Toni Welsh

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.

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 ERβ (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 ERβ 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.

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.

Separation of bioactive prostaglandins and their metabolites by reversed-phase thin-layer chromatography

Prostaglandins are biological lipid mediators that control a variety of physiological and pathophysiological processes. They function as locally acting hormones and are continuously synthesized and inactivated in target tissues by respective biosynthetic and metabolic enzymes. The separation of prostaglandins and their metabolites is crucial for study of their roles in biological systems. In this article, we describe systematic studies of the retention and separation of prostaglandin E2 (PGE2) from its inactive metabolites 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2. We also describe the separation of a second prostaglandin species, PGF2α, from its metabolites 15-keto-PGF2α and 13,14-dihydro-15-keto-PGF2α. Chromatography was performed on RP-18W high-performance thin-layer chromatographic plates with binary mobile phases comprising water and an organic modifier (methanol, ethanol, acetonitrile, acetone, or tetrahydrofuran). The experimental data revealed that at constant temperature, plots of the retention, RM, of the prostaglandins against the mole fraction of organic modifier in the water (XS) were nonlinear. By use of mobile phases of similar eluent strength the effect of temperature on the retention and separation of the solutes was examined. Thermodynamic data, calculated from linear Van’t Hoff plots, indicated that the mechanism of retention for each prostaglandin compound was consistent for the different mobile phases. The separation of PGE2 and PGF2α from each other and from their respective metabolites was rapid and robust in acetonitrile and in acetonitrile—water mobile phases. Interestingly, with 100% acetonitrile as the mobile phase the reversed-phase system apparently performed as a normal-phase system. The chromatographic system described here is applicable to the rapid processing of a large number of samples, which is a requirement for determining the biological metabolism of PGE2 and PGF2α and for studying the kinetics of prostaglandin-inactivating enzymes. The system is also suitable for the prepurification of complex biological samples for subsequent quantification of individual prostaglandins.

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.

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.

PROGESTERONE AND THE CONTROL OF HUMAN PREGNANCY AND PARTURITION

Almost 80 years ago George Corner and colleagues provided the first evidence that progesterone maintains pregnancy and that it does so, at least in part, by promoting myometrial relaxation. In the 1950s, Arpad Csapo proposed the “progesterone block hypothesis”, which posits that progesterone maintains pregnancy by promoting myometrial relaxation and that its withdrawal initiates a cascade of hormonal interactions that transforms the myometrium to a highly contractile state leading to the onset of labour. Csapo later proposed that contractility of the pregnant myometrium is determined by the balance between relaxation induced by progesterone and contraction induced by a cohort of signals including oestrogens, uterine distention and stimulatory uterotonins such as prostaglandins (PGs) and oxytocin (OT). According to this “seesaw” hypothesis, progesterone promotes myometrial relaxation by directly inducing relaxation and/or by inhibiting the production of, or myometrial responsiveness to, stimulatory uterotonins. These landmark concepts, though derived from studies of experimental animals, form the foundation for current understanding of progesterones role in the physiology of human pregnancy. Remarkable progress has been made over the last 20–30 years in understanding the signal transduction pathways through which steroid hormones affect target cells. This knowledge has broadened the scope of Csapos original paradigms and we are now beginning to unravel the specific signaling pathways and molecular interactions by which progesterone affects human myometrium and how its actions are controlled at the functional level. This is important for the development of progestin-based therapeutics for the prevention or suppression of preterm labour and preterm birth. Here we review recent progress in understanding the mechanisms by which progesterone sustains pregnancy and in particular how it promotes myometrial relaxation, how its relaxatory actions are nullified at parturition, and the hormonal interactions that induce progesterone withdrawal to determine the timing of human birth.