Infection and disruption of placental multidrug resistance (MDR) transporters: Implications for fetal drug exposure.

P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP/ABCG2) are efflux multidrug resistance (MDR) transporters localized at the syncytiotrophoblast barrier of the placenta and protect the conceptus from drug and toxin exposure throughout pregnancy. Infection is an important modulator of MDR expression and function. This review comprehensively examines the effect of infection on the MDR transporters, P-gp and BCRP in the placenta. Infection PAMPs such as bacterial lipopolysaccharide (LPS) and viral polyinosinic-polycytidylic acid (poly I:C) and single-stranded (ss)RNA, as well as infection with Zika virus (ZIKV), Plasmodium berghei ANKA (modeling malaria in pregnancy - MiP) and polymicrobial infection of intrauterine tissues (chorioamnionitis) all modulate placental P-gp and BCRP at the levels of mRNA, protein and or function; with specific responses varying according to gestational age, trophoblast type and species (human vs. mice). Furthermore, we describe the expression and localization profile of Toll-like receptor (TLR) proteins of the innate immune system at the maternal-fetal interface, aiming to better understand how infective agents modulate placental MDR. We also highlight important gaps in the field and propose future research directions. We conclude that alterations in placental MDR expression and function induced by infective agents may not only alter the intrauterine biodistribution of important MDR substrates such as drugs, toxins, hormones, cytokines, chemokines and waste metabolites, but also impact normal placentation and adversely affect pregnancy outcome and maternal/neonatal health.

Mid-pregnancy poly(I:C) viral mimic disrupts placental ABC transporter expression and leads to long-term offspring motor and cognitive dysfunction.

Limited information is available about the effect of mid-pregnancy viral infections on the placental expression of efflux transporters and offspring behavior. We hypothesized that maternal exposure to polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA viral mimic, would impair placental cell turnover, the expression of selected ABC transporters and adult offspring behavior. C57BL/6 mice were administered poly(I:C) (10 mg/Kg;ip) or vehicle at gestational day (GD) 13.5 (mid-pregnancy). Dams were euthanized for blood collection 4 h after injection, fetal and placental collection at GD18.5 or allowed to deliver spontaneously at term. At GD 13.5, poly(I:C) induced an acute pro-inflammatory response characterized by an increase in maternal plasma levels of IL-6, CXCL-1 and CCL-2/MCP-1. At GD 18.5, poly(I:C) decreased cell proliferation/death in the labyrinthine and increased cell death in the junctional zones, characterizing a disruption of placental cell turnover. Abca1 and Abcg1 immunolabelling was decreased in the labyrinthine zone, whereas Abca1, Abcg1 and breast cancer resistance transporter (Bcrp) expression increased in the junctional zone. Moreover, adult offspring showed motor and cognitive impairments in the Rotarod and T-water maze tests. These results indicate that viral infection during mid-pregnancy may disrupt relevant placental efflux transporters, as well as placental cell turnover and offspring behavior in adult life.

Prenatal stress and epigenetics.

In utero exposure to environmental stress in both animals and humans could result in long-term epigenome alterations which further lead to consequences for adaptation and development in the offspring. Epigenetics, especially DNA methylation, is considered one of the most widely studied and well-characterized mechanisms involved in the long-lasting effects of in utero stress exposure. In this review, we outlined evidence from animal and human prenatal research supporting the view that prenatal stress could lead to lasting, broad and functionally organized signatures in DNA methylation which, in turn, could mediate exposure-phenotype associations. We also emphasized the advantage of using stressor from quasi-randomly assigned experiments. Furthermore, we discuss challenges that still need to be addressed in this field in the future.

Breast cancer resistance protein (Bcrp/Abcg2) is selectively modulated by lipopolysaccharide (LPS) in the mouse yolk sac.

Bacterial infection alters placental ABC transporters expression. These transporters provide fetal protection against circulating xenobiotics and environmental toxins present in maternal blood. We hypothesized that lipopolysaccharide (LPS-bacterial mimic) alters the yolk sac morphology and expression of key ABC transporters in a gestational-age dependent manner. Yolk sac samples from C57BL/6 mice were obtained at gestational ages (GD) 15.5 and GD18.5, 4 or 24 h after LPS exposure (150ug/kg; n = 8/group). Samples underwent morphometrical, qPCR and immunohistochemistry analysis. The volumetric proportions of the histological components of the yolk sac did not change in response to LPS. LPS increased Abcg2 expression at GD15.5, after 4 h of treatment (p < 0.05). No changes in Abca1, Abcb1a/b, Abcg1, Glut1, Snat1, Il-1ß, Ccl2 and Mif were observed. Il-6 and Cxcl1 were undetectable in the yolk sac throughout pregnancy. Abca1, breast cancer resistance protein (Bcrp, encoded by Abcg2) and P-glycoprotein (P-gp/ Abcb1a/b) were localized in the endodermal (uterine-facing) epithelium and to a lesser extent in the mesothelium (amnion-facing), whereas Abca1 was also localized to the endothelium of the yolk sac blood vessels. LPS increased the labeling area and intensity of Bcrp in the yolk sac's mesothelial cells at GD15.5 (4 h), whereas at GD18.5, the area of Bcrp labeling in the mesothelium (4 and 24 h) was decreased (p < 0.05). Bacterial infection has the potential to change yolk sac barrier function by affecting Bcrp and Abcg2 expression in a gestational-age dependent-manner. These changes may alter fetal exposure to xenobiotics and toxic substances present in the maternal circulation and in the uterine cavity.

Dysregulation of placental ABC transporters in a murine model of malaria-induced preterm labor.

Malaria in Pregnancy (MiP) is characterized by placental accumulation of Plasmodium-infected erythrocytes, intrauterine growth restriction (IUGR) and preterm delivery (PTD). Placental ATP-binding cassette (ABC) transporters mediate the efflux of nutrients, cytokines and xenobiotics. The expression and activity of these transporters are highly responsive to infection. We hypothesized that MiP would perturb the expression of placental ABC transporters, promoting PTD. Peripheral blood, spleens, livers and placentas of pregnant mice, infected with Plasmodium berghei ANKA on gestational day (GD) 13.5, were collected and analyzed on GD18.5. The primary consequences of human MiP, including IUGR, PTD (20%) and placental inflammation, were recapitulated in our mouse model. Electron microscopy revealed attenuated presence of labyrinthine microvilli and dilated spongiotrophoblasts -granular endoplasmic reticulum cisternae. Additionally, a decrease in placental Abca1 (ABCA1), Abcb1b (P-glycoprotein), Abcb9 and Abcg2 (BCRP) expression was observed in MiP mice. In conclusion, MiP associated with PTD impairs placental ABC transporters' expression, potentially modulating placental nutrient, environmental toxin and xenobiotic biodistribution within the fetal compartment, and may, at some degree, be involved with pregnancy outcome in MiP.

Endocrine and paracrine regulation of birth at term and preterm.

We have examined factors concerned with the maintenance of uterine quiescence during pregnancy and the onset of uterine activity at term in an animal model, the sheep, and in primate species. We suggest that in both species the fetus exerts a critical role in the processes leading to birth, and that activation of the fetal hypothalamic-pituitary-adrenal axis is a central mechanism by which the fetal influence on gestation length is exerted. Increased cortisol output from the fetal adrenal gland is a common characteristic across animal species. In primates, there is, in addition, increased output of estrogen precursor from the adrenal in late gestation. The end result, however, in primates and in sheep is similar: an increase in estrogen production from the placenta and intrauterine tissues. We have revised the pathway by which endocrine events associated with parturition in the sheep come about and suggest that fetal cortisol directly affects placental PGHS expression. In human pregnancy we suggest that cortisol increases PGHS expression, activity, and PG output in human fetal membranes in a similar manner. Simultaneously, cortisol contributes to decreases in PG metabolism and to a feed-forward loop involving elevation of CRH production from intrauterine tissues. In human pregnancy, there is no systemic withdrawal of progesterone in late gestation. We have argued that high circulating progesterone concentrations are required to effect regionalization of uterine activity, with predominantly relaxation in the lower uterine segment, allowing contractions in the fundal region to precipitate delivery. This new information, arising from basic and clinical studies, should further the development of new methods of diagnosing the patient at risk of preterm labor, and the use of scientifically based strategies specifically for the management of this condition, which will improve the health of the newborn.

Expression and localisation of breast cancer resistance protein (BCRP) in human fetal membranes and decidua and the influence of labour at term.

Breast cancer resistance protein (BCRP) is a multidrug resistant ABC transport protein (ABCG-2). It extrudes a wide range of substrates, including many chemotherapy drugs, steroids and folate. It is present in many cancers, as well as normal tissues, in particular barrier tissues such as the blood-brain barrier, the intestine, blood vessels and the human placenta. Human fetal membranes (amnion and chorion laeve) provide the barrier between the maternal uterine environment and the fetus. In the present study, we defined the expression and localisation of BCRP mRNA and protein in human fetal membranes (amnion and chorion) and attached decidua obtained before and following labour at term. BCRP protein and mRNA was expressed in all tissues examined and the levels of expression were not altered by labour. BCRP was localised to the amnion epithelial cells, chorion trophoblast cells and decidua stromal cells, as well as the endothelial cells of maternal blood vessels in the decidua, but was absent from mesenchymal cells. In the amnion epithelium, BCRP protein was localised to the apical surface, cytoplasm and membrane between cells. In the chorion trophoblast and decidua stromal cells, BCRP protein was localised to the plasma membrane. However, in the chorion trophoblast, BCRP protein was also highly expressed in the nucleus. The level of BCRP protein in the membranes was comparable to that in the placenta. These high levels raise the possibility that this transporter plays an important role in the physiological function of the tissues.

Breast cancer resistance protein (Bcrp1/Abcg2) in mouse placenta and yolk sac: ontogeny and its regulation by progesterone.

Breast Cancer Resistance Protein (BCRP), a recently-discovered transporter belonging to ABC superfamily, is highly expressed within the labyrinth of the placenta, the primary site of exchange between the maternal and fetal circulation. It has been proposed to function as an efflux pump protecting the fetus from a wide range of xenobiotics. It has also been recently shown that the yolk sac, in addition to the placenta, may be involved in transport of certain substances to and from the fetus. We hypothesised that there are changes in placental Bcrp1 (the mouse orthologue of human BCRP) expression during pregnancy and that these correlate with changes in progesterone production that occur in late gestation. We also hypothesised that Bcrp1 is expressed in the yolk sac, and that levels change with advancing gestation. Either whole concepti, or placenta and yolk sac, were collected from pregnant mice and analysed at embryonic (E) day 9.5, 12.5, 15.5 and 18.5 (term approximately E19.5). Peak expression of Bcrp1 mRNA was detected using in situ hybridisation within the placenta at E9.5 and the yolk sac at E12.5. There was a significant decrease thereafter in both tissues (p<0.001). In contrast, expression of Bcrp1 protein as assessed by immunohistochemistry and Western immunoblots did not change significantly during gestation either in the placenta nor the yolk sac, and no sex difference in Bcrp1 protein expression in either tissue was observed at E12.5. Daily progesterone treatment starting at E14.5 and continuing until E18.5 significantly increased maternal progesterone levels, but did not elicit any changes in the Bcrp1 mRNA or Bcrp1 protein expression either in the placenta or the yolk sac. Significant expression of Bcrp1 protein in fetal tissue was evident at the end of gestation, while expression in the fetal brain endothelium was evident as early as E12.5. We suggest that the placenta and the yolk sac, both of which express Bcrp1, may limit fetal exposure to the potentially adverse effects of xenobiotics including therapeutic drugs which the mother may be exposed to during pregnancy. The significant decrease in Bcrp1 mRNA expression in both the yolk sac and the placenta from mid to late gestation may be counter-balanced by an increase in Bcrp1 expression in fetal organs involved in absorption, excretion and protection.

Prenatal glucocorticoid exposure alters hypothalamic-pituitary-adrenal function in juvenile guinea pigs.

The neurodevelopmental consequences of prenatal glucocorticoid exposure are not well-understood, particularly in species that give birth to neuroanatomically mature offspring. In the present study, we hypothesised that repeated prenatal glucocorticoid administration would alter hypothalamo-pituitary-adrenal (HPA) function in juvenile guinea pig offspring. Pregnant guinea pigs were injected with betamethasone (1 mg/kg) or vehicle on gestational days 40, 41, 50, 51, 60 and 61 (six doses). Prenatal glucocorticoid exposure abolished the pituitary-adrenal response to maternal separation in juvenile males, but had no effect in female offspring. Indeed, female offspring (vehicle and betamethasone) did not mount a significant HPA response to separation at 10 days of age. Although there were no effects of prenatal glucocorticoid exposure on hippocampal or hypothalamic corticosteroid receptor expression or corticotrophin-releasing factor (CRF) mRNA, there were significant effects in the pituitary and adrenal; again males were more affected than females. Prenatal glucocorticoid exposure increased pituitary pro-opiomelanocortin and CRF receptor mRNA, and markedly decreased adrenocortical CYP17 mRNA. In conclusion, repeated prenatal glucocorticoid exposure has profound influences on HPA function and regulation in the juvenile guinea pig, and this involves altered regulation at the level of the pituitary and adrenal cortex. Furthermore, juvenile males appear to be more vulnerable to the effects of prenatal glucocorticoid exposure than females.

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.

Chronic prenatal ethanol exposure increases glucocorticoid-induced glutamate release in the hippocampus of the near-term foetal guinea pig.

Exposure to high cortisol concentration can injure the developing brain, possibly via an excitotoxic mechanism involving glutamate (Glu). The present study tested the hypothesis that chronic prenatal ethanol exposure (CPEE) activates the foetal hypothalamic-pituitary-adrenal axis to produce high cortisol exposure in the foetal compartment and alters sensitivity to glucocorticoid-induced Glu release in the foetal hippocampus. Pregnant guinea pigs received daily oral administration of ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding from gestational day (GD) 2 until GD 63 (term, approximately GD 68) at which time they were euthanised, 1 h after their final treatment. Adrenocorticotrophic hormone (ACTH) and cortisol concentrations were determined in foetal plasma. Basal and electrically stimulated Glu and gamma-aminobutyric acid (GABA) efflux in the presence or absence of dexamethasone (DEX), a selective glucocorticoid-receptor agonist, were determined ex vivo in foetal hippocampal slices. Glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and N-methyl-D-aspartate (NMDA) receptor NR1 subunit mRNA expression were determined in situ in the hippocampus and dentate gyrus. In the near-term foetus, CPEE increased foetal plasma ACTH and cortisol concentrations. Electrically stimulated glutamate, but not GABA, release was increased in CPEE foetal hippocampal slices. Low DEX concentration (0.3 microM) decreased stimulated glutamate, but not GABA, release in both CPEE and control foetal hippocampal slices. High DEX concentration (3.0 microM) increased basal release of Glu, but not GABA, in CPEE foetal hippocampal slices. GR, but not MR, mRNA expression was elevated in the hippocampus and dentate gyrus, whereas NR1 mRNA expression was increased in the CA1 and CA3 fields of the foetal hippocampus. These data demonstrate that CPEE increases high glucocorticoid concentration-induced Glu release in the foetal hippocampus, presumably as a consequence of increased GR expression. These effects of CPEE, coupled with increased glutamate release and increased NMDA receptor expression, may predispose the near-term foetal hippocampus to GR and Glu-NMDA receptor-mediated neurodevelopmental toxicity.

Regulation of Multidrug Resistance P-Glycoprotein in the Developing Blood-Brain Barrier: Interplay between Glucocorticoids and Cytokines.

P-glycoprotein (P-gp) encoded by Abcb1 provides protection to the developing brain from xenobiotics. P-gp in brain endothelial cells (BECs) derived from the developing brain microvasculature is up-regulated by glucocorticoids and inhibited by pro-inflammatory cytokines in vitro. However, little is known about how prenatal maternal glucocorticoid treatment can affect Abcb1/P-gp function and subsequent cytokine regulation in foetal BECs. We hypothesised that glucocorticoid exposure increases Abcb1/P-gp in the foetal brain microvasculature and enhances the sensitivity of Abcb1/P-gp in BECs to the inhibitory effects of cytokines. BECs isolated from dexamethasone- or vehicle-exposed foetal guinea pigs were cultured and treated with interleukin-1ß, interleukin-6 or tumour necrosis factor-α, and Abcb1/P-gp expression and function were assessed. Prenatal dexamethasone exposure significantly increased Abcb1/P-gp expression/activity and cytokine receptor levels in BECs of the foetal brain microvasculature. Foetal dexamethasone exposure in vivo also increased the subsequent responsiveness of BECs to pro-inflammatory cytokines in vitro. In conclusion, maternal treatment with synthetic glucocorticoids appears to prematurely mature P-gp mediated drug resistance at the foetal BBB in vivo and profoundly impact the subsequent responsiveness of P-gp to pro-inflammatory cytokines in the foetal BEC. The significance of these findings to foetal brain protection against xenobiotics and other P-gp substrates in vivo requires further elaboration. However, the results of the present study may have implications for human pregnancy and foetal brain protection, particularly in cases of preterm birth combined with infection.

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.

Central regulation of the hypothalamic-pituitary-adrenal axis during fetal development in the Guinea-pig.

We have previously shown that the foetal guinea-pig hypothalamic-pituitary-adrenal (HPA) axis is activated near the time of parturition and that this is associated with changes in limbic glucocorticoid receptors (GR) and mineralocorticoid receptors. In the present study, we hypothesized that the foetal hypothalamic paraventricular nucleus (PVN) and pituitary contribute significantly to foetal HPA drive but that these areas remain sensitive to negative feedback by circulating glucocorticoids in late gestation. However, we observed decreased corticotrophin-releasing hormone mRNA expression in the PVN and decreased pro-opiomelanocortin (POMC) mRNA levels in the anterior pituitary with advanced gestational age. The reduction in POMC mRNA expression was likely the result of negative feedback via circulating glucocorticoids because GR mRNA was unchanged during development in the foetal pituitary. Furthermore, we found that maternally administered glucocorticoids significantly decreased foetal pituitary POMC mRNA expression in a dose-dependent manner at gestational day (gd) 62 with male foetuses being more sensitive to these effects. These findings show that the foetal HPA axis remains highly sensitive to glucocorticoid feedback even as plasma adrenocorticotropic hormone and cortisol levels are elevated at the end of gestation.

Chronic prenatal ethanol exposure alters glucocorticoid signalling in the hippocampus of the postnatal Guinea pig.

The present study tested the hypothesis that chronic prenatal ethanol exposure causes long-lasting changes in glucocorticoid signalling in postnatal offspring. Pregnant guinea pigs were treated with ethanol (4 g/kg maternal body weight/day), isocaloric-sucrose/pair-feeding or water throughout gestation, and maternal saliva cortisol concentration was determined 2 h after treatment at different stages of gestation. Electrically-stimulated release of glutamate and GABA, in the presence or absence of dexamethasone, as well as glucocorticoid and mineralocorticoid receptor mRNA expression, was determined in the hippocampus and prefrontal cortex of adult offspring of treated pregnant guinea pigs. Maternal saliva cortisol concentration increased throughout pregnancy, which was associated with increased foetal plasma and amniotic fluid cortisol concentration. Ethanol administration to pregnant guinea pigs increased maternal saliva cortisol concentration during early and mid-gestation. In late gestation, ethanol administration did not increase saliva cortisol concentration above that induced by pregnancy. Chronic prenatal ethanol exposure had no effect on stimulated glutamate or GABA release, but selectively prevented dexamethasone-mediated suppression of stimulated glutamate release, and decreased expression of mineralocorticoid, but not glucocorticoid, receptor mRNA in the hippocampus of adult offspring. These data indicate that maternal ethanol administration leads to excessively increased maternal cortisol concentration that can impact negatively the developing foetal brain, leading to persistent postnatal deficits in glucocorticoid regulation of glutamate signalling in the adult hippocampus.

Regulation of the hypothalamo-pituitary-adrenocortical axis in fetal sheep.

Development of the fetal hypothalamo-pituitary-adrenal (HPA) axis is required for normal fetal life and subsequent neonatal health. Activation of the fetal pituitary gland results in the synthesis and release of glucocorticoids from the adrenal cortex. Glucocorticoids promote maturation of several organ systems, are important in responses of the fetus to stress, and are involved in the initiation of parturition in several species. The expression of hypothalamic and pituitary genes associated with HPA function is apparent early in gestation in fetal sheep, although the endocrine changes associated with maturation and parturition do not occur until the last fifth of gestation. In this connection, the fetal HPA axis can be activated by treatment with hypophysiotrophic factors or moderate stress throughout gestation. This review focuses on the development of neuroendocrine mechanisms controlling HPA function during fetal life.

Glucocorticoids and serotonin alter glucocorticoid receptor mRNA levels in fetal guinea-pig hippocampal neurons, in vitro.

The hypothalamic-pituitary-adrenal (HPA) axis is susceptible to programming during fetal life. Such programming occurs, at least partially, at the level of the hippocampus. The hippocampus plays a central role in regulation of the HPA axis and release of endogenous glucocorticoids, via mediation of glucocorticoid negative feedback. Fetal exposure to synthetic glucocorticoids can permanently alter glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) levels within the hippocampus, and serotonin is thought to be involved in this process. In the present study, we hypothesised that dexamethasone, cortisol and serotonin exposure would modify GR mRNA expression within fetal guinea-pig hippocampal cultures. Cultures were derived from 40-day-old guinea-pig fetuses, and were exposed to 0, 1, 10 and 100 nM dexamethasone, cortisol or serotonin for 4 days. Expression of GR and MR mRNA was examined by in situ hybridisation followed by high-resolution silver emulsion autoradiography. Four-day exposure to dexamethasone (P < 0.05; 100 nM) or cortisol (P = 0.08; 100 nM) downregulated the expression of GR mRNA within neurons. There was no change in the expression of MR mRNA levels following cortisol treatment. Exposure to serotonin (100 nM) significantly increased GR mRNA levels in hippocampal neurons. We conclude that synthetic and endogenous glucocorticoids, as well as serotonin, can influence GR expression during hippocampal development and in this way may act to permanently programme HPA function.

Differential regulation of proopiomelanocortin messenger ribonucleic acid in the pars distalis and pars intermedia of the pituitary gland after prolonged hypoxemia in fetal sheep.

POMC messenger RNA (mRNA) is synthesized and translated to form ACTH in the pars distalis and ACTH-related peptides in the pars intermedia of the pituitary in fetuses of species such as sheep. In the fetal sheep, maturation of hypothalamo-pituitary-adrenal (HPA) function occurs during late pregnancy accompanied by increased levels of POMC mRNA in the pars distalis, and contributes to the stimulus for birth. Activation of fetal HPA function also occurs with short term hypoxemia. However, the effects of more prolonged fetal hypoxemia, which may predispose to premature parturition, on POMC mRNA levels in the pars distalis and pars intermedia are not known. We studied the effects of sustained hypoxemia (48 h) in the absence of acidosis on fetal HPA responses at two times in late gestation, days 126-130 and days 134-136 (term = approximately 145 days). Hypoxemia was induced by lowering the fraction of oxygen in the maternal inspired gas mixture. Fetal arterial oxygen tension fell by 6-8 mm Hg, without a change in fetal arterial pH or carbon dioxide tension. Pituitary POMC mRNA was localized and quantified by in situ hybridization. At both gestational ages, hypoxemia caused a transient elevation in fetal plasma ACTH, with a maximum response at +2 h, and a sustained elevation in circulating cortisol. Cortisol responses were greater in the older fetuses. Changes in plasma cortisol were not associated with alterations in the plasma corticosteroid-binding capacity or in levels of hepatic corticosteroid-binding globulin mRNA. Pituitary POMC mRNA showed a regional distribution in the pars distalis, and mean levels increased with gestational age. After 48 h of hypoxemia, POMC mRNA levels had increased in the pars distalis. The regional distribution was unaffected by the hypoxemic insult. In contrast, POMC mRNA levels in the pars intermedia decreased after hypoxemia, and the reduction was greater in the older fetuses. We conclude that in response to prolonged hypoxemia, there is differential regulation of POMC mRNA levels in the pars distalis and pars intermedia of the pituitary in fetal sheep. The magnitude of these responses also changes with gestational age. The increase in POMC mRNA levels in the pars distalis at 48 h of hypoxemia, despite a rise in plasma cortisol and without a change in corticosteroid-binding globulin, suggests altered regulation of HPA function at this time.

Molecular regulation of the hypothalamo-pituitary-adrenal axis in streptozotocin-induced diabetes: effects of insulin treatment.

Increased hypothalamo-pituitary-adrenocortical (HPA) activity in diabetes is likely important in the development of some pathologies associated with the disorder. We hypothesized that central regulation of HPA activity differs among normal, streptozotocin (STZ)-diabetic, and insulin-treated diabetic rats. Blood glucose, ACTH, and corticosterone were elevated, 8 d after inducing diabetes. Insulin treatment normalized these parameters. Plasma norepinephrine was similar in all groups, but epinephrine was lower in STZ-diabetic and higher in insulin-treated rats vs. normals. Increased ACTH with diabetes corresponded with increased hypothalamic CRH mRNA, but no change in pituitary POMC mRNA. With insulin-treatment, CRH mRNA remained elevated, and POMC mRNA was unaltered. Hippocampal MR mRNA expression was dramatically increased with diabetes and, moreover, was not normalized by insulin. No differences in GR mRNA were detected between normal and STZ-diabetic rats. However, insulin treatment increased GR mRNA levels in the paraventricular nucleus and pituitary. We postulate that, in STZ-diabetes: 1) increased HPA activity is caused by increased central drive at and/or above the level of the paraventricular nucleus and is associated with decreased epinephrine; and 2) normalized pituitary-adrenal activity with insulin may be caused by the compensatory increase in GR mRNA allowing glucocorticoid-mediated suppression of ACTH secretion despite the residual increase in central HPA activity. Thus, insulin apparently restored HPA activity at and below the pituitary but, surprisingly, not above it.

15-hydroxyprostaglandin dehydrogenase: implications in preterm labor with and without ascending infection.

There is evidence that intrauterine infection, which stimulates PG synthesis may play a role in the pathogenesis of some preterm labor. Local tissue concentrations of PGs are controlled not only by the rate of synthesis, but also by catabolism, which is regulated by 15-hydroxyprostaglandin dehydrogenase (PGDH). We hypothesized that a decrease of PGDH activity could contribute to an increase in PG output at the time of preterm labor (PTL) especially in association with infection. We measured PGDH activity with a zero order kinetic enzymatic assay, PGDH messenger ribonucleic acid by in situ hybridization and PGDH distribution and localization with immunohistochemistry in human placenta and fetal membranes from women at term before (n = 10) or after (n = 16) labor compared to preterm labor at less than 36 weeks without (n = 16) and with (n = 11) chorioamnionitis. PGDH activity in chorion was significantly lower in PTL than at term and was further reduced when PTL was associated with inflammation. Immunoreactive PGDH and PGDH messenger ribonucleic acid localized predominantly to chorionic trophoblasts at term and were reduced in PTL women with or without infection. These effects were not observed in the placenta. Loss of PGDH with infection was associated with infiltration of chorion by polymorphonuclear leukocytes, resulting in a compromised structural integrity, although the amniotic epithelium was generally intact. We conclude that a reduction in PGDH in the human fetal membranes may occur in some cases of preterm labor and may contribute to an increase in net PG accumulation and drive to myometrial contractility.