Pregnant Rat Uterus Research Articles

Antiinflammatory Effects of an Ethyl Acetate Extract ofEuphorbia royleana

The antiinflammatory activity of the ethyl acetate extract (EA) from the residue of an 85% alcohol extract of the latex of plant Euphorbia royleana was tested in mice and rats. In acute tests EA displayed marked antiinflammatory activity. Significant antiarthritic activity was observed in subacute and chronic models of formaldehyde-induced arthritis, adjuvant-induced developing and established arthritis. It produced inhibition of the exudate volume and the leucocyte migration in rats and vascular permeability in mice. EA showed a poor inhibitory effect on the granuloma formation by cotton pellet and croton oil. In in vitro studies EA inhibited the spontaneous contractions of the isolated pregnant rat uterus and oxytocin-induced contractions of virgin rat uterus. It had a very low ulcerogenic potential. The oral LD 50 in both rats and mice was more than 2500 mg/kg.

Interaction between nitric oxide and prostaglandin E2 pathways in pregnant rat uteri

We examined the possible interrelationship between nitric oxide (NO) and cyclooxygenase (COX) products in the uterus during pregnancy and labor. Results indicate that 1) rat uteri during labor, at term, demonstrated a 69% decrease in nitrite production and a 217% increase in prostaglandin E2 (PGE2) production compared with day 18 of pregnancy; 2) interleukin-1 beta (IL-1 beta) induced a pronounced elevation of both nitrites and PGE2 in rat uteri; 3) diethylenetriamine/NO, a donor of NO, induced a significant increase of PGE2 production by the uterus in a dose-dependent manner; 4) NG-nitro-L-arginine methyl ester, an inhibitor of NO synthesis, markedly inhibited IL-1 beta-induced nitrite and PGE2 in rat uteri; this inhibitory action was reversed by coincubation with L-arginine; 5)exogenous PGE2 significantly inhibited IL-1 beta-induced, but not constitutive, nitrite production; and 6) inhibition of endogenous PGE2 by indomethacin substantially increased IL-1 beta-induced nitrite production. Thus the interaction between NO and COX pathways might be important in the regulation of uterine contractility during pregnancy and labor.

Post‐coital Contraceptive Action of Pueraria tuberosa DC. Extract in Rats

Biochemical and physiological alterations have been observed in the ovary and uterus of pregnant rats treated with an extract of Pueraria tuberosa DC. Its administration from day 1 to 5 post-coitally caused a significant increase in the wet weight, protein and glycogen contents. Activity of acid phosphatase and ATPase was depleted sharply on days 4 and 5, but the activity of alkaline phosphatase was elevated on days 4 and 5. Ovaries of treated rats showed an increased number of follicles of different age. Cells of the corpora lutea showed hypertrophy. Uterine histoarchitecture appeared nonreceptive on the day of implantation. No deciduoma was observed on day 5. These changes have been correlated with the antiimplantation action of the butanol extract.

Post-coital Contraceptive Action ofPueraria tuberosaDC. Extract in Rats

Biochemical and physiological alterations have been observed in the ovary and uterus of pregnant rats treated with an extract of Pueraria tuberosa DC. Its administration from day 1 to 5 post-coitally caused a significant increase in the wet weight, protein and glycogen contents. Activity of acid phosphatase and ATPase was depleted sharply on days 4 and 5, but the activity of alkaline phosphatase was elevated on days 4 and 5. Ovaries of treated rats showed an increased number of follicles of different age. Cells of the corpora lutea showed hypertrophy. Uterine histoarchitecture appeared nonreceptive on the day of implantation. No deciduoma was observed on day 5. These changes have been correlated with the antiimplantation action of the butanol extract.

Congener-specific effects of pcbs on contractions of pregnant rat uteri

Both increased and decreased gestation lengths have been reported following exposures to polychlorinated biphenyl (PCB) mixtures and congeners. Because oscillatory uterine contractions are essential for parturition, we hypothesized that the disparate findings on gestation length may be the result of distinct PCB congener-specific actions on oscillatory uterine contractions. This study examined the acute effects of PCB congeners on isometric contractions of isolated pregnant uteri and the structure-activity relationship for individual congeners. After cumulative exposure to individual PCB congeners (0.5 μM to 150 μM), oscillatory contractions were: 1) not altered by 2,4,5,2′,4′,5′-hexachlorobiphenyl, 3,4,5,3′,4′-pentachlorobiphenyl, or 3,4,3′,4′-tetrachlorobiphenyl; 2) significantly inhibited by 4-hydroxy-2′,4′,6′-trichlorobiphenyl; and 3) markedly increased by 2,4,6-trichlorobiphenyl and 2,4,2′,4′-tetrachlorobiphenyl, when compared to solvent controls. The uteri were more sensitive to PCB congeners with ortho-substituted light chlorination than those highly chlorinated, or those interacting with the Ah-receptor.

Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus

We have shown previously that the epithelial cells of human endometrium produce CRH. The biological role of endometrial CRH is not yet known. Among other things, CRH appears to be involved in the inflammatory process, acting as an autocrine/paracrine proinflammatory regulator. Since the reaction of endometrium to the invading blastocyst has characteristics of an aseptic inflammatory reaction, we have hypothesized that endometrial CRH may participate in the inflammatory phenomena taking place at the implantation site of blastocyst. In the present study we demonstrate a higher content of immunoreactive (IR)-CRH and CRH mRNA in the implantation sites of early pregnant rat uterus compared to the inter-implantation regions. Specifically we have found that : a) rat uterus contained a 1.3 kb CRH transcript, similar or identical in size to that present in human placenta, b) the size of the IR-CRH present in uterine extracts was similar to authentic hypothalamic CRH, c) Northern blot analysis showed that the content of CRH mRNA in uterus at the implantation sites was 3.5 fold higher compared to that in the inter-implantation regions and finally, d) immunohistochemical localization of IR-CRH in early pregnant rat uterus revealed positive staining of the luminal epithelial cells in both implantation and inter-implantation uterine regions, while decidualized stromal cells were positive only at the implantation sites. Our data suggest that endometrial CRH may play a role in the implantation of blastocyst.

Identification of a hyperpolarization-activated inward current in uterine smooth muscle cells during pregnancy

1. 1. Rat uterine smooth muscle possesses spontaneous contraction and automaticity. This study was exerted to examine one of the underlying currents that may be involved in the generation of spontaneous activity, the hyperpolarization-activated inward current. 2. 2. Whole-cell patch clamp experiments were performed on freshly-isolated single longitudinal smooth muscle cells of pregnant rat uterus (18-day gestation). The holding potential was −30 mV, and long-duration (3 sec) hyperpolarizing pulses were applied to −40 to −120 mV. Experiments were performed at room temperature (22°C). 3. 3. A hyperpolarization-activated inward current ( I f ) was produced at the larger hyperpolarizing steps. Its current density (at −120 mV) was −1.03 ± 0.31 pA/pF ( n = 5). The average cell capacitance was 64.3 ± 12.3 pF ( n = 8). The threshold potential for activation of I f was about −70 mV, and the reversal potential was −18.6 ± 2.1 mV ( n = 4). In the presence of Cs + (3 mM), the I f current (at −120 mV) was decreased by 76.5 ± 2.1% ( n = 5, P < 0.01). 4. 4. These results indicate that the Cs +-sensitive hyperpolarization-activated inward current is present in the longitudinal muscle cells of pregnant rat uterus. This I f current may contribute to some extent to the electrogenesis of the spontaneous activity.

Alternative splicing and differential targeting of fibroblast growth factor receptor 1 in the pregnant rat uterus

Recent studies suggest that steroid effects on uterine cell proliferation may be moderated by polypeptide growth factors. We now provide evidence that high affinity fibroblast growth factor (FGF) receptors are present temporally and spatially in the pregnant rat uterus (days 4-6) to support the idea that basic FGF action occurs via binding to its high affinity FGF receptor 1 (FGFR1). Reverse transcription-polymerase chain amplification indicates that both the full-length transcript and an alternatively spliced messenger RNA are present in the uterus. Western immunoblot analysis confirms that rat uterine membrane proteins contain two receptor isoforms, and these receptors bind basic FGF with high affinity and specificity. Immunolocalization of FGFR1 revealed receptor-positive cells in both the uterine stroma and epithelia on days 4-6 of pregnancy. However, the receptor was differentially localized in the disparate cell types. The nuclei of stromal cells were positive for FGFR1, whereas epithelial cell nuclei were negative. Together, these results suggest that FGF signal transduction in uterine stromal cells is mediated by activation of FGFR1.

Alternative splicing and differential targeting of fibroblast growth factor receptor 1 in the pregnant rat uterus.

Recent studies suggest that steroid effects on uterine cell proliferation may be moderated by polypeptide growth factors. We now provide evidence that high affinity fibroblast growth factor (FGF) receptors are present temporally and spatially in the pregnant rat uterus (days 4-6) to support the idea that basic FGF action occurs via binding to its high affinity FGF receptor 1 (FGFR1). Reverse transcription-polymerase chain amplification indicates that both the full-length transcript and an alternatively spliced messenger RNA are present in the uterus. Western immunoblot analysis confirms that rat uterine membrane proteins contain two receptor isoforms, and these receptors bind basic FGF with high affinity and specificity. Immunolocalization of FGFR1 revealed receptor-positive cells in both the uterine stroma and epithelia on days 4-6 of pregnancy. However, the receptor was differentially localized in the disparate cell types. The nuclei of stromal cells were positive for FGFR1, whereas epithelial cell nuclei were negative. Together, these results suggest that FGF signal transduction in uterine stromal cells is mediated by activation of FGFR1.

Changes in kinetic properties of oxytocin receptors in longitudinal muscle membranes of rat uterus during gestation

Receptors of oxytocin were found to occur in the membrane fraction obtained from longitudinal muscle of pregnant rat uterus. The affinity of the membrane receptor for oxytocin increased through an increase in the association rate and a decrease in the dissociation rate during gestation. The membrane oxytocin receptor concentrations rose almost three-fold from Day 15 to Day 21. A transition of oxytocin receptors from a single class of independent sites to site-site interacted multiple binding sites, which most likely exhibit positive cooperativity during the last seven days of gestation, was observed. These results suggest that changes in the dynamics of uterine oxytocin receptors also play an important role in the onset of labor.

A low-affinity estrogen-binding site in pregnant rat uteri: analysis and partial purification.

We have identified a low-affinity (type II) estrogen-binding site (EBS) that is expressed at high levels during pregnancy in rat uteri. Although this activity was detectable in nonpregnant rat uteri, it was present in amounts (0.094 pmol/g of uteri) that were severalfold lower than the high-affinity type I estrogen receptor (0.57 pmol/g of uteri). During pregnancy, at 19-20 days of gestation, the low-affinity type II EBS became the major (> or = 88%) estrogen-binding site in rat uteri. The increase in the level of low-affinity EBS (7.9 pmol/g) in uteri was approximately 85-fold with an approximately 20-fold increase in the specific activity (0.39 pmol/mg) of this form, whereas the high-affinity form remained relatively unchanged. We report here a method of purification of type II EBS from pregnant rat uteri and present an analysis of its DNA and steroid-binding properties. Estradiol-binding studies and Scatchard analysis showed that the type II EBS had an apparent estradiol-binding affinity of > or = 24 nM. Gel filtration and SDS/PAGE analysis indicated that the type II EBS was a monomeric 73-kDa protein. The estradiol binding remained apparently uninhibited in the presence of a large excess of tamoxifen, nafoxidine, or dihydrotestosterone. Estradiol, diethylstilbestrol, and quercitin (a type II EBS-specific inhibitor) competed efficiently. The purified low-affinity EBS did not have sequence-specific DNA-binding activity with the estrogen-responsive element, which indicated that it differs in function from the type I estrogen receptor.

The effect of platelet activating factor antagonist (BN 52021) on pregnancy duration and collagen content in the pregnant rat uterus and cervix.

In order to study the role of Platelet Activating Factor (PAF) in the mechanism of parturition and connective tissue metabolism in the pregnant rat uterus, we investigated the influence of the Platelet Activating Factor Receptor Antagonist (BN 52021) on gestation duration and collagen concentration in pregnant rat uterus and cervix at term. Total and soluble collagen as well as dry tissue/wet tissue ratio were measured in the BN 52021 treated group (n = 9) and in the control group (n = 13). Gestation duration was evaluated in a group receiving BN 52021 (n = 10) and in a control group (n = 19) receiving pure solvent. There were no significant differences in water content. The total and soluble collagen concentration was also similar to the control in both the uterus and the cervix. The only significant difference was observed in the soluble collagen/total collagen ratio in the uterus. BN 52021 administration did not alter the gestational period. Basing on this experiment we suggest that not all the phospholipid metabolites activated by phospholipase A2 significantly influence the duration of pregnancy and connective tissue metabolism in the pregnant rat uterus.

Protein kinase C stimulates Ca2+ current in pregnant rat myometrial cells

The factors that regulate the voltage-dependent Ca2+ channels in pregnant uterine smooth muscle cells have not been elucidated, including any roles for protein kinase C (PKC). Therefore, the role of PKC in the regulation of the slow (L type) Ca2+ channels was examined in myometrial cells isolated from late pregnant (18-19 day) rat uterus, using the nystatin-perforated whole-cell voltage clamp. A PKC activator, phorbol 12,13-dibutyrate (PDB), increased the L-type Ca2+ current (ICa(L)). Bath application of PDB (0.03 and 0.3 microM) increased the peak amplitude of ICa(L) by 21 +/- 14% (n = 6) and 37 +/- 8% (n = 9, p < 0.01), respectively. PDB did not change the holding current or shift the current-voltage relationship for ICa(L). The PKC inhibitors, H-7 (20 microM) or staurosporine (10 nM), reversed the effect of PDB. These results indicate that PKC may play a role in regulating Ca2+ channel function in pregnant rat myometrial cells and, therefore, may be involved in control of uterine contraction.

Immunohistochemical localization of procollagen types I and III during placentation in pregnant rats by type-specific procollagen antibodies.

To examine the sequential localizations of procollagen Types I (Pro I) and III (Pro III) during chorioallantoic placental formation in pregnant rats, we prepared polyclonal anti-rat Pro I- and III-specific antibodies. Biochemical analysis of a fraction containing [14C]-glycine-incorporated collagen from pregnant rat uteri showed that collagen Types I and III were actively synthesized during placental development. We examined 8-, 9.5-, 13-, and 20-day gestation rat uteri immunohistochemically. At Days 8 and 9.5, in the basal decidua facing the fetal cytotrophoblastic giant cell layer and implantation site, the immunoreactivity for Pro I was higher than that for Pro III. On Day 13, the enlarged myometrium and cytotrophoblastic cell layer showed increased immunoreactivity for Pro III. Unexpectedly, polygonal trophoblastic cells invading and modifying the maternal central artery showed intense immunoreactivity for Pro III. On Day 20, the fetal mesenchyme, large fetal blood vessels, and subendothelial stroma, including fetal blood capillaries, were more immunoreactive to Pro III antibody than to Pro I antibody in the labyrinth. Pro I and III synthesis and processing appear to be developmentally regulated and may be related to control of the microenvironment for supporting the fetus, control of the maternal blood supply stabilizing the fetoplacental physiological functions, and parturition.

Effect of parathyroid hormone-related protein (PTHrP) on the contractility of the myometrium and localization of PTHrP in the uterus of pregnant rats.

The effect of parathyroid hormone-related protein (PTHrP) on the contractility of uterine segments taken from pregnant rats and the localization of PTHrP in the uterus during pregnancy were investigated. PTHrP(1-34) had a potent inhibitory effect on spontaneous contractions of the longitudinal layer of uterine myometrium taken from rats at day 4 of pregnancy (IC50 1.6 nmol l-1). In low calcium De Jalon's solution, it also decreased base-line tension (IC50 1.5 nmol l-1) in a concentration-dependent manner. The effect of PTHrP(1-34) on uterine motility decreased as pregnancy progressed until day 13, after which PTHrP(1-34) had no measurable effect on uterine contractility. In contrast, PTHrP(1-34) had no effect on the contractions of the circular smooth muscle of the uterus at any stage of pregnancy. PTHrP(50-69) had no effect on the contractility of either muscle layer of the myometrium. A temporal pattern of staining for PTHrP in the uterus of pregnant rats was observed, and the changes in the staining patterns in the endometrium and myometrium were different in each layer. These data suggest that PTHrP may have at least two distinct roles in the uterus, a relaxing action on longitudinal muscle that depends on the hormonal state of the rat and a novel effect either intraluminally or within the endometrial layer.

Synthesis and secretion of an estrus stage-specific protein by rat uterus.

A specific protein with an estimated molecular weight of 260 kDa was found to be synthesized and secreted into the incubation medium by rat uterus only during the estrus stage of the cycle. This secreted uterine protein was designated as estrus stage-specific protein (ESP). ESP was not produced by pregnant, lactating or immature pup rat uteri. Estradiol administered to ovariectomized rats induced production of ESP which was blocked by the antiestrogen, ICI 182, 780. The present results show that the synthesis and secretion of ESP is regulated by estradiol and this protein maybe involved in blastocyst implantation.

Tenascin in pregnant and non-pregnant rat uterus: unique spatio-temporal expression during decidualization.

Tenascin is a large extracellular matrix (ECM) protein generally restricted to developing embryonic tissues and tissues undergoing remodelling in the adult, but it has been suggested that tenascin is immunomodulatory. In this study we have used indirect immunofluorescence to investigate the distribution of tenascin within the uteri of pregnant and non-pregnant rats. The non-pregnant uterus expressed tenascin in the stroma around the glands, but only the subluminal stroma showed cyclical variations. Dependence of the latter on progesterone was verified in ovariectomized animals supplemented with steroid hormones. During early pregnancy, the uterine circular smooth muscle maintained an intense expression of tenascin. In the decidualizing endometrium, tenascin expression showed dramatic changes that reflected the regionalization of the decidualization process. On Day 6 (blastocyst attachment), tenascin immunoreactivity was prominent in the primary decidualization zone immediately surrounding the conceptus. On Day 7, expression had increased within the secondary decidua that had developed throughout the antimesometrial endometrium. By Day 8, the antimesometrial decidua showed diminished tenascin immunoreactivity; expression became largely confined to the ECM of the developing decidua in the mesometrial region, where levels were high. On Day 10, the well-developed mesometrial decidua was negative. Interestingly, tenascin expression was up-regulated in the walls of the vessels in the metrial gland within the mesometrial triangle beginning on Day 10 of pregnancy. From these studies we conclude that tenascin expression may be an important reflection of the dynamics involved with tissue restructuring during early pregnancy and may play a role in immunomodulation, since expression of this protein in the mesometrial decidua coincides with the presence of differentiating natural killer cell lineage lymphocytes in the same region.

Expression of calbindin-D 9k in the early pregnant rat uterus: Effects of RU 486 and correlation to estrogen receptor mRNA

Calbindin-D 9k (CaBP-9k) is a calcium binding protein expressed at high levels in the rat uterus. The CaBP-9k gene carries an estrogen response element which is involved in the steroid hormone regulation of the gene during the estrous cycle and gestation. The present study was aimed at determining expression of the gene during the first half of pregnancy and to assess the role of progesterone (P 4) and the estrogen receptor (ER). Expression of CaBP-9k mRNA was determined by Northern blot analysis during the first 10 days of pregnancy. On pregnancy day 1 (P1), CaBP-9k mRNA levels were relatively high. On P2, 3 and 5 CaBP-9k mRNA decreased to the detection limit using 10 μg total RNA probed with a random primed cDNA. On P10, CaBP-9k transcripts began to reappear at levels of about 30% of P1. Expression of β-actin mRNA displayed a continuous increase during this period with a rapid rise of 240% between P2 and P3. The typical increase of P 4 accompanied by moderate changes of estradiol (E 2) was determined in serum of experimental groups. When RU 486 at 10 mg/kg was administered as a single s.c. injection on P3, the CaBP-9k down-regulation was rapidly interrupted and mRNA expression became extremely high. The effect was seen maximally at 24 h post injection and was maintained at 48 and 72 h. Expression of β-actin mRNA was increased only moderately at 24 h and was unchanged at 48 and 72 h. Serum P 4 remained unaffected by the treatment and E 2 displayed a slight increase. When ER mRNA was quantified by reverse transcription/PCR techniques a more than 300% increase was detected in the RU 486 treated rats. These data implicate that P 4 in early pregnancy is responsible for down regulation of the CaBP-9k gene in uterus via an indirect effect involving the expression of the estrogen receptor.

Possible roles of insulin and insulin-like growth factors in rat preimplantation development: investigation of gene expression by reverse transcription-polymerase chain reaction.

The sensitive mRNA phenotyping technique of reverse transcription-polymerase chain reaction was used to demonstrate that insulin receptor mRNA is present in rat embryos during the preimplantation period. In addition, mRNA encoding insulin-like growth factor (IGF) type I and type II receptors have also been detected in rat preimplantation embryos. IGF-I mRNA was not detected in preimplantation embryos but was found in oviducts and uteri of prepubertal and early pregnant rats. IGF-II mRNA was present in both embryos and in oviducts and uteri during the preimplantation period. These findings suggest that insulin and IGF-I could influence early embryo development in endocrine or in paracrine fashions, whereas IGF-II may have an additional autocrine mode of action in affecting preimplantation embryos in rats.

Comparative enzyme histochemistry of the early and term rat decidua with special attention to decidual regression

As the early rat decidua is believed to fulfil functions other than the late or basal decidua, the question as to whether this difference is reflected in decidual cell metabolism was investigated. Using cryosections of pregnant rat uteri of the 10th, 15th and 21st gestational day, activities of oxyradical-forming enzymes and hydrolases were analysed histochemically. The enzyme activities of decidual stromal cells and fibroblasts of the metrial gland exhibited three main fluctuations. One group of enzyme activities did not change during gestation, a second group decreased or disappeared, and a third group increased or was expressed in the late decidua only. Enzymes of the purine and polyamine pathway, including oxyradical-forming oxidases, were absent from early mesometrial decidual cells, but were highly active in the late regressing decidua and metrial gland. Some acid hydrolases and neutral proteases became active in the mature decidua. The possibility that purine-degrading and oxyradical-forming enzymes support decidual as well as metrial gland regression, and thus placental separation, by direct tissue damage and/or by indirect rupture of lysosomal membranes, inducing the release of acid hydrolases, is considered.