Differentiation of patient-specific void urine-derived human induced pluripotent stem cells to fibroblasts and skeletal muscle myocytes.

Cell-based therapy is a major focus for treatment of stress urinary incontinence (SUI). However, derivation of primary cells requires tissue biopsies, which often have adverse effects on patients. A recent study used human induced pluripotent stem cells (iPSC)-derived smooth muscle myocytes for urethral sphincter regeneration in rats. Here, we establish a workflow using iPSC-derived fibroblasts and skeletal myocytes for urethral tissue regeneration: (1) Cells from voided urine of women were reprogrammed into iPSC. (2) The iPSC line U1 and hESC line H9 (control) were differentiated into fibroblasts expressing FSP1, TE7, vinculin, vimentin, αSMA, fibronectin and paxillin. (3) Myogenic differentiation of U1 and H9 was induced by small molecule CHIR99021 and confirmed by protein expression of myogenic factors PAX7, MYOD, MYOG, and MF20. Striated muscle cells enriched by FACS expressed NCAM1, TITIN, DESMIN, TNNT3. (4) Human iPSC-derived fibroblasts and myocytes were engrafted into the periurethral region of RNU rats. Injected cells were labelled with ferric nanoparticles and traced by Prussian Blue stain, human-specific nuclear protein KU80, and human anti-mitochondria antibody. This workflow allows the scalable derivation, culture, and in vivo tracing of patient-specific fibroblasts and myocytes, which can be assessed in rat SUI models to regenerate urethral damages and restore continence.

Urban young women's preferences for intervention strategies to promote physical and mental health preconception: A Healthy Life Trajectories Initiative (HeLTI).

This study aimed to qualitatively investigate young women's preferences for preconception intervention strategies to promote physical and mental health in a rapidly transitioning, urban setting. Four semi-structured focus group discussions were conducted with young women (n = 29, 18-24 years old) from Soweto, South Africa. Qualitative data were thematically analysed. Two main themes were identified: 1) challenges and needs of intervention beneficiaries; and 2) preferences for intervention strategies (content and delivery). The challenges participants mentioned could be classified as those relating to social pressure, identity, and socioeconomic circumstances. Mental health support appeared to be a greater need than physical health, and this featured in their preferences for intervention content, although a number of physical health topics were also mentioned (healthy eating and contraception). Participants had mixed preferences for intervention materials, ranging from printed to electronic and mobile resources. Their preferences for intervention activities ranged from educational sessions, to fun and interactive practical activities, and activities they could take home. Community health workers (CHWs) were the preferred agent of delivery for interventions, though participants emphasised the importance of CHWs having appropriate interpersonal skills and own life experience. Some women preferred one-on-one sessions with a CHW, while others preferred group sessions. While recognising the value of family sessions, young women were less enthusiastic about this approach. These findings provide valuable formative data for developing effective interventions to optimise young women's preconception health in urban Africa. These contextual realities should be acknowledged when addressing key physical and mental health issues facing young women.

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.

Dynamic changes in maternal decidual leukocyte populations from first to second trimester gestation.

INTRODUCTION: Decidual leukocytes are critical to the development of the fetomaternal interface, regulating tolerance to the semi-allogeneic fetus and vascular transformation of the uterine spiral arteries. Despite the continuation of these processes beyond the first trimester of pregnancy, the second trimester has largely been unstudied, with investigation focusing on early gestation and term tissues. We sought to characterize changes in decidual leukocyte populations from first to second trimester. METHODS: Multicolor flow cytometry was performed on isolated decidual leukocytes from elective terminations of pregnancy between 6 and 20 weeks of gestation for study of first (6-12 weeks) and second trimesters (13-20 weeks). Specific subpopulations were identified by comparison to isotype and fluorescent-minus-one (FMO) controls. RESULTS: Decidual natural killer cells (CD56(+)CD16(-)CD3(-)) did not change in number, although a population of dNK with decreased CD56 brightness was observed in second trimester decidua. CD14(+)HLA-DR(+) macrophage numbers declined from first to second trimester (p = 0.031), yet a CD163(+)CD206(+) subset designating alternatively activated M2-like macrophages increased during the same period (p = 0.015). Intermediate CD205(+) dendritic cells demonstrated significant decline (p = 0.022), but immature CD209(+) and mature CD83(+) dendritic cells did not differ between trimesters. Total CD3(+) and CD3(+)CD4(+) T lymphocytes increased (p = 0.0079, p = 0.0028); CD3(+)CD8(+) T cells trended towards increase but did not differ significantly. CONCLUSION: Several changes in leukocyte subsets are observed in the second trimester that promote a tolerogenic and angiogenic decidual microenvironment through mid-gestation.

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.

Polymorphisms in genes within the IGF-axis influence antenatal and postnatal growth.

Two pregnancy cohorts were used to investigate the association between single-nucleotide polymorphisms (SNPs) in genes within the insulin-like growth factor (IGF)-axis and antenatal and postnatal growth from birth to adolescence. Longitudinal analyses were conducted in the Raine pregnancy cohort (n = 1162) using repeated measures of fetal head circumference (HC), abdominal circumference (AC) and femur length (FL) from 18 to 38 weeks gestation and eight measures of postnatal height and weight (1-17 years). Replications of significant associations up to birth were undertaken in the Generation R Study (n = 2642). Of the SNPs within the IGF-axis genes, 40% (n = 58) were associated with measures of antenatal growth (P ⩽ 0.05). The majority of these SNPs were in receptors; IGF-1R (23%; n = 34) and IGF-2R (13%; n = 9). Fifteen SNPs were associated with antenatal growth (either AC or HC or FL) in Raine (P ⩽ 0.005): five of which remained significant after adjusting for multiple testing. Four of these replicated in Generation R. Associations were identified between 38% (n = 55) of the IGF-axis SNPs and postnatal height and weight; 21% in IGF-1R (n = 31) and 9% in IGF-2R (n = 13). Twenty-six SNPs were significantly associated with both antenatal and postnatal growth; 17 with discordant effects and nine with concordant effects. Genetic variants in the IGF-axis appear to play a significant role in antenatal and postnatal growth. Further replication and new analytic methods are required in order to better understand this key metabolic pathway integrating biologic knowledge about the interaction between IGF-axis components.

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.

Spatiotemporal expression of alpha(1), alpha(3) and beta(1) integrin subunits is altered in rat myometrium during pregnancy and labour.

Integrins are transmembrane extracellular matrix (ECM) receptors composed of alpha- and beta-subunits. Integrins can cluster to form focal adhesions and, because there is significant ECM remodelling and focal adhesion turnover in the rat myometrium during late pregnancy, we hypothesised that the expression of alpha(1), alpha(3) and beta(1) integrin subunits in the rat myometrium would be altered at this time to accommodate these processes. Expression of alpha(1) and beta(1) integrin subunit mRNA was significantly increased on Days 6-23 of pregnancy compared with non-pregnant (NP) and postpartum (PP) time points (P < 0.05). In contrast, alpha(3) integrin subunit mRNA expression was significantly increased on Days 14, 21 and 22 compared with NP, Day 10, 1 day PP and 4 days PP (P < 0.05). A relative gene expression study revealed that, of the integrins studied, the expression of beta(1) integrin mRNA was highest in pregnant rat myometrium. The alpha(1), alpha(3) and beta(1) integrin subunit proteins became immunolocalised to myocyte membranes in situ by late pregnancy and labour in both myometrial muscle layers. Increased alpha(1), alpha(3) and beta(1) integrin gene expression during gestation and the specific detection of these subunits in myocyte membranes during late pregnancy and labour may contribute to the cell-ECM interactions required for the development of a mechanical syncytium.

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 the multidrug resistance P-glycoprotein, (ABCB1 glycoprotein) in the human placenta decreases with advancing gestation.

The multidrug resistance p-glycoprotein (P-gp), encoded by the ABCB1 gene, is a plasma membrane protein that actively extrudes a wide variety of substances from cells. Preliminary studies in mice have shown that the ABCB1/P-gp can protect the fetus from a number of toxic substances. ABCB1/P-gp is expressed in the human placenta and is potentially capable of protecting the fetus from a large number of drugs and toxins, including herbicides and pesticides. The protein can also extrude various steroids including certain glucocorticoids and may therefore play an important role in regulating fetal access of glucocorticoids. The aim of the present study was to examine the expression profile and cellular localization of ABCB1/P-gp in human placenta throughout gestation. We hypothesized that there would be gestational age-related changes in the expression of the protein. ABCB1/P-gp mRNA was measured by Real-Time PCR using specific probes in tissues obtained from 6 weeks gestation to term. ABCB1/P-gp mRNA levels in placental tissue obtained at 6-10 weeks (n=5) and 24-35 weeks (n=5) were significantly higher than in tissues obtained at term (38-41 weeks gestation) by elective C-section (n=6) or following labor (n=6). The profile of ABCB1/P-gp protein levels, quantified using Western analysis, demonstrated a similar decrease with advancing gestation. At all gestational ages ABCB1/P-gp was localized by immunohistochemistry to the syncytiotrophoblast. In term tissues, it appeared to be localized to some areas of the villi and not others. Together, these data indicate that with advancing gestation there is a decrease in the level of ABCB1/P-gp in the human placenta indicating that the fetus may be more susceptible to toxic insults in the latter part of gestation. Further, the reduction in ABCB1/P-gp expression may contribute to the increased transfer of maternal cortisol to the fetus that is known to occur in late gestation.

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.

Gap junctions are required for trophoblast proliferation in early human placental development.

Little is known about the role of gap junctional intercellular communication (GJIC) in human trophoblast differentiation, particularly during the formation of extravillous trophoblast (EVT) cell columns and their subsequent differentiation into invasive cells. We have identified transcripts for five connexin gap junction proteins in the early human placenta (Cx32, Cx37, Cx40, Cx43 and Cx45). Of these, Cx40 and Cx45 proteins immunolocalize to EVT in anchoring cell columns. Cx40 expression is prominent in the anchoring column throughout the first trimester of pregnancy (6-14 weeks gestation). We used first trimester placental villous explant cultures to determine the functional significance of the inhibition of GJIC in EVT cell proliferation and differentiation using two known GJIC inhibitors, carbenoxolone (CBX) and heptanol. The morphology of EVT outgrowths changed dramatically upon GJIC-blockade, from compact and organized outgrowths into a scattered group of rounded individual trophoblast cells, reminiscent of an early invasive phenotype. Furthermore, the inhibition of GJIC in placental explants by CBX or heptanol induced a switch away from the proliferative and towards an invasive EVT phenotype, as evident from (a) the loss of the proliferation marker Ki67 and (b) an increase in the invasive marker alpha1 integrin. We also utilized antisense oligonucleotides to inhibit Cx40 protein expression in placental explants. Cx40 antisense treatment also resulted in the abolishment of outgrowth EVT cell proliferation (as determined by Ki67 immunostaining). Together, these results suggest that gap junctions composed particularly of Cx40 channels are required for the proliferation of EVT cells in anchoring cell columns, and that a loss of GJIC contributes to differentiation to the invasive EVT phenotype.

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).

Expression of prostaglandin I(2) synthase, but not prostaglandin E synthase, changes in myometrium of women at term pregnancy.

Prostaglandins (PGs) act as potent uterotonins at the time of labor. Prostaglandin E synthase (PGES) is responsible for the formation of PGE(2), a uterotonin. PGI(2) is synthesized by the prostaglandin I synthase enzyme (PGIS) and contributes to relaxation in the lower uterine segment. We examined the expression of membrane-bound PGES and PGIS in myometrium from pregnant women during preterm and term labor. Tissues were collected from the lower uterine segment from preterm no labor, preterm labor, term no labor, and term labor patients and used for immunohistochemistry and Western blot analysis using specific antibodies. Immunoreactive (ir-) PGES and PGIS proteins were localized to the cytoplasm of myocytes of the myometrium and vascular smooth muscle cells. Ir-PGES was also detected in vascular endothelial cells. Western blot analyses revealed a predominant protein band of 180 kDa, and a second 16-kDa band for ir-PGES and 56-kDa band for ir-PGIS. There was no significant change in ir-PGES protein (180 or 16 kDa) or mRNA levels with preterm or term labor or gestational age. There was a significant decrease in PGIS mRNA and protein with advancing gestational age. We conclude that the gestational age decrease in the inhibitory PGIS is consistent with lessening of its influence in myometrium at the time of labor. The lack of change in PGES indicates that alterations at other points along the pathway of arachidonic acid metabolism may be of greater importance in affecting local changes in PGE(2).