Sites In Uterus Research Articles

Restraint stress induces uterine microenvironment disorder in mice during early pregnancy through the β2-AR/cAMP/PKA pathway

During pregnancy, uterus undergoes the environment adaptation as part of a program of development. In the world, one in four people worldwide suffer from mental illness, especially pregnant women. β-Adrenergic receptor (β-AR) is an important regulator that converts environmental stimuli into intracellular signals in mice uterus. CD-1 (ICR) mice undergone restraint stress, which was a case in model to simulate the psychological stress. The plasma and implantation sites in uterus were obtained and examined. PCR analysis demonstrated that β2-AR expression levels in embryo day (E) 3, 5 and 7 were kept at a significantly higher level (p < 0.05) under restraint stress and higher than β1-AR and β3-AR in different gestation ages. The β2-AR protein levels were obviously increased (p < 0.05) due to the markedly elevated norepinephrine (NE) concentration (p < 0.05). In our previous study, restraint stress can induce the apoptosis and inflammation. Also, the matrix metalloprotein-9 (MMP-9) was decreased significantly (p < 0.05) under restraint stress. Meanwhile, Caspase3, p-NF-κB p65 and p-ERK1/2 were obviously increased (p < 0.05) in the work. In vitro studies showed that the p-ERK1/2 and Caspase-3 levels were raised (p < 0.05) after β2-AR was activated. However, they were decreased when PKA was blocked. The protein levels of Caspase-3 were reduced when ERK and NF-κB were blocked (p < 0.05). In conclusion, the β2-AR/cAMP/PKA pathway promoted apoptosis and affected the development of the uterus through the ERK and NF-κB signaling pathway. The findings of this study may provide evidence for female reproduction under psychological stress.

76 Clock MUTANT MICE HAVING A DIMINISHED CIRCADIAN CLOCK SHOW ABNORMAL IMPLANTATION

The circadian clock involves co-operative transcription of circadian genes including Clock and regulates circadian rhythms in mammals such as the rhythm of sleep and wakefulness. In reproductive physiologies in mammals, there are several time-dependent events such as progression of the oestrous cycle and embryonic development, and we therefore checked the reproductive characters of mice homozygous for Clock delta-19 mutation (CL) having a diminished circadian clock to elucidate the effect of the circadian clock on reproductive physiologies. Profiles of the oestrous cycle were the same in female (f) wild-type mice (WT) and fCL: oestrous cycles of both fWT and fCL consisted of the same 3 stages, proestrous, oestrous, and metestrous/diestrous stages, and the average lengths of each stage and one oestrous cycle were the same in 10 fWT and 14 fCL. We therefore compared outcomes from the 4 possible mating groups between WT and CL. Average numbers of newborn pups obtained from mating pairs of 14 male (m) WT × fWT, 10 mCL × fWT, 14 mWT × fCL, and 15 mCL × fCL were 13.4 ± 0.8, 12.6 ± 0.4, 12.3 ± 0.7, and 8.6 ± 1.5, respectively, and gradually decreased depending on the number of mutated Clock alleles in mothers and embryos. Since increases in body weights of the mothers during the gestation period were not different in the 4 mating groups and since there were no signs of spontaneous abortion from mid to late gestation, we reasoned that some embryos were lost before or at the time of implantation. Immediately before implantation (88 h after fertilization), neither the number of embryos collected from uteri nor the percentage of embryos that reached the pre-implantation stage (blastocyst stage) differed significantly among mating groups. In contrast, immediately after implantation (160 h after fertilization), the average numbers of implantation sites in mating pairs of 12 mWT × fWT, 11 mCL × fWT, 13 mWT × fCL, and 13 fCL × fCL were 13.0 ± 1.5, 13.1 ± 1.2, 11.7 ± 0.8, and 7.0 ± 1.3, respectively, and also gradually decreased with increase in the number of mutated Clock alleles in mothers and embryos. This decrease was accompanied by a significant lowering of the positions of implantation sites in uteri, a possible cause of the decrease of the number of newborns and implantation sites, and average percentages of embryos implanted in a lower part of the uterus were 48.8, 59.3, 69.7, and 77.7% in mWT × fWT, mCL × fWT, mWT × fCL, and mCL × fCL, respectively. The difference between the mCL × fWT and mWT × fWT groups was statistically significant (P &lt; 0.05), and this difference was thought to be due to the difference in embryonic genotype, specifically between WT and heterozygous embryos. However, the distribution of implantation sites in the mWT × fCL group was significantly smaller than that in the mCL × fWT group (P &lt; 0.05), presumably due to the difference in maternal genotype, specifically between WT and homozygous mutant dams. This study showed involvement of the circadian clock, possibly the maternal and embryonic circadian clock, in implantation among events that occur from fertilization to parturition.

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development.

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.

Role of Ovarian Steroids in Development of Uterine Binding Sites for Prolactin in the Ferret1

The objectives of this study were to investigate 1) the presence of specific prolactin (PRL) binding sites in the ferret uterus, 2) the uterine location of 125I-labeled ovine PRL (oPRL) binding sites, 3) changes in uterine PRL binding sites during pseudo-pregnancy, and 4) regulation of PRL binding sites by ovarian steroids. Binding was determined through use of 125I-oPRL and 300-800 micrograms of protein from the 50,000 x g particulate fraction. Optimal binding occurred within 6 h at 25 degrees C. Scatchard analysis of saturation data revealed a single set of high-affinity (Kd = 4.99 x 10(-11) +/- 0.88 M), low-capacity (22.76 +/- 1.62 fmol/mg) binding sites. Analysis of hormonal specificity revealed that ovine growth hormone (oGH) cross-reacted with oPRL for the uterine binding sites, displacing 38% of the bound ligand. However, no inhibition of 125I-oPRL binding occurred in the presence of a 500-fold excess of bovine thyroid-stimulating hormone (bTSH), ovine LH (oLH), or ovine FSH (oFSH), suggesting hormonal specificity of the binding sites that are located in the luminal and glandular epithelium. Prolactin binding to ferret uterine membranes increased during the first half of pseudopregnancy, plateaued between Days 21 and 28, and then declined. The concentration of PRL binding sites in uteri of ferrets on Day 1 of pseudopregnancy was 4.91 +/- 0.42 fmol/mg of protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of progestin-binding cells in estrogen-treated and untreated neonatal mouse uterus and oviduct: autoradiographic study with [125I]progestin

Progestin-binding sites in uteri and oviducts of estrogen-treated and untreated 8-day-old mice were studied by thaw-mount autoradiography with [125I]progestin. In the untreated uteri, nuclear concentration of radiolabelled progestin was observed in all tissues of the uterus, with strongest nuclear labelling in luminal and glandular epithelia and in stroma. In the estrogen-treated uteri, the degree of labelling was markedly augmented in stroma and muscle, but much reduced in the luminal and glandular epithelia, compared to untreated uteri. In untreated oviducts, nuclear labelling was observed in stroma and muscle in all regions and in epithelium in the isthmic and uterine regions. The epithelium in infundibular and ampullar regions was only scarcely labelled. The estrogen-treatment augmented the labelling in stroma and muscle of the oviduct as in the uterus, but the labelling in epithelium was not affected. These results indicate that estrogen-treatment induces progesterone receptor differentially among tissue compartments both in the uterus and oviduct.

Different regulation of the concentration of estrogen receptors in the rat liver and uterus following ovariectomy

Data concerning the short- and longterm effects of ovariectomy on the levels of estrogen binding sites in the rat uterus and liver are presented. The information increases the understanding of the regulation of estrogen receptor synthesis. The circulating estrogen level is suggested to affect receptor synthesis in the uterus and liver differently. Shortly after gonadectomy (2–20h), an elevation in the concentration of cytoplasmic binding sites in the uterus of 35% was observed, whereas no effect was seen in the liver cell. A longer period of time after ovariectomy (2–3 months) caused a reduction in the number of uterine receptor sites by 74%, whereas in the liver an increase of 84% was detected.

Estrogenic activity of the insecticide chlordecone (Kepone) and interaction with uterine estrogen receptors.

The chlorinated insecticide chlordecone (Kepone) interacts with the estrogen receptor system in the rat uterus in vitro and in vivo. It competes with estradiol for binding to the cytoplasmic receptor in vitro and also induces nuclear accumulation of estrogen receptor sites in uteri in vitro. When injected into immature rats, chlordecone translocates estrogen receptor sites to the uterine nucleus, increases uterine weight, and stimulates the synthesis of the progesterone receptor, an estrogen receptor-mediated process. Its slow onset of action but prolonged duration of interaction with estrogen receptor and stimulation of uterine weight gain and progesterone receptor synthesis indicates that, although it has an affinity for receptor only 0.01-0.04% that of estradiol, its considerable estrogenic activity may likely be derived from its long half-life and bioaccumulative character.

Relationship between cytosol and nuclear oestrogen receptors and oestrogen concentrations in the fetal compartment of guinea-pig

Previous reports from our laboratory as well as the results of the present study show that specific [ 3H]oestradiol binding is present in various organs of the fetal guinea-pig. At the same fetal age ( ~ 50 days of gestation), the concentration of specific [ 3H]oestradiol binding sites per mg DNA in the various fetal tissues can be classed as follows: uterus > lung > brain ⩾ kidney. The [ 3H]oestradiol binding sites in all the tissues studied are capable of binding other oestrogens such as oestrone or oestriol but aldosterone, testosterone, cortisol and progesterone do not compete for [ 3H]oestradioI binding sites. In uterus [ 3H]oestradiol and [ 3H]oestrone were found to bind in equal quantities to the same binding site. Binding of [ 3H]oestradiol is of high affinity in all four tissues ( K D4 C = 2 −8 × 10 −10 M). The sedimentation coefficient of this [ 3H]oestradiol binding component in all four tissues is 8 S in low salt sucrose density gradients and the isoelectric point (pI) as determined by isoelectrofocusing on gel plates is 6–6.2. The concentration of oestradiol and oestrone in fetal plasma and in the tissues were determined by radioimmunoassay. The values in fetal plasma from 50 to 64 days of gestation for oestradiol are 9–11 pg/ml plasma and for oestrone, 94–105 pg/ml plasma. In the uterus, the concentration of both oestrogens is higher than in plasma, in the order of 1–2 ng per g of tissue. These relatively low levels of circulating oestrogen concentration correlate well with the finding that 93.3% of the [ 3H]oestradiol binding sites are in the cytosol fraction. Moreover, the concentration of occupied binding sites in the uterus (9 pmol/g tissue) corresponds well with the concentration of oestrogens in the tissue (7 pmol/g tissue). A possible biological role of oestradiol receptors in fetal uterus has been revealed. Administration of oestradiol to the pregnant guinea-pig has been found to increase very significantly the concentration of specific progesterone binding sites in the fetal uterus.

Properties of two progesterone-binding proteins of the rat uterus

DEAE-cellulose filtration was used to measure high affinity binding sites in uteri of rats during the estrus cycle. The cytosol concentrations (pmol/mg protein ± S.E.M.) of progesterone-specific ( R) sites were: proestrus, 0.9 ± 0.07; estrus, 0.4 ± 0.04; metestrus, 0.4 ± 0.04; diestrus-1, 0.2 ± 0.04; and diestrus-2, 0.6 ± 0.03. This pattern is consistent with the concept that the concentration of R sites is increased by estrogens, known to rise in rat serum on the evening of diestrus-1 and to remain high at proestrus. The respective values for corticosteroid-binding globulin-like ( G) sites were 12 ± 0.2, 8 ± 0.1, 13 ± 0.2, 12 ± 1.1 and 16 ± 0.2. That the lowest number of R sites was at diestrus-1 and of G sites at estrus may indicate different half-lives or regulatory mechanisms for the two proteins. Preliminary data showed that nuclear progesterone binding was largest at proestrus and metestrus. The equilibrium association constant of the R protein for progesterone was measured in preparations purified by spheroidal hydroxylapatite chromatography. The estimates were in the range 2–8 10 8 × M −1. Of several steroids tested, deoxycorticosterone and 20α-dihydroprogesterone competed for G sites while dexamethasone and 5α-dihydroprogesterone did not. Only the latter steroid and deoxycorticosterone showed some affinity for R sites. When uterine nuclei from adrenalectomized-ovariectomized rats injected with either progesterone or corticosterone were added to incubation mixtures containing [ 3H]-progesterone or [ 3H]-corticosterone, significant exchange occurred only between nuclear progesterone and [ 3H]-progesterone, suggesting that nuclei take up only progesterone bound to R sites.

3H]Dihydroergocryptine binding in rat brain

[ 3H]dihydroergocryptine (DHE) appears to bind to α-adrenergic receptor sites in rabbit uterine membranes. We have characterized the binding of [ 3H]DHE to membranes prepared from rat cerebral cortex, α-Adrenergic agents were less potent and dopamine and serotonin, more potent, in displacing brain DHE binding than in uterus. Furthermore brain DHE binding sites demonstrated less stereospecificity for catecholamines than sites in uterus. Dopamine displaced DHE binding with about the same potency in cerebellar and cerebral cortical membranes, but was 10 times as potent in displacing DHE binding in the striatum. The binding of [ 3H]DHE in brain is complex and differs significantly from the rabbit uterus. There are two possible explanations for this discrepancy. [ 3H]DHE may bind a single site in brain with properties differing from known peripheral adrenergic receptors or DHE may bind to multiple sites in brain, sites which may or may not represent other neurotransmitter receptors.

Oestrogen and nuclear binding sites. Determination of specific sites by ( 3 H)oestradiol exchange.

A method was developed for the determination of the number of specific oestradiol-binding sites in the nuclear fraction of oestrogen-sensitive tissues. The method is based on the exchange of [(3)H]oestradiol with non-labelled oestradiol that is bound to nuclear binding sites. The number of specific nuclear binding sites after the injection of 2.5mug of oestradiol, an amount sufficient to saturate all binding sites, is 1.6-1.7pmol per immature uterus. The number of sites occupied after an injection of physiological amounts of oestradiol (0.1mug) was 0.46pmol. The injection of oestradiol results in an increased number of nuclear binding sites in uterus and vagina, but has no effect on kidney or muscle. Injections of testosterone or progesterone failed to increase the number of uterine nuclear binding sites. This method permits an evaluation of the number of oestradiol-binding sites in the nuclear fraction of various tissues as a function of either endogenous oestradiol or non-labelled oestradiol administered by injection.