 10 days or older. 3. A rapid method is described for the detection of the ;induced protein', based on cellulose acetate electrophoresis. Induction of this protein could be demonstrated at the age of 10, 15 and 20 days, but not in 5-day-old rats. 4. In 20-day-old rats the rate of (3)H-labelled amino acid incorporation into protein increased by 3h after oestradiol administration. Incorporation into the different protein fractions reached peak values asynchronously: at 3-4h for acid-insoluble nuclear protein, at 6h for total protein and at about 12h for acid-soluble protein. 5. Treatment with oestradiol failed to stimulate amino acid incorporation into protein in 5- or 10-day-old rats; at the age of 15 to 30 days the hormone caused a significant increase in incorporation into total protein and into both types of nuclear protein. 6. Since the capacity for nuclear binding of oestradiol and for synthesis of the induced protein is demonstrable in the rat uterus before it acquires the ability to respond to the hormone with enhanced general protein synthesis and DNA synthesis, it appears that nuclear binding and the synthesis of the induced protein may be necessary but not sufficient conditions for the trophic action of oestradiol.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17β

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. Formation and properties of an aminoacyl-transfer ribonucleic acid–transferase I complex

1. Transferase I of rat liver binds aminoacyl-tRNA to form a relatively stable complex, which is retained on cellulose nitrate filters. This reaction proceeds at both 0 degrees C and 37 degrees C and is inhibited by GTP. The resulting product is stabilized by GTP and Mg(2+). 2. Only very low quantities of deacylated tRNA are bound by transferase I. 3. Methods are described for the preparative isolation of the transferase I-aminoacyl-tRNA complex from incubation mixtures by using ion-exchange procedures. 4. The transferase I-aminoacyl-tRNA complex becomes readily bound to ribosomes. The presence of Mg(2+) is essential for the binding. GTP stimulates this reaction but is not absolutely required. 5. It is concluded that the formation of the transferase I-aminoacyl-tRNA complex may be the primary reaction in the binding of aminoacyl-tRNA to mammalian ribosomes and that, unlike in bacterial systems, GTP is not absolutely required for this step.     

Histone acetylation and hormone action. Early effects of oestradiol-17β on histone acetylation in rat uterus

The effect of oestradiol treatment on the acetylation of histones of the immature rat uterus has been studied. A 10mug dose of oestradiol causes a 70% increase at 5min and a 140% increase at 10min after administration in the labelling of the histone fraction F2+F3. No effect of oestradiol is seen on the labelling of histones F1 or acidic non-histone chromatin proteins. The oestradiol stimulation is seen in animals pretreated with either cycloheximide or actinomycin D. The stimulation of labelling caused by oestradiol is completely abolished by pretreatment of the animals with the anti-oestrogen, nafoxidine. The stimulation is given by lower doses of oestradiol, by stilboestrol and oestriol, but is not given by testosterone. These results suggest that stimulation of histone acetylation in the uterus is the earliest known effect of the hormone on its target tissue.     

Regulation of Akt expression and phosphorylation by 17β-estradiol in the rat uterus during estrous cycle

Background

Ablation of the low-affinity receptor subunit for leukemia inhibitory factor (LIFR) causes multi-systemic defects in the late gestation fetus. Because corticosterone is known to have a broad range of effects and LIF function has been associated with the hypothalamo-pituitary-adrenal axis, this study was designed to determine the role for LIFR in the fetus when exposed to the elevated maternal glucocorticoid levels of late gestation. Uncovering a requirement for LIFR in appropriate glucocorticoid response will further understanding of control of glucocorticoid function.

Methods

Maternal adrenalectomy or RU486 administration were used to determine the impact of the maternal glucocorticoid surge on fetal development in the absence of LIFR. The mice were analyzed by a variety of histological techniques including immunolabeling and staining techniques (hematoxylin and eosin, Alizarin red S and alcian blue). Plasma corticosterone was assayed using radioimmunoassay.

Results

Maternal adrenalectomy does not improve the prognosis for LIFR null pups and exacerbates the effects of LIFR loss. RU486 noticeably improves many of the tissues affected by LIFR loss: bone density, skeletal muscle integrity and glial cell formation. LIFR null pups exposed during late gestation to RU486 in utero survive natural delivery, unlike LIFR null pups from untreated litters. But RU486 treated LIFR null pups succumb within the first day after birth, presumably due to neural deficit resulting in an inability to suckle.

Conclusion

LIFR plays an integral role in modulating the fetal response to elevated maternal glucocorticoids during late gestation. This role is likely to be mediated through the glucocorticoid receptor and has implications for adult homeostasis as a direct tie between immune, neural and hormone function.     

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.     

Androgen dynamics in vitro in the human prostate gland. Effect of oestradiol-17β

Normal, hyperplastic and adenocarcinomatous human prostatic tissue was perfused in vitro with radioactively labelled androstenedione, testosterone and 5alpha-dihydrotestosterone with and without added oestradiol-17beta. Various parameters of tissue-steroid relationship were measured at the steady state. When oestradiol (0.11 or 0.22mumol/l) was added to the perfusing medium, the entry of the steroids into the tissue and their metabolism was increased in the majority of the glands studied. The ;uptake' of all the steroids varied, in response to the addition of oestradiol, in both normal and adenocarcinomatous glands in a way differing from the response of hyperplastic glands. As a consequence, the tissue clearance of the steroids, particularly of androstenedione and testosterone, increased in normal and adenocarcinomatous glands in the presence of oestradiol, and decreased in the hyperplastic tissues. At a concentration 0.33mumol/l, oestradiol decreased the entry of the steroids in all the tissues studied, while the clearance of steroids tended to decrease. The significance of these findings in terms of the regulation of androgen dynamics in vivo in the normal and diseased human prostate, with particular regard to the response to oestrogen treatment, is discussed.     

Perioestrous patterns of circulating LH,FSH, prolactin and oestradiol-17β in the gilt

Concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) were measured in jugular blood and those of oestradiol-17β (E₂17β) in utero-ovarian blood. Samples were taken from five intact gilts every 15 min for 108 h starting between day 15 and day 18 of the oestrous cycle. In the late luteal/early follicular phase, high pulsatile LH secretion, close to one pulse per hour, was observed. This could be the stimulus necessary for the final maturation of the ovarian follicles.Thereafter, frequency and amplitude of pulses, and the baseline value, decreased and were low at least between 36 and 12 h before the preovulatory LH surge. PRL and FSH concentrations also declined. This was probably due to the increase of oestrogen secretion. As E₂17β concentrations were still high, the surge of LH which was accompanied by increase in FSH and PRL, occurred for approximately 13 to 20 h. While LH and PRL mean levels decreased, FSH concentrations continued to increase. Peaks of PRL were observed during the late luteal/early follicular phase and during the LH discharge. During the period of estrus, each exposure to the boar was immediately followed by one of these peaks, which could play a role in the sexual behavior of the gilt.     

Effect of ethanol on the response of the rat urinary bladder to in vitro ischemia: Protective effect of α-lipoic acid

Purpose: Ethanol exposure has been used to demonstrate the increase of oxidative stress to a variety of tissues. We studied the effect of ethanol on the response of isolated strips of rat bladder to In vitro hypoxia in the absence of glucose (In vitro ischemia). Secondly, we determined if -lipoic acid (LA) could alter the response to ethanol + In vitro ischemia.Methods: Sixty-four rats were used for the these experiments. Each rat was anesthetized and its urinary bladder excised. The bladder body was cut into two longitudinal strips and each strip mounted in individual baths filled with oxygenated Tyrodes solution containing glucose at 37 C. Ethanol (0.3%, 1%, or 3%) was placed in the first six baths (two strips at each concentration). The last two baths did not receive ethanol. Each strip was incubated for 1 h and then stimulated with field stimulation at 2, 8, and 32 Hz. Each strip was stimulated with 10 M carbachol, washed three times with fresh oxygenated buffer and ethanol re-added to their respective baths. Each strip was then stimulated with 120 mM KCl and washed three times as before. Strips were then subjected to 1 h In vitro ischemia (incubation in the absence of glucose with Tyrodes equilibrated with nitrogen instead of oxygen). During the ischemic period, each strip was stimulated for 5 s every 10 min by 32 Hz FS to simulate hyperreflexia. At the end of the hour, the tissues were incubated for an additional hour in the presence of oxygen + glucose and subjected to a second series of stimulations as before. At all times, ethanol was maintained in baths 1–6. In set 2, 1% ethanol was added to the first six baths. LA was added to every other bath, and the experiments performed as mentioned earlier.Results: (a) Ethanol at 0.3% or 1% had no effect on the contractile responses prior to exposure to In vitro ischemia; 3% was inhibitory. (b) In vitro ischemia mediated a significant decrease in the contractile responses to all forms of stimulation except for carbachol. (c) Ethanol mediated a dose-response enhancement of the contractile dysfunctions caused by In vitro ischemia. (d) LA completely reversed the effects of ethanol on contractile responses following In vitro ischemia except for carbachol.Conclusions: The results demonstrate that direct exposure to ethanol significantly enhanced contractile dysfunctions mediated by In vitro ischemia followed by re-oxygenation and that the presence of LA significantly inhibits this effect of ethanol. (Mol Cell Biochem 271: 133–138, 2005)This material is based upon work supported in part by the Office of Research and Development, Department of Veterans Affairs, and NIH RO-1-DK067114     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

Rate of synthesis of βL-lipovitellin in the liver of immature chicks treated with 17β estradiol

Using a combination of radioimmunoprecipitation and SDS-polyacrylamide gel electrophoresis of the immunoprecipitate we studied the rate of synthesis of the heavy chain of β-lipovitellin in the liver of immature chicks. In male and female chicks the base-line synthesis of β_L-lipovitellin¹ was about 30 molecules per minute and per cell. Four days after a single injection of 40 mg estradiol/kg, as many as 48,000 molecules of β_L-lipovitellin were synthesized per minute and per diploid liver cell. The increase in the rate of β_L-lipovitellin synthesis could be correlated with an increase in membrane bound mRNA coding for β_L-lipovitellin.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.