Localization of histamine in uterine structures

By means of luminescent-histochemical method of Cross, Even, Rost histamine is revealed in all uterine structures. Visual and fluorometric data demonstrate uneven distribution of histamine in the organ's structures. A high content of histamine is specific for macrophages and mast cells, less high--in tegmental epithelium and endometrial glands. A low level of histamine have endometrial stroma, smooth myocytes, cells of the serous membrane and vessels. Basing on the literature data, concerning various sensitivity of the uterine tissues to estrogens and regarding effect of the estrogens upon histamine metabolism in the uterine and regarding interconnection of the histamine receptors in the uterus and the estrogens, a suggestion is made that various contents of histamine in the uterine structures depend on various amount of the histamine receptors in them and on different abilities of the uterine tissues to inactivate histamine. The ability of macrophages to accept free forms of bioamines, as it is described in the literature, evidently can be spread to the uterine macrophages, where a high content of histamine is revealed.     

Hormonal control of implantation in guinea pigs

In the guinea pig, for which implantation is supposedly progesterone-dependent, actual hormonal requirements were assessed by measuring the levels of circulating estradiol and progesterone and correlating them with their content in the ovaries and uterus, and uterine concentrations of their receptors prior to, during, and immediately after implantation. Ovarian and uterine content and plasma levels of estradiol and progesterone, as well as uterine cytosolic receptors of these two hormones, were high at proestrus. Up to day 3 of pregnancy, estradiol remained high in peripheral plasma, ovarian and uterine tissues, but reached low levels at the time of implantation. The levels of progesterone showed a gradual increase in plasma and ovaries till the time of implantation, with the embryonic site of the uterus accumulating more of progesterone compared to estradiol. As pregnancy progressed, a gradual translocation of cytosolic to nuclear receptors occurred, both with estradiol and progesterone receptors. Comparing the receptor values for estradiol at each uterine site showed no significant alterations between embryonic and interembryonic cytosolic receptors. While significantly high levels of nuclear estradiol receptor were found at the inter-embryonic site on day 9 of pregnancy, the cytosolic and nuclear progesterone receptor concentrations were greater at the embryonic site on the same day. These findings demonstrated that the uterus is adequately exposed to estradiol and progesterone prior to ovulation and again in early pregnancy (day 1-3), thus facilitating implantation in the guinea pig (on days 7-8).     

The occurrence of steroid-free, "activated" estrogen receptor in target cell nuclei

New techniques for the adrenalectomy of pigs and for the isolation of pig uterus nuclei are described. The isolated nuclei were analysed for their content of estradiol and of estradiol receptor. The concentration of the latter exceeded that of the hormone in extracts of uterine nuclei from ovariectomized pigs by ratios of 1.8--10.3. Substantial amounts of both monomer and "activated" dimer receptor but no estradiol at all were extracted from uterine nuclei of ovariectomized/adrenalectomized pigs. The mechanism of action of steroid hormones is discussed on the basis of these results.     

Effect of estradiol on estrogen receptor expression in rat uterine cell types

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Multistage functional system amplifying and spreading the effect of estradiol in rat uterus

Summary Estradiol is demonstrated to induce histidine decarboxylase, and histamine is shown to activate adenylate cyclase in rat uterus. Histamine and cyclic 3,5-AMP mimic the effects of estradiol in that they enhance RNA synthesis, induce glycolytic enzymes and uterus imbibition. The data suggest that estradiol enhances by induction of histidine decarboxylase the formation of histamine, the latter activates adenylate cyclase providing accumulation of cyclic 3,5-AMP, which, probably, induces glycolytic enzymes through phosphorylation of chromatin proteins, and mediates other estradiol effects. The chain of successively acting enzymes and mediators constitutes, obviously, a cascade amplifying estradiol action. Since histamine is known to act as an intercellular mediator, attempts were made to find out the distribution of estradiol histamine and cyclic 3,5-AMP among uterus cells. Autoradiography has shown that [³H]-estradiol is bound by the nuclei of myometrium cells, [³H]-histamine was found above the cytoplasm of these cells, [³H]-cyclic 3,5-AMP is selectively bound by the cells of capillary endothelium of the uterus. The estradiol mediators seem to spread effect of hormone on cells of different types which form together a kind of multicellular functional system.     

Unique sensitivity to alpha-galactosylceramide of NKT cells in the uterus

It was previously reported that NKT cells, which are mainly present in the liver of mice, are also present in the uterus and that these uterine NKT cells are associated with abortion under overactivated conditions. In this study, we further examined their phenotypic and functional properties. In parallel with the progression of pregnancy, the number of uterine lymphocytes increased. This increase accompanied an increase in the number of TCRalphabeta(+) T cells and NKT cells in the uterus, although the number of NKT cells decreased at late pregnancy. Approximately one-third of the TCRalphabeta(+) cells were NKT cells at the early pregnant stage, while this ratio decreased toward late pregnancy. These uterine NKT cells were found to respond to alpha-galactosylceramide (alpha-GalCer) differently in comparison with liver NKT cells. In contrast to the apoptotic response of liver NKT cells on day 1 after alpha-GalCer injection, uterine NKT cells expanded prominently without such apoptosis. The majority of liver NKT cells were CD4(+). In contrast, almost all of the uterine NKT cells were double negative CD4(-)8(-). However, similar to liver NKT cells, uterine NKT cells used an invariant chain of Valpha14Jalpha281 gene for TCRalpha. The resistance against apoptosis might be due to the high expression of bcl-2 on uterine NKT cells after alpha-GalCer activation. Other evidence was that macrophages which existed in the pregnant uterus carried an activation marker, CD69, and could potentially produce many cytokines by their activation. The present results suggest that uterine NKT cells and surrounding macrophages are quite different in function from those in the liver.     

Multiphasic regulatory system mediating the effect of estradiol in rat uterus]

Estradiol is shown to induce histidine decarboxylase and histamine to activate adenylate cyclase in the rat uterus. Cyclic AMP like histamine simulates the effect of estradiol, intensifying RNA synthesis and inducing glycolytic enzymes and uterus inhibition. It was found by autoradiography that 3H-estradiol is accepted by the nuclei of some myometrium cells, 3H-histamine by their cytoplasm and 3H-cAMP is selectively bound by endothelium cells of the uterus capillaries. The estradiol messengers (histamine and cAMP) seem to mediate hormonal effect of some uterus heterofunctional cells forming a kind of multicellular functional system.     

Dissociation by colchicine of the wet weight and the cGMP responses to estradiol in immature rat uterus

We previously reported on a positive correlation between two effects of estrogen on rat uterus, namely the early increases in cGMP and in water content of the organ suggesting that they were under the control of the same hormone sensitive regulatory process or linked by a cause to effect relationship. Up to now we were unable to find experimental conditions that would dissociate the two responses. In this work, immature Wistar rats were treated with colchicine (50 micrograms/animal) given at the same time as estradiol-17 beta (1 microgram/animal) or with estradiol alone. The experiments showed: (1) that the estradiol induced increase in uterine wet weight that occurs during the first 8 h after hormone injection was completely suppressed in the presence of colchicine indicating that it might depend on an intact microtubular system and (2) that, by contrast, the estrogen-induced increase in uterine cGMP remained unaffected by the colchicine presence. These data allow to conclude that the cGMP response to estradiol can be dissociated from the wet weight response and, therefore, that it is not controlled by the latter. From this and from data in the literature the hypothesis is proposed that the increase in uterine cGMP content might trigger the wet weight response, this possibly through a positive action on some microtubular function.     

Radioautographic study of the localization of 3H-estradiol, 3H-histamine, and 3H-cyclic adenosine monophosphate in rat uterus]

The method of radioautography has demonstrated that 3H-estradiol adheres to the nuclei of some myometrium cells, 3H-histamine is accepted by the cytoplasm of the majority of the myometrium cells, 3H-cyclic-AMP is selectively bound by the endothelial cells of capillaries and small vessels in all layers of the uterus. From the data obtained it is possible to conclude that estradiol and its mediators, histamine and cyclic-AMP, specifically interact with different cells and with different cell structures.     

酪氨酸对大鼠子宫胞浆雌,孕激素受体的影响

采用放射受体分析法,测定了动情周期不同阶段及去卵巢大鼠子宫胞浆雌二醇和孕酮受体含量,并观察了子宫腔内注射酪、丝、苏三种氨基酸对子宫胞浆雌。醇、孕酮受体含量的影响。结果表明：（１）Ｌ－酪氨酸对动情前期、动情期、间情期大鼠子宫胞浆雌二醇和孕酮受体都具有明显的降低作用。（２）Ｌ－酪氨酸也降低去卵巢大鼠子宫胞浆雌二醇和孕酮含量,即这一作用不是通过影响卵巢激素分泌实现的。（３）Ｌ－苏氨酸仅可降低动情期和间情期大鼠子宫胞浆孕酮受体含量,而对相应周期雌二醇受体没有明显作用。（４）Ｌ－丝氨酸和Ｌ－苏氨酸对去卵巢大鼠子宫胞浆雌二醇和孕酮受体均无影响。     

Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.     

Insulin signaling in the liver and uterus of ovariectomized rats treated with estradiol

We used rat hepatic and uterine tissues to examine the impact of estradiol (E2) on insulin (INS) signaling. Ovariectomized (OVX) female Wistar rats were treated with E2 (20 microg/kg b.wt., i.p.) and used for the experiment 6h after E2 administration. To highlight E2 effects on tyrosine phosphorylation of INS receptor (IR) and INS receptor substrates (IRSs) and IRSs association with p85 subunit of phosphatidylinositol 3-kinase (PI3-K) in the context of INS signaling, E2-treated OVX rats were also injected with INS (20 IU, i.p.), 30 min before the experiment. Treatment with E2 did not change the levels of plasma INS and glucose (Glu). However, it significantly decreased the free fatty acid (FFA) level and increased uterine weight. Furthermore, the results show that E2 had no effect on the content of hepatic IR protein, but significantly increased IR protein content in the uterus and decreased IR tyrosine phosphorylation in both the liver and uterus. Compared to the control, hepatic IRS-1 and IRS-2 were significantly decreased and increased, respectively, after E2 treatment. Protein content of both molecules, IRS-1 and IRS-2, was increased in uterine tissue after E2 administration. Protein content of the p85 subunit of PI3-K and that of protein kinase B (Akt) were increased in the uterus, with no changes in the liver. The results suggest that E2 treatment induces tissue-specific changes in INS signaling. The consequences of E2 treatment on INS signaling molecules are more apparent in the uterus, but their physiological relevance for INS action is probably greater in the liver.     

Blastocyst H(2) receptor is the target for uterine histamine in implantation in the mouse

The process of implantation is a 'two-way' interaction between the blastocyst and uterus. It has long been suspected that histamine is an important mediator in embryo-uterine interactions during implantation, but its source, targets and mechanism of actions remained undefined. We have recently demonstrated that uterine epithelial cells are the source of histamine, which peaks on day 4 of pregnancy (the day of implantation) in the mouse. In searching for its target and site of action, we discovered that preimplantation blastocysts, which express histamine type 2 receptor (H(2)), is the target for histamine action. Using multiple approaches, we demonstrate herein that uterine-derived histamine interacts with embryonic H(2) receptors in a paracrine fashion to initiate the process of implantation.     

Steroidal control mechanism of cell proliferation in mouse uterine epithelium

The percentage of labeled cells in the uterine luminal epithelium of cycling mice showed the different zonal distributions at each stage of estrous cycle after cumulative labeling with 3H-thymidine for 36 hr. It was estimated that the proliferating fraction in the epithelium at proestrus, estrus, metestrus, and diestrus was 100%, 100%, 40% and 5%, respectively. The percentage of labeled cells in the uterine luminal epithelium of cycling mice treated with progesterone remained below 10% level for at least 20 hr after injections of progesterone. Total labeling was attained in the uterine epithelium of castrated mice by the administration of estradiol-17beta. On the other hand, the cell proliferation in the uterine epithelium of castrated mice treated with estradiol and progesterone was markedly suppressed and the percentage of labeled cells remained approximately at 35%. The remaining cell population, however, still showed the mitotic potency when mice received estradiol. It is suggested from this study that the effect of progesterone is to suppress the epithelial cell proliferation and transfer cells into resting cell fraction which is still evoked to proliferate as the effect of estradiol and that a key factor controlling epithelial proliferation in mouse uterus during the estrous cycle is proliferating fraction rather than cell cycle time.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

Estrogen responsiveness of fetal guinea pig uterine cells in culture

Cells from the uterus of the guinea pig fetus have been grown as a monolayer culture in serum-containing medium. Cells from the first subculture showed high concentrations of progesterone receptor (PR; 9.3-13.8 pmol/mg DNA) even after 9 days in medium containing charcoal-treated serum and estradiol did not induce any further increase. The antiestrogens, tamoxifen and monohydroxytamoxifen, both had an inhibitory effect which could be overcome by estradiol. The progestins, progesterone and R5020, as well as the antiprogestin, RU38486, also decreased the PR concentration. Estrogen receptor (ER) levels did not vary with the compounds tested but were found to be low compared to concentrations found in the fetal guinea pig uterus at 55-65 days of gestation. None of the compounds tested had any effect on the growth of the fetal uterine cells so that the modulation of PR concentrations was dissociated from the regulation of cell growth. It is concluded that estrogens are necessary but not sufficient factors in the control of PR levels in fetal uterine cells. The establishment of a culture system for separate types of fetal uterine cells will permit us to study in vitro the factors involved in the growth effects of estrogens and the control of PR synthesis.     

The effects of sequential administration of 17 beta-estradiol on the synthesis and secretion of specific proteins in the immature rat uterus

We report here results of a study of the effect of sequential administration of 1 microgram 17 beta-estradiol in vivo on the incorporation of L-[35S]methionine into specific proteins in vitro in the immature rat uterus. One-dimensional SDS-PAGE analysis of labeled secreted uterine proteins and cellular proteins extracted from the luminal epithelial and from the stroma plus myometrial uterine fractions revealed that estradiol preferentially stimulated the synthesis of 110 K, 74 K and 66 K secreted proteins, 180 K and 110 K epithelial proteins and a 175 K stroma-myometrial protein among others, while it decreased the relative rate of synthesis of a 32.5 K secreted protein and a 70 K stroma-myometrial protein. The 110 K protein, a secreted luminal epithelial protein whose labeling in vitro dramatically increased greater than 60-fold per mg endometrial DNA after in vivo estrogen stimulation, may be a useful marker for studying estrogen action in the luminal epithelium of the immature rat uterus. Comparison of the secreted proteins labeled at 28 h (4 h after a second injection) and at 54 h (6 h after a third injection) revealed that estradiol effected a sequential change in the pattern of synthesis of secreted uterine proteins in vitro. Comparison of the number and magnitude of changes in the synthesis of specific proteins in the luminal epithelium and the stroma plus myometrium revealed that protein synthesis in the luminal epithelium is clearly more responsive to estradiol and readily distinguishable from the responsiveness of the stroma plus myometrium.     

天花粉对小鼠子宫肥大细胞超微结构的影响

本研究用雌性中国１号小鼠１２只,分实验组和对照组,实验组分别腹腔注射天花粉后处死,取子宫组织作超薄切片,电镜观察。在天花粉作用下,常见子宫肥大细胞脱颗粒,呈功能活化状态,并与子宫平滑肌细胞紧密相邻。提示肥大细胞可能通过脱颗粒释放组胺促进子宫平滑肌的收缩。同时,也见肥大细胞与子宫结缔组织中的成纤维细胞,淋巴细胞等密切接触,提示成纤维细胞和淋巴细胞与天花粉作用下子宫肥大细胞的数量增多有关。     

Formation of specific proteins and peptides in the rabbit uterus induced by estradiol

It has been shown that specific proteins and peptides are synthetized in uterus cells during 1-4 h after estradiol administration into rabbits. These compounds side by side with peroxidase may serve as biological marker of estradiol action in target-tissue.