ATP-binding cassette transporters in reproduction: a new frontier.

BACKGROUND: The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS: This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS: ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as 'gatekeepers' at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1. CONCLUSIONS: The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors.

Effect of oxygen on multidrug resistance in the first trimester human placenta.

INTRODUCTION: The multidrug resistance proteins, P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP, encoded by ABCG2) are highly expressed in the first trimester placenta. These transporters protect the fetus from exposure to maternally derived toxins and xenobiotics. Since oxygen is a regulator of multidrug resistance in various tissues, we hypothesized that changes in oxygen tension alter placental ABCB1/P-gp and ABCG2/BCRP expression in the first trimester. METHODS: Placental specimens were collected from first (n = 7), second (n = 5) and term pregnancies (n = 5). First trimester placental villous explants were incubated (24 or 48 h) in different oxygen tension (3-20%). ABCB1, ABCG2 and VEGFA mRNA expression levels were assessed by RT-PCR and protein was localized by IHC. RESULTS: ABCB1 is expressed most highly in the first trimester placenta (p < 0.05), whereas ABCG2 expression does not change significantly over pregnancy. P-gp and BCRP staining is present in the syncytiotrophoblast and in cytotrophoblasts. ABCG2 mRNA is increased in hyperoxic (20%) conditions after 48 h (p < 0.05). In contrast, hypoxia (3%) did not change ABCB1 mRNA expression but significantly increased VEGFA mRNA (p < 0.05). Hypoxia resulted in increased BCRP staining in cytotrophoblasts and in the microvillous membrane of the syncytium. Whereas, hypoxia resulted in increased P-gp staining in proliferating cytotrophoblasts. CONCLUSION: We conclude that placental multidrug resistance expression, specifically ABCG2, is regulated by oxygen tension in the first trimester. It is possible that changes in placental oxygen supply are capable of altering fetal drug exposure especially during early pregnancy.

Fat mass and obesity-associated obesity-risk genotype is associated with lower foetal growth: an effect that is reversed in the offspring of smoking mothers.

Fat mass and obesity-associated (FTO) gene variants are associated with childhood and adult obesity; however, the influence of FTO polymorphisms on foetal growth is unknown. Associations between the FTO variant rs9939609 and the foetal growth trajectories, maternal pregnancy weight gain, anthropometric measures at birth and body mass index (BMI) at age 14 years were assessed in 1079 singleton-birth Australian Caucasians. Analyses were repeated in 3512 singleton-birth Dutch Caucasians. The rs9939609 obesity-risk AA genotype was associated with symmetrical intrauterine growth restriction; an effect reversed in mothers who smoked during pregnancy. The effect increased over time and was modified by maternal smoking for head circumference (P = 0.007), abdominal circumference (P = 0.007), femur length (P = 0.02) and estimated foetal weight (P = 0.001). The modification of the association between the AA genotype and birth anthropometrics by maternal smoking was consistent across birth weight (P = 0.01) and birth length (P = 0.04) and neonatal day 2 anthropometry. Consistent associations were replicated in the Generation R cohort. Maternal pregnancy weight gain matched the pattern of birth weight and was independent of placental weight. In adolescents, the AA genotype was associated with increased BMI-adjusted-for-age in males (P = 0.00009), but no effect was detected in females. A variant in the FTO gene influences foetal growth trajectories in the third trimester, early postnatal growth and adiposity in adolescence. Maternal smoking during pregnancy reversed the direction of association of rs9939609 on foetal growth, which was probably mediated by maternal energy intake. The detection of genetic variants associated with foetal growth has the potential to identify novel molecular mechanisms underlying growth and targeted early life intervention.

Heparin promotes soluble VEGF receptor expression in human placental villi to impair endothelial VEGF signaling.

BACKGROUND: Severe preeclampsia is characterized by hypertension, renal injury and placental dysfunction. Prothrombotic disorders are discovered in 10-20% of women with preeclampsia, providing the rationale for prescribing low-molecular-weight heparin (LMWH) in future pregnancies. Heparin has diverse molecular actions and appears to reduce the recurrence risk of preeclampsia in women without prothrombotic disorders. The placenta-derived anti-angiogenic splice-variant protein soluble vascular endothelial growth factor (VEGF) receptor-1 (sFLT1) is strongly implicated in the pathogenesis of the underlying endothelial dysfunction. As the placental syncytiotrophoblast is the principal source of sFLT1, we tested the hypothesis that heparin suppresses placental sFLT1 secretion. METHODS AND RESULTS: First trimester placental villi exposed to LMWH (0.25-25 IU mL(-1)) in an in vitro explant model significantly increased the expression and release of sFLT1 by the syncytiotrophoblast into culture media, reducing phosphorylation of FLT1 and KDR receptors in cultured human umbilical vein endothelial cells. This response was significantly diminished in placental villi from healthy term pregnancies. Placental villi from severely preeclamptic pregnancies had a higher baseline sFLT1 release, compared with first trimester placental villi and did not respond to LMWH treatment. LMWH promoted villous cytotrophoblast proliferation (BrdU incorporation) and impaired syncytial fusion-differentiation, causing syncytiotrophoblast apoptosis (by caspase 3&7 activity and TUNEL staining) and necrosis (ADP/ATP ratio). CONCLUSION: LMWH promotes sFLT1 synthesis and release from first trimester placental villi in a manner similar to that of severely preeclamptic placental villi, which antagonizes VEGF signaling in endothelial cells. These effects in part are mediated by an interaction between heparin and the cytotrophoblasts that regenerates the overlying syncytiotrophoblast responsible for sFLT1 secretion into the maternal blood.

HER1 signaling mediates extravillous trophoblast differentiation in humans.

This study examines the role of HER1 signaling in the differentiation of proliferative extravillous trophoblast (EVT) into invasive EVT. Using the JAR choriocarcinoma cell line and placental villous explants as experimental models and immunohistochemical assessment of protein markers of EVT differentiation (downregulation of HER1 and Cx40 and upregulation of HER2 and alpha1 integrin), we show that the ability of decidual conditioned medium (DCM) to induce HER1/2 switching was abrogated in the presence of the HER1 antagonist, AG1478. Similarly, epidermal growth factor (EGF) treatment resulted in the downregulation of HER1 and an upregulation of HER2 expression, whereas co-incubation of EGF with AG1478 inhibited this response. However, EGF did not downregulate Cx40 or induce migration of EVT. In contrast, heparin-binding epidermal-like growth factor (HBEGF) stimulated dose-dependent JAR cell migration, which was inhibited by both AG1478 and AG825 (HER2 antagonist). Western blot analysis of HER1 activation demonstrated that HBEGF-mediated phosphorylation of the HER1 Tyr992 and Tyr1068 sites, while EGF activated the Tyr1045 site. Moreover, HBEGF induced a stronger and more sustained activation of both the mitogen-activated protein kinase and phosphoinositol 3 kinase (PIK3) signaling pathways. Migration assays using a panel of signaling pathway inhibitors demonstrated that the HBEGF-mediated migration was dependent on the PIK3 pathway. These results demonstrate that HBEGF-mediated HER1 signaling through PIK3 is an important component of EVT invasion.

EGF modulates trophoblast migration through regulation of Connexin 40.

In this study we show that decidua conditioned media (DCM) downregulate Connexin 40 (C x 40) expression in extravillous trophoblast (EVT) outgrowths and can promote EVT differentiation to the invasive phenotype resulting in switching of integrin and EGF receptor expression. This suggests that growth factors secreted by the decidua, such as EGF, mediate trophoblast migration/invasion and may do so by modulating C x 40 expression and function. To test this hypothesis we have utilized migration assays using cell lines expressing C x 40. Migration assays were performed with Jeg-3, Jeg-3 overexpressing C x 40 (JpUHD) and JAR cells seeded on fibronectin-coated inserts with 8 microm pores and incubated in the absence or presence of serum-starved decidual cells. Cell migration was only observed in the presence of DCM. Conversely overexpression of C x 40 in Jeg-3 cells resulted in inhibition of cell migration as compared to wild-type control. Addition of DCM to cultured JAR cells resulted in the downregulation of C x 40 protein. EGF is known to stimulate trophoblast migration/invasion and was detected in DCM; therefore, we investigated the action of EGF on C x 40. EGF (10 ng/mL) resulted in the downregulation of C x 40 in the JAR cell line. However, EGF had no effect on JAR cell migration. We conclude that decidual secretion of growth factors, such as EGF, may act to prime trophoblast for migration/invasion through modulation of connexin expression and function.

Expression of breast cancer resistance protein (BCRP/ABCG2) in human placenta throughout gestation and at term before and after labor.

Breast cancer resistance protein, BCRP, is a multidrug resistance protein that is highly expressed in the human placenta. In cancer tissues, this protein actively extrudes a wide variety of chemically and structurally unrelated chemotherapeutic drugs and other compounds. Studies in mice have shown that in the absence of BCRP activity in the placenta, there is a 2-fold increase in the uptake in BCRP substrates into fetus. This suggests that in the placenta, BCRP extrudes compounds that would otherwise cross the syncytiotrophoblast cells into fetal circulation. The purpose of this study was to examine the expression and localization of BCRP in the human placenta throughout gestation. Tissues from 6-13, 16-19, 24-29, 32-35, and 38-41 weeks of gestation were used. Real time RT-PCR analysis demonstrated that the mRNA levels of BCRP in the placenta do not change significantly as gestation progressed. However, Western blot analysis revealed that the protein levels increased towards the end of gestation. We demonstrated that BCRP is localized to the syncytiotrophoblast of the placenta and in some fetal blood vessels within the placenta. Tissues from the early stages of pregnancy (6-13 weeks) showed fewer BCRP positive blood vessels than term tissues (38-41 weeks).

Intramolecular interactions between the AF3 domain and the C-terminus of the human progesterone receptor are mediated through two LXXLL motifs.

The human progesterone receptor (PR) exists in two major forms, PRA and PRB, which differentially regulate gene transcription in a cell- and promoter-specific manner. The molecular mechanisms underlying this differential transcriptional activity have been attributed to the presence of a unique AF3 domain within PRB that may result in the two isoforms adopting different protein conformations. We demonstrate here that in myometrial cells, PRB exhibits strong progesterone-dependent transcriptional activity that is dependent on the presence of two LXXLL motifs within the AF3 domain. In vitro and in vivo protein interaction assays indicate that these motifs mediate the direct interaction between the AF3 domain and C-PR in a progesterone-dependent manner. Mutation of either of the LXXLL motifs or deletion of the last 30 amino acids within the C-terminus disrupts this interaction and progesterone-dependent transcriptional activity of PRB. Members of the p160 family of co-activators (such as GRIP-1) also interact with C-PR through their LXXLL motifs. However, GRIP-1 does not compete with AF3 but rather acts to synergize these two transactivation domains. Our data suggest that a failure to form an appropriate AF3-C-terminus interaction results in an inability of co-activators to induce maximal PR-dependent transactivation. The absence of an AF3 domain within PRA may account for its inability to activate progesterone-responsive genes, as well as its actions as a dominant trans-repressor.

Effects of cortisol and oestradiol on hepatic 11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor proteins in late-gestation sheep fetus.

In the late-gestation sheep, increased fetal plasma cortisol concentration and placental oestradiol (E(2)) output contribute to fetal organ maturation, in addition to the onset of parturition. Both cortisol and E(2) are believed to regulate the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which interconverts bioactive 11-hydroxy glucocorticoids and their inactive 11-keto metabolites. 11beta-HSD1, abundantly expressed in fetal liver, operates primarily as a reductase enzyme to produce bioactive cortisol and thus regulates local hepatic glucocorticoid concentrations. Cortisol acts through the glucocorticoid receptor (GR) present in the liver. In this study, we examined the effects of cortisol and E(2) on hepatic 11beta-HSD1 and GR in the liver of chronically catheterized sheep fetuses treated with saline (n=5), cortisol (1.35 mg/h; n=5), saline+4-hydroxyandrostendione, a P450 aromatase inhibitor (4-OHA; 1.44 mg/h; n=5), or cortisol+4-OHA (n=5). Cortisol infusion resulted in increased plasma concentrations of fetal cortisol and E(2); concurrent administration of 4-OHA attenuated the increase in plasma E(2) concentrations. Using immunohistochemistry, we showed that fetal hepatocytes expressed both 11beta-HSD1 and GR proteins. Cortisol treatment increased GR in both cytosol and nuclei of hepatocytes; concurrent administration of 4-OHA was associated with distinct nuclear GR staining. Western blot revealed that cortisol, in the absence of increased E(2) concentrations, significantly increased concentrations of 11beta-HSD1 (34 kDa) and GR (95 kDa) proteins. 11beta-HSD1 enzyme activity was measured in the liver microsomal fraction in the presence of [(3)H]cortisone (10(-)(6) M) or [(3)H]cortisol (10(-)(6) M) and NADPH (reductase activity) or NADP(+) (dehydrogenase activity) respectively. 11beta-HSD1 reductase activity was significantly greater in the presence of cortisol. In summary, we found that, in sheep during late gestation, cortisol increased both 11beta-HSD1 and GR in the fetal liver, and these effects were accentuated in the absence of increased E(2).

Understanding preterm labor.

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis. In fetal sheep, increased cortisol output during pregnancy regulates prostaglandin H synthase type 2 (PGHS2) expression in the placenta in an estrogen-independent manner, resulting in increased levels of PGE2 in the fetal circulation. Later increases in maternal uterine expresssion of PGHS2 require elevations of estrogen and lead to increased concentrations of PGF2alpha in the maternal circulation. Thus, regulation of PGHS2 at term is differentially controlled in fetal (trophoblast) and maternal (uterine epithelium) tissue. This difference may reflect expression of the glucocorticoid receptor (GR), but not estrogen receptor (ER), in placental trophoblast cells. In women, cortisol also contributes to increased PG production in fetal tissues through upregulation of PGHS2 (amnion and chorion) and downregulation of 15-OH PG dehydrogenase (chorion trophoblasts). The effect of cortisol on chorion expression of PGDH reverses a tonic stimulatory effect of progesterone, potentially through a paracrine or autocrine action. We have interpreted this interaction as a reflection of "progesterone withdrawal" in the primate, in relation to birth. Other agents, such as proinflammatory cytokines, similarly upregulate PGHS2 and decrease expression of PGDH, indicating the presence of several mechanisms by which labor at term or preterm may be initiated. These different mechanisms need to be considered in the development of strategies for the detection and management of the patient in preterm labor.

Gadolinium chloride inhibits pulmonary macrophage influx and prevents O(2)-induced pulmonary hypertension in the neonatal rat.

Newborn rats exposed to 60% O(2) for 14 d demonstrated a bronchopulmonary dysplasia-like lung morphology and pulmonary hypertension. A 21-aminosteroid antioxidant, U74389G, attenuated both pulmonary hypertension and macrophage accumulation in the O(2)-exposed lungs. To determine whether macrophage accumulation played an essential role in the development of pulmonary hypertension in this model, pups were treated with gadolinium chloride (GdCl(3)) to reduce lung macrophage content. Treatment of 60% O(2)-exposed animals with GdCl(3) prevented right ventricular hypertrophy (p < 0.05) and smooth muscle hyperplasia around pulmonary vessels, but had no effect on morphologic changes in the lung parenchyma. In addition, GdCl(3) inhibited 60% O(2)-mediated increases in endothelin-1, 8-isoprostane, and nitrotyrosine residues. Organotypic cultures of fetal rat distal lung cells were subjected to cyclical mechanical strain to assess the potential role of GdCl(3)-induced blockade of stretch-mediated cation channels in these effects. Mechanical strain caused a moderate increase of endothelin-1 (p < 0.05), which was unaffected by GdCl(3), but had no effect on 8-isoprostane or nitric oxide synthesis. A critical role for endothelin-1 in O(2)-mediated pulmonary hypertension was confirmed using the combined endothelin receptor antagonist SB217242. We concluded that pulmonary macrophage accumulation, in response to 60% O(2), mediated pulmonary hypertension through up-regulation of endothelin-1.

Parathyroid hormone-induced up-regulation of connexin-43 messenger ribonucleic acid (mRNA) is mediated by sequences within both the promoter and the 3'untranslated region of the mRNA.

The gap junction protein connexin 43 (Cx43) mediates communication between osteoblasts and is important for maximal PTH responsiveness. We examined the role of the Cx43 promoter and messenger RNA 3' untranslated region (UTR) in conferring responsiveness to PTH in the rat osteosarcoma cell line UMR-106. PTH induced a 4-fold increase in luciferase activity of a reporter construct containing 1.6 kb 5' of the transcription start site. Deletion analysis of the promoter localized responsive sequences to between -31 to +1 bp. PTH treatment of transgenic mice containing the 1.6 kb promoter luciferase construct induced increases in luciferase and Cx43 immunoreactivity in bone cells underlying the tibial growth plate. The full Cx43 3'UTR conferred a 3-fold response to PTH when placed 3' of a CMV-luciferase construct. Deletion analysis localized responsive sequences to between 2510 and 3132 of the 3'UTR. Cloning of this segment 5' of the CMV promoter disrupted the PTH response, indicating this sequence does not function as an enhancer. Sequences within the promoter conferred responsiveness to forskolin, whereas the 3'UTR responded to both TPA and forskolin. These data indicate that PTH responsive sequences are present in the Cx43 promoter and 3'UTR, suggesting that transcriptional and posttranscriptional pathways operate to regulate PTH-induced Cx43 expression in osteoblast cells.

Regulation of connexin43 expression by c-fos and c-jun in myometrial cells.

Labor is associated with a dramatic increase in the myometrial expression of connexin43 (Cx43) which is thought to mediate myocyte contractile coupling. The transcription factor c-fos is also dramatically increased prior to the onset of labor and is therefore a potential regulator of Cx43. The promoter region of Cx43 contains a conserved activator protein-1 (AP-1) site, which binds dimers of Fos and Jun proteins. We constructed expression vectors for c-Fos and c-Jun to investigate the role of these transcription factors in the regulation of Cx43 expression. These expression vectors were then co-transfected into SHM (syrian hamster myocyte) cells with a Cx43 promoter-Luciferase vector. The combinations of c-Fos and c-Jun proteins activated the Cx43 promoter while c-Jun alone had no effect on Cx43 promoter activity. Mutation of the AP-1 site was found to reduce this responsiveness. These data indicate that the transcription factors c-Fos and c-Jun are important in the regulation of Cx43 expression.

Prostaglandin production at the onset of ovine parturition is regulated by both estrogen-independent and estrogen-dependent pathways.

A current hypothesis of ovine parturition proposes that fetal adrenal cortisol induces placental E2 production, which, in turn, triggers intrauterine PG production. However, recent evidence suggests that cortisol may directly increase PG production in trophoblast-derived tissues. To separate cortisol-dependent and estrogen-dependent PG production in sheep intrauterine tissues, we infused singleton, chronically catheterized fetuses beginning on day 125 of gestation (term, 147-150 days) with 1) cortisol (1.35 mg/h; n = 5); 2) cortisol and 4-hydroxyandrostendione, a P450aromatase inhibitor (4-OHA: 1.44 mg/h; n = 5); 3) saline (n = 5); or 4) saline and 4-OHA (n = 5). Fetal and maternal arterial blood samples were collected at 12-h intervals starting 24 h before infusion and continuing during treatment for 80 h or until active labor. Uterine contractility was measured by electromyogram recording of myometrial activity. Plasma E2, progesterone (P4), PGE2, and 13,14-dihydro- 15-keto-PGF2alpha were quantified by RIA. PGHS-II messenger RNA (mRNA) and protein expression were determined by in situ hybridization and Western blot analysis, respectively. Data were analyzed by ANOVA (P < or = 0.05). Labor-type uterine contractions were present after 68 h of cortisol infusion and had increased significantly by 80 h. Labor-type uterine contractions were induced after 68 h of cortisol plus 4-OHA infusion, but the contraction frequency remained less than that in the cortisol-treated animals. Fetal cortisol infusion increased fetal and maternal plasma E2 concentrations and decreased the maternal plasma P4 concentration significantly; concurrent 4-OHA infusion attenuated the increase in fetal and maternal plasma E2, but not the decrease in maternal plasma P4. The fetal plasma PGE2 concentration increased after both cortisol and cortisol plus 4-OHA infusion. The maternal plasma 13,14-dihydro-15-keto-PGF2alpha concentration rose after fetal cortisol infusion, but not after cortisol plus 4-OHA infusion. Placental trophoblast PGHS-II mRNA and protein expression were increased significantly after both cortisol and cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein expression increased after cortisol infusion, but not after cortisol plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine activity pattern, and intrauterine PGHS-II expression were not altered in either control group. We conclude that these data suggest distinct pathways of intrauterine PG synthesis: a cortisol-dependent/E2-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an E2-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2alpha and appears necessary for uterine activity and parturition.

Changes in expression of platelet-derived growth factor and its receptors in the lungs of newborn rats exposed to air or 60% O(2).

PDGF-related gene expression has been well characterized during fetal rat lung development and adult rat lung injury, but not during normal postnatal lung growth or injury. Lung expression of the mRNA for PDGF-A, -B, -alpha R, and -beta R and immunoreactive PDGF-AA, -BB, -alpha R, and -beta R were assessed in rat pups raised in air or 60% O(2) for up to 14 d after birth. Expression of mRNA and immunoreactive ligand did not correlate for pups raised in air. Immunoreactive PDGF-alpha R and -beta R, but not PDGF-AA and -BB, were evident throughout the lung at birth. Both PDGF-AA and -BB were evident in airway epithelium, PDGF-BB in alveolar epithelial cells and PDGF-AA was widely distributed in parenchymal tissue at 4 d. PDGF-alpha R was localized to airway epithelium, and PDGF-beta R to subendothelial perivascular regions and to airway and alveolar epithelium at 4 d. Immunoreactive PDGF ligands all declined after 4 d. Intraperitoneal injection of neutralizing antibodies or truncated soluble receptors to PDGF-BB reduced lung DNA synthesis in air. Exposure to 60% O(2) significantly increased mRNA for PDGF-B, -beta R, and -alpha R, but not PDGF-A, relative to air-exposed lung at various time points after birth. PDGF-A, -B, and -alpha R immunoreactivities in these lungs were reduced and delayed, consistent with a global inhibition of lung growth. Pups exposed to 60% O(2) had a similar distribution of PDGF-beta R to that seen in air, except that at 14 d PDGF-beta R was distributed throughout the lung parenchyma. We conclude that PDGF ligands and receptors are important for normal postnatal lung growth and that their expression is delayed by O(2) exposure.

Temporal expression of prostaglandin H synthase type 2 (PGHS-2) and P450(C17)in ovine placentomes with the natural onset of labour.

Labour in the sheep is preceded by increased tissue and plasma prostaglandin (PG) concentrations, and PGs could potentially contribute to the regulation of P450(C17)in placental tissue. Therefore, we determined the cellular localization and temporal pattern of expression of P450(C17)and prostaglandin H synthase type 2 (PGHS-2), the primary PG synthetic enzyme, in intrauterine tissues from three groups of pregnant ewes at term; animals not in labour (NIL;n=5; 140-145 days of gestation), animals in early labour (EL;n=6; 143-149 days) and animals in active labour (L;n=6; 145-149 days). Allocation of animals into the three groups was based on continuous monitoring and assessment of myometrial contractile activity (EMG) and changes in the intrauterine pressure (IUP). Levels of mRNA encoding PGHS-2 and P450C17 were determined by in situ hybridization. Localization and levels of immunoreactive (ir-) P450(C17)and ir-PGHS-2 protein were determined by immunohistochemistry and Western blotting. PGHS-2 mRNA and ir-PGHS-2 were already elevated in placentomes of NIL animals and did not increase further with the progression of labour, whereas P450C17 mRNA increased progressively with labour, and ir-P450C17 rose significantly only in animals in active labour. The rise in P450C17 expression corresponded temporally to a progressive increase in maternal plasma concentration of oestradiol. We suggest that the temporal relationship and subsequent co-localization of PGHS-2 and P450(C17)proteins in the uninucleate trophoblast cells of the placentomes are consistent with the possibility that placental PGs could act to enhance placental output of oestrogen leading to labour and delivery.

Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia.

During early pregnancy, placentation occurs in a relatively hypoxic environment which is essential for appropriate embryonic development. Intervillous blood flow increases at around 10-12 weeks of gestation and results in exposure of the trophoblast to increased oxygen tension (PO2). Prior to this time, low oxygen appears to prevent trophoblast differentiation towards an invasive phenotype. In other mammalian systems, oxygen tension effects are mediated by hypoxia inducible factor-1 (HIF-1). We found that the ontogeny of HIF-1alpha subunit expression during the first trimester of gestation parallels that of transforming growth factor-beta3 (TGFbeta3), an inhibitor of early trophoblast differentiation. Expression of both molecules is high in early pregnancy and falls at around 10 weeks of gestation when placental PO2 levels are believed to increase. Antisense-induced inhibition of HIF-1alpha inhibited the expression of TGFbeta3, and stimulated extravillous trophoblast (EVT) outgrowth and invasion. Of clinical significance we found that TGFbeta3 expression was increased in pre-eclamptic placentae when compared to age-matched controls. Significantly, inhibition of TGFbeta3 by antisense oligonucleotides or antibodies restored the invasive capability to the trophoblast cells in pre-eclamptic explants. We speculate that if oxygen tension fails to increase, or trophoblasts do not detect this increase, HIF-1alpha and TGFbeta3 expression remain high, resulting in shallow trophoblast invasion and predisposing the pregnancy to pre-eclampsia. Effective fetal-maternal interactions during early placentation are critical for a successful pregnancy. Optimal placental perfusion requires the controlled invasion of trophoblast cells deep into the decidua to the spiral arteries. Trophoblast stem cells, also referred to as cytotrophoblast cells, reside in chorionic villi of two types, floating and anchoring villi. Floating villi, which represent the vast majority of chorionic villi, are bathed in maternal blood and primarily perform gas and nutrient exchange for the developing embryo. During early placentation, cytotrophoblast cells in the floating villi proliferate and differentiate by fusing to form the multinucleate syncytiotrophoblast layer. Cytotrophoblast cells in anchoring villi either fuse to form the syncytiotrophoblast layer, or break through the syncytium at selected sites and form multilayered columns of non-polarized extravillous trophoblast cells, which physically connect the embryo to the uterine wall (Figure 1). The extravillous trophoblast cells invade into the uterine wall as far as the first third of the myometrium and its associated spiral arteries, where they disrupt the endothelium and the smooth muscle layer and replace the vascular wall. This results in the conversion of the narrow calibre arteries into distended uteroplacental arteries, thereby increasing blood flow to the placenta and allowing an adequate supply of oxygen and nutrients to the growing fetus. The invasive activity of the extravillous trophoblast cells is at a maximum during the first trimester of gestation, peaking at around 10-12 weeks and declining thereafter. Insufficient invasion contributes to the development of pre-eclampsia, which often results in fetal intrauterine growth restriction, maternal hypertension and proteinuria. In contrast, unrestricted invasion is associated with premalignant conditions, such as invasive mole, and with malignant choriocarcinoma. Invading trophoblast cells undergo striking and rapid changes in cellular functions that are temporally and spatially regulated along the invasive pathway (Figure 1) (Cross, Werb and Fisher, 1994. The formation of the anchoring villi is accompanied by changes in synthesis and degradation of extracellular matrix proteins and their receptors, and changes in the spatial distribution of extracellular matrix proteins, as well as changes in the expression of adhesion molecules (Damsky, Fitzgerald and

Expression and regulation of the messenger ribonucleic acid encoding the prostaglandin F(2alpha) receptor in the rat myometrium during pregnancy and labor.

OBJECTIVE: We examined expression of messenger ribonucleic acid encoding the prostaglandin F(2alpha) receptor in the rat myometrium throughout late gestation and its regulation by progesterone and mechanical stretch. STUDY DESIGN: Normal pregnant rats were killed on gestational day 15, 22, or 23 (during labor) or 1 day post partum. The effects of progesterone on prostaglandin F(2alpha) receptor messenger ribonucleic acid levels were investigated by daily injections of progesterone (4 mg) from day 20 of normal gestation or from day 17 in rats bilaterally ovariectomized on day 17. To investigate the effects of myometrial stretch, unilaterally pregnant rats underwent either sham surgery or placement of a polyvinyl tube 3 mm in diameter in the nongravid uterine horn on day 15 or 18 and were killed 5 days later. Prostaglandin F(2alpha) receptor messenger ribonucleic acid levels in the myometrium were determined by a semiquantitative reverse transcription-polymerase chain reaction method. RESULTS: Myometrial prostaglandin F(2alpha) receptor messenger ribonucleic acid levels significantly increased during both term and ovariectomy-induced preterm labor. This increase was blocked by progesterone. In rats with unilateral pregnancies prostaglandin F(2alpha) receptor messenger ribonucleic acid levels in the nongravid horns were similar to those in the contralateral gravid horns on day 20 and during labor regardless of whether they were stretched by a 3-mm tube. CONCLUSION: Increased myometrial expression of prostaglandin F(2alpha) receptor messenger ribonucleic acid during term and preterm labor is temporally associated with progesterone withdrawal but is not dependent on mechanical stretch of the myometrium.

Localization of prostaglandin synthase type-1 (PGHS-1) mRNA and prostaglandin synthase type-2 (PGHS-2) mRNA in ovine myometrium and endometrium throughout gestation.

Increased prostaglandin production by tissues in the sheep uterus and placenta are thought to be important for the onset of parturition. In the sheep placenta, this is most likely due to increased expression of prostaglandin synthase type-2 (PGHS-2) rather than prostaglandin synthase type-1 (PGHS-1). However, there is no information concerning expression of PGHS isoenzymes in maternal uterine tissues during pregnancy. Therefore, the purpose of the present study was to examine the expression of PGHS-1 and PGHS-2 in the sheep myometrium and endometrium during late gestation using in situ hybridization and immunohistochemistry. Using (35)S-labelled oligonucleotide probes, which give specific hybridization signals in other tissues, we localized PGHS-2 mRNA to endometrial epithelium, and apparently to other cells in both endometrium and myometrium. This artefactual signal was still present with 100-fold excess unlabelled oligonucleotide probe and with sense probes, but was resolved with the use of (33)P-oligonucleotides. Using (33)P-labelled oligonucleotide probes we could not detect either PGHS-1 or PGHS-2 mRNA in myometrium, and found expression only of PGHS-2 mRNA in endometrium. PGHS-2 mRNA localized to the endometrial epithelium and was undetectable in glandular epithelium. The level of PGHS-2 expression rose significantly between days 80 and 85 of pregnancy and term, and this corresponded to the appearance of immunoreactive PGHS-2 protein, measured by immunohistochemistry, in the endometrial epithelium. Therefore we conclude that (33)P-labelled probes are preferred for detection of mRNAs encoding PGHS-2 in ovine uterine tissues. Expression of PGHS-2 mRNA is greater than that of PGHS-1, increases during gestation, and predominates in the endometrial epithelium, consistent with the site of PGHS-2 protein localization.