Progesterone receptor expression declines in the guinea pig uterus during functional progesterone withdrawal and in response to prostaglandins.

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.

Mechanisms leading to increased risk of preterm birth in growth-restricted guinea pig pregnancies.

Intrauterine growth restriction (IUGR) is a risk factor for preterm labor; however, the mechanisms of the relationship remain unknown. Prostaglandin (PG), key stimulants of labor, availability is regulated by the synthetic enzymes, prostaglandin endoperoxidases 1 and 2 (PTGS1 and 2), and the metabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (HPGD). We hypothesized that IUGR increases susceptibility to preterm labor due to the changing balance of synthetic and metabolizing enzymes and hence greater PG availability. We have tested this hypothesis using a surgically induced IUGR model in guinea pigs, which results in significantly shorter gestation. Myometrium, amnion, chorion, and placentas were collected from sham operated or IUGR pregnancies, and PTGS1 and HPGD protein expression were quantified throughout late gestation (>62 days) and labor. The PTGS1 expression was significantly upregulated in the myometrium of IUGR animals, and chorionic HPGD expression was markedly decreased (P < .01 and P < .001, respectively). These findings suggest a shift in the balance of PG production over metabolism in IUGR pregnancies leads to a greater susceptibility to preterm birth.

15-Hydroxyprostaglandin dehydrogenase expression and localization in guinea pig gestational tissues during late pregnancy and parturition.

Prostaglandins are key components of the parturition cascade; however, the mechanisms that regulate prostaglandin concentrations in the uterus during pregnancy are largely unknown. The purpose of this study was to determine the intrauterine expression of the chief prostaglandin-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase (PGDH), during gestation and labor in the guinea pig, an animal model in which the endocrine control of pregnancy and parturition is analogous to that of women. PGDH messenger RNA (mRNA) abundance decreased significantly in the visceral yolk sac membrane (VYS, the anatomical equivalent of the human chorion laeve) and the amnion throughout the last third of pregnancy. PGDH protein was robustly expressed in the VYS epithelium and mesoderm, correlated strongly with PGDH mRNA levels and exhibited a nadir at term prior to labor onset. PGDH protein was not detected in the amnion. PGDH mRNA and protein levels in the placenta and myoendometrium were variable throughout late gestation. In the placenta, PGDH protein was concentrated in the parietal yolk sac membrane (PYS) lining the placental surface and in placental blood vessels. We observed strong expression of PGDH protein in the endometrial epithelium with comparably little expression in the myometrium. These data indicate that metabolic inactivation of prostaglandins in the pregnant guinea pig uterus takes place in the VYS, PYS, and endometrium. Decreased PGDH expression in the fetal membranes may contribute to the increase in intrauterine prostaglandin concentrations at term, stimulating the onset of labor.

Estrogen receptor (ER) expression and function in the pregnant human myometrium: estradiol via ERα activates ERK1/2 signaling in term myometrium.

Estrogens are thought to promote labor by increasing the expression of pro-contraction genes in myometrial cells. The specific estrogen receptors ((ERs: ERα and ERß (also known as ESR1 and ESR2)) and G protein-coupled receptor 30 (GPR30; also known as G protein-coupled estrogen receptor 1)) and signaling pathways that mediate these actions are not clearly understood. In this study, we identified the ERs expressed in the pregnant human myometrium and determined a key extranuclear signaling pathway through which estradiol (E(2)) modulates expression of the gene encoding the oxytocin receptor (OXTR), a major pro-contraction protein. Using quantitative RT-PCR, we found that ERα and GPR30 mRNAs were expressed in the human pregnant myometrium while ERß mRNA was virtually undetectable. While mRNA encoding ERα was the predominant ER transcript in the pregnant myometrium, ERα protein was largely undetectable in myometrial tissue by immunoblotting. Pharmacological inhibition of 26S proteasome activity increased ERα protein abundance to detectable levels in term myometrial explants, however, indicating rapid turnover of ERα protein by proteasomal processing in the pregnant myometrium. E(2) stimulated rapid extranuclear signaling in myometrial explants, as evidenced by increased extracellularly regulated kinase (ERK1/2) phosphorylation within 10 min. This effect was inhibited by pre-treatment with an ER antagonist, ICI 182 780, indicating the involvement of ERα. Inhibition of ERK signaling abrogated the ability of E(2) to stimulate OXTR gene expression in myometrial explants. We conclude that estrogenic actions in the human myometrium during pregnancy, including the stimulation of contraction-associated gene expression, can be mediated by extranuclear signaling through ERα via activation of the ERK/mitogen-activated protein kinase pathway.

Nuclear progesterone receptor expression in the human fetal membranes and decidua at term before and after labor.

To explore how progesterone affects human pregnancy, we identified the progesterone target cells within the fetal membranes (amnion, chorion, and decidua) at term by assessing the extent of expression and localization of the nuclear progesterone receptors, progesterone receptor-A and progesterone receptor-B. Fetal membranes (separated into amnion and chorion-decidua) were obtained after term cesarean deliveries performed before (n = 7) and after (n = 7) labor onset. Nuclear progesterone receptor expression was determined by the abundance of nuclear progesterone receptor mRNAs (by quantitative reverse transcriptase-polymerase chain reaction) and proteins (by western blotting). Localization of nPRs was determined by immunohistochemistry. Progesterone receptor-A and progesterone receptor-B mRNA and protein levels were highest in the chorion-decidua and did not change in association with labor. Nuclear progesterone receptor mRNAs and proteins were barely detectable in amnion. Nuclear progesterone receptor immunostaining was detected only in the nucleus of decidual cells. These findings suggest that the decidua, and not the amnion and chorion, is a direct target for nuclear progesterone receptor-mediated progesterone actions during human pregnancy.

Steroid hormone control of myometrial contractility and parturition.

The precise temporal control of uterine contractility is essential for the success of pregnancy. For most of pregnancy, progesterone acting through genomic and non-genomic mechanisms promotes myometrial relaxation. At parturition the relaxatory actions of progesterone are nullified and the combined stimulatory actions of estrogens and other factors such as myometrial distention and immune/inflammatory cytokines, transform the myometrium to a highly contractile and excitable state leading to labor and delivery. This review addresses current understanding of how progesterone and estrogens affect the contractility of the pregnancy myometrium and how their actions are coordinated and controlled as part of the parturition cascade.

Optimization of a solid phase extraction procedure for prostaglandin E2, F2 alpha and their tissue metabolites.

The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.

Nuclear progesterone receptors in the human pregnancy myometrium: evidence that parturition involves functional progesterone withdrawal mediated by increased expression of progesterone receptor-A.

CONTEXT: We examined whether human parturition involves functional progesterone withdrawal mediated by changes in myometrial expression of progesterone receptors (PRs)-A and -B. OBJECTIVE: Our objectives were to: 1) measure PR-A and PR-B protein levels in human pregnancy myometrium and determine whether the PR-A to PR-B ratio changes with advancing gestation and labor onset; and 2) determine how changes in the PR-A to PR-B ratio affect myometrial cell progesterone responsiveness. DESIGN: PR protein levels and cellular localization were measured by Western blotting and immunohistochemistry, respectively, in lower uterine segment uterine wall tissue from preterm (<37 wk; not laboring; n = 5) and term (37-40 wk; not in labor: n = 6; in labor: n = 5) cesarean delivery. The capacity for PR-A and PR-B, alone and in combination, to mediate genomic progesterone responsiveness measured by the activity of a progesterone-responsive reporter plasmid was examined by artificially modulating their levels in the PHM1-31 myometrial cell line. RESULTS: PR-A and PR-B immunostaining was detected only in the nucleus of myometrial cells. The PR-A to PR-B protein ratio was 0.49 +/- 0.082 (mean +/- sem) in preterm tissue; increased to 1.03 +/- 0.071 (P < 0.001) in nonlaboring term tissue; and increased further to 2.65 +/- 0.344 (P < 0.001) in laboring term tissue. Only PR-B mediated progesterone-induced transcriptional activity. PR-A had no effect alone but markedly decreased PR-B-mediated progesterone responsiveness. CONCLUSIONS: Functional progesterone withdrawal in human parturition may be mediated by an increase in the myometrial PR-A to PR-B ratio due to increased PR-A expression.

Virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation.

OBJECTIVES: We aimed to characterise and quantify the incidence of common infectious agents in acute exacerbations of chronic obstructive pulmonary disease (COPD) requiring ventilation, with a focus on respiratory viruses. DESIGN: An epidemiological study conducted over 3 years. SETTING: A 12-bed intensive care unit (ICU). PARTICIPANTS: ICU patients over 45 years of age with a primary diagnosis of COPD exacerbation requiring non-invasive ventilation (NIV) or ventilation via endotracheal tube (ETT). MATERIALS AND METHODS: Nasopharyngeal aspirates (NPA) and posterior pharyngeal swabs (PS) were tested for viruses with immunofluorescence assay (IFA), virus culture (VC) and polymerase chain reaction (PCR). Paired virus and atypical pneumonia serology assays were taken. Blood, sputum and endotracheal aspirates were cultured for bacteria. RESULTS: 107 episodes in 105 patients were recorded. Twenty-three (21%) died within 28[Symbol: see text]days. A probable infectious aetiology was found in 69 patient episodes (64%). A virus was identified in 46 cases (43%), being the sole organism in 35 cases (33%) and part of a mixed infection in 11 cases (10%). A probable bacterial aetiology was found in 25 cases (23%). There was no statistically significant difference in clinical characteristics or outcomes between the group with virus infections and that without. CONCLUSION: Forty-six (43%) of the patients with COPD exacerbation requiring mechanical ventilation had a probable viral pathogen. Prodromal, clinical and outcome parameters did not distinguish virus from non-virus illness. PCR was the most sensitive whilst virus culture was the least of virus assays.

Activation of the lutropin/choriogonadotropin receptor in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase by a pathway that involves Src family kinases.

We show that activation of the endogenous or recombinant lutropin/choriogonadotropin receptor (LHR) in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated, we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577, but it does not affect the phosphorylation of Tyr397, Tyr861, or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes, and overexpression of either of these two tyrosine kinases enhances the LHR-mediated phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Galpha-subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways.

Prostaglandin H2 synthase-1 and -2 expression in guinea pig gestational tissues during late pregnancy and parturition.

Increased intrauterine prostaglandin (PG) production is crucial for the initiation of parturition. To investigate the mechanisms controlling intrauterine PG synthesis, we examined the expression of the key PG biosynthetic isoenzymes, PG-H2 synthase (PTGS)-1 and -2, in the amnion, visceral yolk sac (VYS), placenta and myo-endometrium of pregnant guinea pigs. This animal model was chosen because the hormonal milieu of pregnancy and the role of PGs in the hormonal control of parturition are similar to those in the human. PTGS1 mRNA abundance, measured by real-time RT-PCR, increased in the amnion and the placenta during the last third of gestation. During labour, PTGS1 mRNA levels decreased precipitously in all four tissues. PTGS1 protein abundance, assessed by immunoblotting, increased to high levels in the amnion and the placenta by the end of pregnancy and remained high during labour. PTGS2 mRNA expression was higher in the placenta than in the other tissues, but did not change before and during labour. PTGS2 protein expression decreased in the placenta and remained low in the other tissues during labour. Immunohistochemistry showed pervasive PTGS1 protein expression in the amnion and strong expression in the parietal yolk sac membrane (PYS) covering the placenta. PTGS2 was expressed in the PYS and the endometrium. The PTGS inhibitor piroxicam, administered in doses that inhibited PTGS1 but not PTGS2, significantly prolonged gestation. These data suggest that PGs generated by intrauterine PTGS1 are involved in the timing of birth in guinea pigs. The induction of PTGS1 in the amnion and the PYS is a critical event leading to labour in guinea pigs and models analogous changes in the human gestational tissues before labour.