Browsing by Subject "Uterus"
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Item Characterization and estrogen regulation of uterine growth factor activity(Texas Tech University, 1988-08) Beck, CandaceEstradiol-17B has been shown to stimulate glucose transport, measured by phosphorylation of 2-deoxyglucose, and stimulate DNA synthesis, as measured by 3H-thymidine incorporation, In rat uterus in vivo. Attempts to demonstrate these responses in uterine cells in primary culture and in uterine tumor cells In culture have been unsuccessful. These observations led to the hypothesis that some responses to estradiol are mediated through the local action of peptide factors. Acid extracts of rat, bovine and rabbit uterus stimulated glucose transport and DNA synthesis in uterine tumor cells and in primary cultures of rat uterine cells. The stimulation of glucose transport was of the same magnitude (1.5-to 3.0-fold) and followed the same time course (maximum stimulation at 2-3 h) as estradiol stimulation in vivo. Uteri from estradiol treated rats contained 4 times more glucose transport-stimulating activity as did control rat uteri. The activity was acid and heat stable, was inactivated by trypsin, but not removed by dextran-coated charcoal treatment. The activity eluted in 6-12 kDa range on Sephadex G-50. DNA synthetic activity in rat uterine homogenates was elevated 3-fold within 18-24 h after estradiol injection and remained elevated with subsequent Injections. The growth-promoting activity was acid and heat stable, was reduced by trypsin but not reduced by treatment with dextran-coated charcoal. Gel filtration showed molecular weight heterogeneity with activity eluting at MW 10,000-30,000. The effect of purified growth factors on DNA synthesis in primary cultures of rat uterine cells was examined. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-B (TGFB) had no effect on 3H-thymidine incorporation under optimal conditions of incorporation. Relaxin and multiplication stimulating activity (MSA) demonstrated a stimulatory effect only at high concentrations, 207% of control at 100 |ag/ml and 175% of control at 100 ng/ml, respectively. Insulin stimulated incorporation 350% at 100 ng/ml, insulinlike growth factor-l (IGF-I) stimulated incorporation 300-400% at 10-100 g/ml, and platelet-derived growth factor (PDGF)stimulated incorporation 450% at 3 units/ml. When the positive effectors (insulin, IGF-I, MSA, and PDGF) were analyzed either combined or individually in the presence of uterine extract, the level of stimulation was greater than the maximum stimulation bserved with extract alone and approached that seen with 10% serum. Uterine extracts from estradiol treated and control rats were analyzed for IGF-I by radioimmunoassay. IGF-I was elevated 500-1000% in estradiol treated extracts relative to control levels. In summary, estradiol increases the growthpromoting activity and glucose transport-stimulating activity of uterine extracts. IGF-I was a positive stimulator of DNA synthesis in primary uterine cultures and was elevated in uterine extracts. This growth factor may be involved in the stimulation of DNA synthesis In rat uterus by estradiol.Item Estradiol stimulation of glucose transport in rat uterus(Texas Tech University, 1985-08) Meier, Daniel AlanA study of the mechanism of the estradiol-mediated increase in glucose transport in the uteri of ovariectomized rats was undertaken. An essential first step in this study was the characterization of the glucose transport process using plasma membrane vesicles. Uterine plasma membrane preparations were obtained by centrifugation on discontinuous sucrose gradients. The specific activity of the plasma membrane marker 5'-nucleotidase was increased 10-fold while the specific activity of an endoplasmic reticulum marker glucose-6-phosphatase was increased 3-fold. D-Glucose transport into plasma membrane vesicles was inhibited by sulfhydryl reagents, phloretin, and cytochalasin B. Uptake was prevented by high osmotic pressures. The Km of glucose transport was 12.2 +_ 1.1 mM. Transport was unaffected by sodium and was energy independent. 2-Deoxyglucose transport was determined in uteri of rats grouped by stages of the reproductive cycle, i.e., diestrus 1, diestrus 2, proestrus and estrus. The rate of 2-deoxyglucose transport was highest in proestrus and lowest in diestrus 1. The increase in glucose transport in ovariectomized rats was half-maximal at approximately 5 ng estradiol/rat and reached the maximal 2 to 3-fold response after 2 hours whether measured in whole tissue or in uterine plasma membrane vesicles. Estrone and estriol treatment resulted in a similiar 2-fold increase in glucose transport while progesterone and dihydrotestosterone had no effect. Injection of protein synthesis inhibitors cycloheximide and emetine resulted in an increase in the basal glucose transport rate while having no effect on the estradiolstimulated increase in glucose transport. Treatment with the transcriptional inhibitor actinomycin D resulted in a slight increase in glucose transport with no effect on the estradiol-stimulated increase in glucose transport. Antiestrogen treatment (nafoxidine and tamoxifen) resulted in a 85-90 % decrease in the total estradiol binding sites in the cytosol but did not effect the estradiol stimulated increase in 2-deoxyglucose transport in uterine tissue. Insulin injection (0.2 mg/rat) resulted in a 40 % increase in 2-deoxyglucose transport in 24 h-starved rats in contrast to the 200 % increase seen with estradiol treatment. Insulin and estradiol treatment together were not additive in regard to the increase in 2-deoxyglucose transport in tissue. Estradiol treatment did not change binding of [1251 ] insulin to uterine plasma membranes. Estradiol treatment resulted in a 3-fold increase in the Vmax with no apparent change in the Km for 2-deoxyglucose transport. Also, estradiol treatment did not result in an increase in the amount of glucose transporters in uterine plasma membranes as measured by antibodies raised against the glucose transporter protein from human erythrocytes. In summary, estradiol stimulates the rate of glucose transport in rat uterus by increasing the rate by which the transporter protein moves glucose across the plasma membrane .Item Parathyroid hormone and uterine contraction(Texas Tech University, 1981-08) Shew, Ronald LewisThe purpose of the present investigation was to examine the effect of synthetic bovine parathyroid hormone [bPTH-(l-34)] on the contraction of rat uterine horns iii vitro. The studies were designed: 1) to examine the effect of bPTH-(.l-34) on uterine contraction stimulated by various contractile agonists as well as by electrical stimulation, 2) to determine whether the uterine response to bPTH-(l-34) treatment was specific or merely due to the addition of polypeptide molecules to the in vitro tissue bath, 3) to investigate the effect of oxidation of bPTH-(l-34) molecule on the observed uterine response to the hormone, 4) to determine if the action of bPTH-(,l-34) on stimulated uterine contraction was direct or indirect and 5) to examine the possible mechanism by which bPTH-(.l-34) alters uterine contraction. Synthetic bPTH- (1-34) significantly diminished the magnitude of uterine contraction initiated by oxytocin, prostaglandin F-a (PGF a), acetylcholine (Ach) or electrical stimulation. In contrast, bovine serxim albumin (BSA), corticotropin inhibiting peptide (CIP) and salmon calcitonin (sCT) did not reduce oxytocin stimulated uterine contraction. Synthetic bPTH-(1-34) obtained from two different sources both diminished the magnitude of uterine contraction stimulated by oxytocin. Oxidation of bPTH-(1-34) by hydrogen peroxide destroyed the ability of the hormone to diminish oxytocin stimulated uterine contraction. However the effect of bPTH-(.l-34) was not blocked or reversed by anticholinergic (atropine), antiadrenergic (a-phentolamine, g-propranolol), or antihistaminergic (H1-pyrilamine, H2-cimetidine) drugs. Similarly, indomethacin, a prostaglandin synthetase inhibitor, also did not inhibit the hormone's effect. The effect of bPTH-(l-34) was enhanced by theophylline and 1-methyl-3-i3obutylxanthine (MIX) and was partially destroyed with imidazole. In addition, N 0 -dibutyryl-adenosine-3', 5'-cyclic phosphate also reduced oxytocin stimulated contraction. Finally, bPTH-(l-34) was shown to increase the levels of radioimmunoassable cyclic-3', 5'-adenosine monophosphate (cAI'lP) in uterine tissue. These results suggest that: 1) bPTH-(l-34) is capable of reducing uterine contraction stimulated by a variety of contractile agonists, 2) the effect of bPTH-(1-34) on uterine contraction was not caused by a contaminant in the hormone preparation nor due to a general action of polypeptides on uterine contraction in vitro, 3) alteration of the structural configuration of bPTH-(l-34) by H_0_ oxidation effectively abolishes the effect on uterine contraction, 4) the bPTH-(.l-34) effect is due to the direct action of the hormone on uterine tissue, and 5) the effect of bPTH- (1-34) may be mediated by cyclic-AMP.Item The role of ovine betaretroviruses in uteroplacental function(2009-06-02) Dunlap, Kathrin AnsonEndogenous retroviruses (ERVs) account for a substantial portion of the genetic pool of every animal species (e.g. ~ 8% of the human genome). Despite their overwhelming abundance in nature, many questions on the basic biology of ERVs are unanswered. Sheep harbor approximately 20 copies of endogenous betaretroviruses (enJSRVs), which are related to an exogenous oncogenic virus, Jaagsiekte sheep retrovirus (JSRV). Therefore, they are an attractive model for investigation of the potential beneficial roles of ERVs in reproductive biology. Studies were conducted to determine: 1) expression of enJSRVs envelope (env) and HYAL2 mRNAs in the ovine uterus and conceptus (embryo/fetus and extraembryonic membranes) throughout gestation; 2) regulation of enJSRVs expression by progesterone; and 3) the role of enJSRVs in regulating peri-implantation placental growth and differentiation. Study One determined the localization of enJSRVs env and HYAL2 mRNAs throughout gestation. Results demonstrate that alterations in expression of enJSRVs and HYAL2 in the sheep uterus and placenta suggest the probability of a variety of physiological roles in implantation and placentation. Partial sequencing of the transcriptionally active enJSRVs from ovine uteroplacental tissues revealed expression of multiple enJSRV loci. Study Two assessed the influence of progesterone, interferon tau, and pregnancy stage on enJSRVs expression, as an effort to understand factors that may regulate enJSRVs. Results of this study support the hypothesis that expression of enJSRVs is modulated by progesterone, but not IFN? in vivo. Study Three provides for enJSRVs regulating trophectoderm growth and differentiation in the peri-implantation conceptus. Blocking conceptus enJSRVs Env expression compromised pregnancy by retarding trophoblast outgrowth and differentiation. Inhibition of enJSRVs Env in vitro also reduced proliferation of mononuclear trophectoderm cells. Consequently, these results demonstrate that enJSRVs Env regulates trophectoderm growth and differentiation in the ovine conceptus, strongly supporting the biological significance of ERVs in placental evolution and animal reproduction Collectively, these studies illustrate that enJSRVs play an integral role in success of pregnancy. While the definitive roles of the enJSRVs have not yet been elucidated, it is evident that enJSRVs are an important component of the ovine genome and that they influence recognition and maintenance of pregnancy and placental formation.Item The roles of estradiol-17 beta and prolactin in uterine gland development in the neonatal ewe(Texas A&M University, 2005-11-01) Carpenter, Karen DeniseEndometrial glands are required for adult uterine function and develop post-natally in mammalian species. Therefore, studies were conducted using neonatal ewes as a model to determine: 1) the roles of estradiol-17-alpha and estrogen receptor-alpha (ER-beta) in endometrial gland development; 2) the role of ovaries in endometrial gland development; 3) the role of prolactin in endometrial gland development; and 4) factors regulating prolactin receptor expression in endometrial glands. Study one determined the effects of neonatal exposure of ewes to estradiol-17-alpha valerate (EV); EM-800, an ER-beta antagonist; or CGS-20267, an aromatase inhibitor on endometrial gland development. Results indicate E2-17-alpha does not regulate endometrial gland differentiation or development. Additionally, ER-beta does not regulate primary differentiation of glandular epithelium, but does influence coiling and branching morphogenesis of endometrial glands. Study two determined the effects of ovariectomy on endometrial gland morphogenesis. Results suggest that the ovary and, thus, an ovarian-derived factor(s) regulate, in part, the coiling and branching of endometrial glands. Expression of subunits of activin, follistatin, and inhibin in the neonatal ovine ovary in addition to modulation of the components of the activin/follistatin system in the uterus of ovariectomized ewes supports the hypothesis that the ovarian factors that influence endometrial adenogenesis in the neonatal ewe may be activin, follistatin, and/or inhibin. Studies three and four determined the role of prolactin in endometrial adenogenesis in the neonatal ewe. Studies in which either hypoprolactinemia or hyperprolactinemia were induced indicate that prolactin regulates ovine endometrial adenogenesis in the neonatal ewe. The aim of study five was to determine transcription factors that regulate the glandular epithelium specific expression of prolactin receptor. Prolactin receptor exon 2 was cloned and sequenced, but no identifiable exon 1 or promoter was found. Additionally, many bovine contigs containing portions of the prolactin receptor gene were identified suggesting the bovine genome will be a useful tool as it becomes more complete. These results indicate ER-beta, prolactin and prolactin receptor, along with an unidentified ovarian factor(s), influence endometrial gland development in the neonatal ewe; however, exposure of the neonatal ewe to exogenous estradiol-17-alpha prevents differentiation and development of endometrial glands.