Estrogen interconversions in the induced cycle in female rhesus monkeys

To determine the extractions and interconversions of estrone and estradiol across and within the uterus, [3H]estradiol and [14C]estrone were infused at a constant rate in six ovariectomized female rhesus (Macaca mulatta) monkeys. Studies were done on Days 9, 14, and 23 of artificial menstrual cycles induced by the timed insertion and removal of Silastic capsules of estradiol and progesterone. Measurements of estrogen radioactivity were made from peripheral arterial blood and uterine venous blood as well as from endometrial biopsy samples. A significant increase occurred in the conversion of estradiol to estrone measured within the uterus on Day 23 compared to Days 9 and 14. The conversion of estrone to estradiol, measured within the uterus, fell progressively from Day 9 to Day 23, but this decrease was not significant. The extractions and interconversions across the uterus, and the overall interconversions of estrone and estradiol were not significantly different on Days 9, 14, or 23 of the cycle. Thus, we have been able to confirm in vivo the increase in the activity of the 17 beta-hydroxysteroid dehydrogenase, the enzyme responsible for estradiol to estrone interconversions, shown earlier by studies done in vitro. However, the increase in 17 beta-hydroxysteroid activity in the uterus is not reflected in the overall interconversions of estrone and estradiol as reflected by measurements in peripheral arterial blood.     

Effect of iodide on estrogen-induced uterine peroxidase

Iodide administered in the drinking water for 5–7 days increased the activity of estradiol-induced uterine peroxidase in the immature rat. This effect was specific for iodide and could not be mimicked by chloride, bromide, thiocyanate, perchlorate or iodate. Sodium iodide also increased peroxidase activity in the parotid gland but had no effect on glucose-6-phosphate dehydrogenase in the uterus, thyroid or parotid even though estradiol produced a 2-fold increase in the activity of this enzyme in the uterus. ¹²⁵I was taken up more readily by the uterus than by muscle but this process was not influenced by prior treatment of the animals with estrogen. The in vitro effect of sulfhydryl reagents on uterine peroxidase was also investigated and proposals made for possible mechanisms of action of iodide on this enzyme in the intact animal.     

Regulation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase activity in rat uterine tissues

Initiation of uterine DNA synthesis and mitosis in response to estrogen appears to depend upon the stimulation of protein synthesis. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase could have a key function in controlling uterine mitosis through its control of mevalonic acid and cholesterol synthesis as the rate-limiting enzyme in their synthetic pathways. These studies were initiated to examine the kinetics of the uterine increases in HMG-CoA reductase activity in response to estradiol. In the uterus of the ovariectomized mature rat, estradiol increased levels of enzyme activity in both the luminal epithelium and stroma-myometrium up to 12 h after estradiol treatment. Levels of HMG-CoA reductase activity decreased after 12 h in the luminal epithelium and further increased in the stroma-myometrium. Previous studies have shown that estradiol does not increase DNA synthesis and mitosis in the stroma-myometrium of the uterus of the ovariectomized mature rat. Since estradiol increased HMG-CoA reductase activity in both the luminal epithelium and stroma-myometrium, we conclude that even though increased HMG-CoA reductase activity may be a prerequisite for increased DNA synthesis, increases in uterine HMG-CoA reductase activity are not necessarily followed by increased DNA synthesis.     

Effect of estrogens on phosphofructokinase activity of uterus, liver and kidney in rat and hamster

The effect of estradiol-17β on the phosphofructokinase (PFK) activity of uterus, liver and kidney in rat and hamster has been studied. 16 hours after a dose of 10 μg estradiol/100 g body weight, there are no differences in the uterotrophic responses of rats and hamsters and the increase in uterine PFK activity is similar in both animals. 48 hours after two doses of 100 μg estradiol/100 g body weight, the uterotrophic response is slightly higher in hamster than in rat, but rat uterus shows a greater increase of PFK activity than does hamster uterus.Hepatic and renal PFK activities in hamsters of both sexes are not modified 48 hours after two doses of 100 μg estradiol/100 g body weight. These results indicate that in hamster and rat, PFK is under estrogenic control in uterus and not in liver and kidney.     

Uterine glutathione reductase activity: modulation by estrogens and progesterone

The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF2 alpha the treatment with progesterone (4 mg X day-1, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol-17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE2, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE2 catabolism into 15-keto-PGE2 as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.     

In vivo estrogenic and antiestrogenic activity of phenolphthalein and derivative compounds

The estrogenic activity of phenolphthalein and other related triphenylmethane dyes was evaluated in vivo in the immature rat uterus. Phenolphthalein behaved as a partial agonist of estradiol in stimulating the growth of rat uterus. Other specific estrogenic effects of the dye included an increase of the uterine DNA content, histological changes and induction of estrogen-modulated secretory proteins. The progressive introduction of side chains in the triphenylmethane skeleton concomitantly decreased the estrogenic activity. Triphenylmethanes competed with [3H]estradiol for the binding to the estrogen receptor in vitro, the relative binding affinity being correlated with the estrogenic potency observed in vivo. Phenolphthalein also showed antiestrogenic activity that could be overcome by increasing the dose of estradiol.     

Participation of the estradiol receptor in the hormonal inhibition of cyclic nucleotide phosphodiesterase in the uterus

The role of estradiol receptor was studied in the inhibitory effect of hormone on the cyclic nucleotide phosphodiesterase from immature Wistar rat uterus. It was shown that the preparative separation of the enzyme and hormone receptor by ultracentrifugation in isokinetic sucrose density gradient results in a 2.5-3-fold decrease of the estradiol effect on phosphodiesterase. This effect is completely restored after adding the separated estradiol receptor to the phosphodiesterase devoid of it. The effect of estradiol on the phosphodiesterase activity depends on a degree of receptor component aggregation: the action of estradiol on the enzyme intensities after transformation of receptor into the dissociated form (4S) and removes in the presence of the receptor component associated form (8S).     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF_2α the treatment with progesterone (4 mg.day⁻¹, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol- 17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE₂, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE₂ catabolism into 15-keto- PGE₂ as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Differential effects of sex steroids on uterine and renal ODC activity in ovariectomized rats

Many hormones are known to induce the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme for polyamine biosynthesis, in their target tissues. Using ovariectomized rats, we have compared the effects of sex hormones on ODC activity in the uterus and the kidney which contain estrogen and androgen receptors. The results show that 1) both estrogen and androgen stimulate renal ODC activity, 2) estrogen but not androgen effectively increases ODC activity in the uterus, 3) estrogen at higher dosage can stimulate renal ODC activity to an extent similar to that in the uterus, 4) daily treatment with estradiol for 5 days results in the desensitization of uterine ODC activity, but not that of renal ODC activity to the hormonal stimuli. Although both uterus and kidney are targets of sex hormones, our results indicate that estrogen and androgen have differential influences on the ODC activity in these two organs.     

Preparation of 14,15-secoestra-1,3,5(10)-trien-15-ynes, inhibitors of estradiol dehydrogenase

The conversion of estrone to 14,15-secoestratrien-15-ynes, inactivators of estradiol dehydrogenase from human term placenta, is described. The optically pure precursor 7-acetoxy-octahydro-2-phenanthrenecarboxylic acid methyl ester is prepared from estrone in five steps and 40% yield. The unsubstituted propargylic secoestratriene diol, a mechanism-based inactivator of estradiol dehydrogenase, and the corresponding acetylenic ketone, an affinity label inactivator of the same enzyme, arise from the phenanthrene ester in three and four steps. The propargylic secoestratriene diol also competes with [3H]estradiol for binding to calf uterus estrogen receptor and possesses weak uterotrophic activity.     

Androgen- and estrogen-dependent regulation of insulin-degrading enzyme in subcellular fractions of rat prostate and uterus

Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus.     

Characteristics and analysis of uterine electromyographic activity in pregnant cows

The electromyographic activity of the cow uterus in the last trimester of pregnancy was investigated. The investigation was performed on 12 animals and the electrical activity was recorded in the last trimester of pregnancy during 11 different periods until the delivery. The duration of the action potential bursts (APB) recorded during the first 7 periods, was small. It did not exceed 2 seconds. A significant increase, however, was recorded at 7 to 9 days before the labor and it involved all the investigated areas in the uterus. The number of APB of the gravid horn was significantly higher than that recorded at other locations in the uterus with the exception of the day of delivery. A significant correlation was found between the number of APB and the level of magnesium in blood serum. The levels of 17-beta estradiol and progesterone were similar during all studied periods with the exception of the last week, in which a dramatic fall in estradiol level and a significant increase in the progesterone concentration were observed. The results showed that it is possible to distinguish three different phases of electric activity in the cow uterus during the last trimester of pregnancy. The features of these phases were discussed.     

Opposite regulation of cAMP concentration in the quail oviduct and the mouse uterus by tamoxifen. Correlation with estrogen-antagonist and estrogen-agonist activity

The ability of estradiol and tamoxifen to regulate cAMP levels and cAMP phosphodiesterase activities has been determined in the quail oviduct and in the mouse uterus. In the quail, tamoxifen (1 mg/kg daily for 3 days) had no effect on oviducal growth but significantly increased cAMP concentration (+49%). Injected concurrently with estradiol, tamoxifen completely inhibited oviduct growth as well as the increase of cAMP phosphodiesterase activity induced by the hormone alone and increased cAMP concentration (+229% over estradiol treated group). In the mouse, estradiol and tamoxifen displayed uterotrophic activity and increased cAMP phosphodiesterase activity. In both groups, cAMP concentration was greatly reduced (−76% in estradiol treated group; −86% in tamoxifen treated group). The opposite regulation of cAMP levels in the quail oviduct and the mouse uterus by tamoxifen reflected large differences in the contribution of calmodulin-dependent and -independent forms of phosphodiesterase to the hydrolysis of cAMP in the two models and the fact that tamoxifen stimulated the activity of the calmodulin-independent isoenzyme, while it competitively inhibited the activation of the calmodulin-dependent isoenzyme by calmodulin. Several lines of evidence strongly suggest that the regulation of cAMP levels is involved in growth-inhibiting or growth-promoting activity of tamoxifen.     

Expression of peptidylarginine deiminase in the uterine epithelial cells of mouse is dependent on estrogen.

The effects of the steroid hormones estrogen and progesterone on peptidylarginine deiminase protein-L-arginine iminohydrolase, EC 3.5.3.15) levels in adult ovariectomized mouse uterus were studied. The amount of the enzyme in the uterus was considerably diminished by ovariectomy. When the mice were injected with a variety of estrogenic compounds, 17 beta-estradiol-3-benzoate, which was the most potent stimulator of uterine cell proliferation among the estrogens tested, dramatically elevated the enzyme formation of the uterus in a dose- and time-dependent fashion. Results of immunohistochemistry with the antiserum against mouse peptidylarginine deiminase demonstrated that the induction of the enzyme by the estradiol exclusively occurred at the luminal and glandular epithelia, corresponding with the previous findings in the normal estrous cycle. Furthermore, administration of the estradiol significantly increased the content of mRNA coding for peptidylarginine deiminase in uterus, indicating the evidence of regulation in pretranslation. On the other hand, progesterone alone did not restore the enzyme level of the ovariectomized mouse, but moderated the action of estrogen when given in concert with estrogen. Thus, the expression of peptidylarginine deiminase in luminal and glandular epithelia of mouse uterus is controlled by the amount of the steroid hormones estrogen and progesterone.     

Effect of ovarian hormones on glycosyltransferase activities in the endometrium of ovariectomized rats.

Some of the properties of galactosyl- and sialyltransferases present in rat endometrial tissue were investigated. The enzyme activities were found to be partly membrane-bound and partly in soluble form. The galactose enzyme was also present in uterine secretions. The specific activities of both galactosyl- and sialyltransferases were greatly enhanced in endometrium of ovariectomized rats following 17 β-estradiol injections, although the enzyme activities in the liver remained unaffected. Mixing experiments with the homogenates of endometrium from control and estradiol-treated rats failed to suggest the presence of any “activator” or “inhibitor” of the enzymes. Diethylstilbesterol, estrone, and estriol also stimulated galactosyl- and sialyltransferase activities, whereas testosterone stimulated sialyltransferase only. Prolactin administration had no effect on either of the enzymes. The effect of estradiol on both enzymes was shown to be dose-dependent and the specific activities of the enzymes started to increase about 6 hr after hormone administration, reaching a peak around 48 h. Progesterone, on its own, had no effect on the galactosyltransferase in ovariectomized rat endometrium but effectively prevented the stimulatory effect of estradiol. When estradiol-primed rats were treated with progesterone, it was found that very small doses of progesterone resulted in decrease of galactosyltransferase activity. In such animals sialyltransferase activity was stimulated by a low concentration of progesterone which was followed by inhibition at higher concentrations. These effects of ovarian hormones on glycosyltransferase activities in endometrium are compatible with earlier reports on the effects of these hormones on glycoprotein and glycosaminoglycan levels in rabbit uterus after ovariectomy (7). Regulation of glycosyltransferase activities in endometrium induced by estradiol and progesterone may bear some relationship to the “receptive” state of the uterus for blastocyst implantation.     

Collagen proline hydroxylase activity in the uterus of the rat during rapid collagen synthesis in vivo

The activity of collagen proline hydroxylase in the 27,000g supernatant of the uterus was compared in the normal 20-day-old rat and in the adult rat 21 days after ovariectomy. The cofactor requirements of this enzyme were shown to be qualitatively the same as the enzyme from rat liver and skin. The specific activity of collagen proline hydroxylase in the uterus of the immature rat is approximately 250% higher than that of the ovariectomized animal. Although the total protein of the uterus of the ovariectomized rat is much greater, the total activity of this enzyme is 50% higher in the uterus of the immature rat. The daily administration of 5 μg estradiol-17β for 4 consecutive days to either animal results in a significant increase in the activity of collagen proline hydroxylase. Enzyme activity increases significantly 24 hr after the first dose of estradiol-17β and remains elevated in a reproducible pattern throughout the experimental period. Other estrogens including estriol, estrone, diethylstilbestrol, and ethynylestradiol-3-methyl ether also increase significantly the activity of collagen proline hydroxylase in the uterus of the immature rat. The activity of collagen proline hydroxylase was compared in the 27,000g supernatant of uterus of the immature and ovariectomized rat in a dose-response study with estradiol-17β and there appears to be little, if any, difference in total enzyme capacity. These results suggest that the failure of collagen to accumulate in the uterus of the ovariectomized rat administered estradiol-17β is unrelated to a low activity of collagen proline hydroxylase.     

Modulation of quail oviduct adenylate cyclase activity by estradiol and progesterone

Oviduct adenylate cyclase activity of the quail was measured by radiochemical analysis following different hormonal treatments. A single injection of estradiol benzoate (EB) to immature female quails resulted in a prereplicative surge of adenylate cyclase activity. A second surge of enzyme activity was observed during the proliferative phase induced by EB. Estradiol-17 alpha, estrone, estriol and testosterone were ineffective. Tamoxifen completely inhibits the growth-promoting effect of EB and the second surge of adenylate cyclase activity but does not inhibit the prereplicative increase of enzyme activity. This prereplicative increase of adenylate cyclase activity was also observed, even in the absence of increased plasma estradiol, when estradiol-17 beta (E2) was perfused through the hepatic portal vein. Moreover, E2 had no effect on enzyme activity when added directly to the oviduct homogenate preparation, at concentrations ranging from 10(-9) to 10(-7) M. In response to progesterone injection, oviduct adenylate cyclase activity followed a different pattern, beginning its increase after 3 h and remaining elevated up to 24 h. The activation by estradiol was independent of the presence of guanylylimidodiphosphate. Moreover, the enzyme was more sensitive to forskolin at submaximal concentration in estradiol treated birds than in control. These results demonstrate that transient activation of adenylate cyclase at the early stages of the action of estradiol does not occur through the classic nuclear receptor-gene activation pathway or a membrane receptor mediated process, but involves an indirect pathway, yet to be defined.     

Behavior of mouse placental and uterine estrogen sulfotransferase during chromatography and other procedures

The estrogen sulfotransferase activity of high-speed supernatants of mouse placenta and uterus behaves on conventional and high-performance liquid chromatographic gel filtration as an enzyme species with a molecular weight of the order of 30 000. This is so whether the cytosols are freshly prepared or have been stored at -20 degrees C before chromatography. The presence of thiol groups or EDTA has no effect on the elution pattern. The partially purified enzyme is extremely unstable and is poorly recovered by (NH4)2SO4 fractionation. Some stabilization can be achieved in the presence of 0.1 microM estradiol. Chromatofocusing of cytosols results in the elution of one or two sulfotransferase peaks, depending upon experimental conditions such as the presence or absence of thiol groups. These peaks act upon both estrone and estradiol as substrates. Chromatofocusing by fast protein liquid chromatography (FPLC) in the absence of thiol groups results in the elution of one sulfotransferase peak whose activity can be detected only when thiol groups are present during enzyme assay.