Development of prostaglandin endoperoxide synthase expression in the ovine fetal central nervous system and pituitary.

In this study, we tested the hypothesis that prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and PGHS-2) are expressed throughout the latter half of gestation in ovine fetal brain and pituitary. Hypothalamus, pituitary, hippocampus, brainstem, cortex and cerebellum were collected from fetal sheep at 80, 100, 120, 130, 145days of gestational age (DGA), 1 and 7days postpartum lambs, and from adult ewes (n=4-5 per group). mRNA and protein were isolated from each region, and expression of prostaglandin synthase-1 (PGHS-1) and -2 (PGHS-2) were evaluated using real-time RT-PCR and western blot. PGHS-1 and -2 were detected in every brain region at every age tested. Both enzymes were measured in highest abundance in hippocampus and cerebral cortex, and lowest in brainstem and pituitary. PGHS-1 and -2 mRNA's were upregulated in hypothalamus and pituitary after 100 DGA. The hippocampus exhibited decreases in PGHS-1 and increases in PGHS-2 mRNA after 80 DGA. Brainstem PGHS-1 and -2 and cortex PGHS-2 exhibited robust increases in mRNA postpartum, while cerebellar PGHS-1 and -2 mRNA's were upregulated at 120 DGA. Tissue concentrations of PGE(2) correlated with PGHS-2 mRNA, but not to other variables. We conclude that the regulation of expression of these enzymes is region-specific, suggesting that the activity of these enzymes is likely to be critical for brain development in the late-gestation ovine fetus.

Inhibition of brain prostaglandin endoperoxide synthase-2 prevents the preparturient increase in fetal adrenocorticotropin secretion in the sheep fetus.

Maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. Recently, we proposed that prostaglandin generation within the fetal central nervous system is critical for the modulation of hypotension-induced fetal ACTH secretion. The present study was designed to test the hypothesis that the preparturient increase in fetal ACTH secretion is dependent upon fetal central nervous system prostaglandin synthesis mediated by the activity of prostaglandin endoperoxide synthase (PGHS)-2 (cyclooxygenase-2) in the fetal brain. We performed two studies in chronically catheterized fetal sheep. In the first study, we infused nimesulide or vehicle intracerebroventricularly (i.c.v) into singleton fetal sheep and collected blood samples until spontaneous parturition. Nimesulide significantly delayed parturition, and inhibited fetal ACTH and proopiomelanocortin secretion but did not prevent the preparturient increase in fetal plasma cortisol concentration. In the second study, we used twin fetuses. One fetus received intracerebroventricular nimesulide and the other intracerebroventricular vehicle. Nimesulide reduced brain tissue concentrations of prostaglandin estradiol, while not affecting plasma prostaglandin E(2) concentrations, demonstrating an action restricted to the fetal brain. Nimesulide reduced PGHS-2 mRNA and increased PGHS-2 protein, while not altering PGHS-1 mRNA or protein in most brain regions, suggesting an effect of the inhibitor on PGHS-2 turnover and relative specificity for PGHS-2 in vivo. We conclude that the preparturient increase in fetal ACTH and proopiomelanocortin is dependent upon the activity of PGHS-2 in the fetal brain. However, we also conclude that the timing of parturition is not solely dependent upon ACTH in this species.

Development of ER-alpha and ER-beta expression in the developing ovine brain and pituitary.

Fetal neuroendocrine development in late gestation is critical for maintenance of fetal homeostasis, growth, and readiness for birth. We designed the present study to identify the regional patterns of expression of the two main isoforms of the estrogen receptor, ER-alpha and ER-beta, in the developing ovine fetal brain. Fetal (80, 100, 120, 130, and 145 days gestation), neonatal (1 and 7 days), and adult sheep were euthanized and the following tissues were collected: pituitary, hypothalamus, hippocampus, cerebral cortex, and brainstem. Both ER's are expressed in the ovine brain as early as 80 days gestation, and the expression of both receptors appears to be developmentally regulated. We conclude that both forms of the estrogen receptor are expressed in fetal brain and pituitary throughout the latter half of gestation.

Genomic analysis of neuroendocrine development of fetal brain-pituitary-adrenal axis in late gestation.

The present study was performed to identify the changes in genomic expression of critical components of the hypothalamus-pituitary-adrenal (HPA) axis in the second half of gestation in fetal sheep. We isolated mRNA from pituitary, hypothalamus, hippocampus, and brain stem in fetal sheep at 80, 100, 120, 130, and 145 days of gestation and 1 and 7 days after delivery (n = 4-5/group). Using real-time RT-PCR, we measured mRNA expression levels of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), serum- and glucocorticoid-induced kinase-1 (sgk1), proopiomelanocortin (POMC), CRF, and arginine vasopressin (AVP). Both MR and GR were highly expressed in pituitary and hippocampus; in all tissues GR was more highly expressed than MR. AVP was more highly expressed than CRF in hypothalamus. MR, GR, and sgk1 expression were increased postnatally in brain stem, and sgk1 expression was increased postnatally in hypothalamus. GR expression was reduced in pituitary in term fetuses compared with younger ages. Hypothalamic CRF expression was increased at the end of gestation compared with younger ages, and AVP expression was increased in newborn lambs. Pituitary POMC was increased at 100 days of gestation compared with 80 days; hypothalamic POMC was increased at 120 days. Overall, the results demonstrate the expression of both MR and GR in brain regions important for control of the HPA axis. Decreases in expression of GR in pituitary at the end of gestation might contribute to the decreased corticosteroid negative feedback sensitivity at term in this species.

Developmental expression of chemokine receptor genes in the human fetus.

BACKGROUND: Chemokines induce cell motility during embryogenesis by activating specific receptors. While the orchestration of organogenesis is complex and requires the interaction of many morphoregulatory molecules that lead to coordinated organ development, limited knowledge exists regarding the human developmental biology of chemokines and their receptors. Such information on chemokine receptor expression could potentially enhance our understanding of organogenesis in the normal human fetus. AIM: To determine the distribution of the CXC receptors (CXCR-1, CXCR-2, CXCR-3, and CXCR-4) and SDF-1 in human fetuses. SUBJECTS: Tissues from human fetuses 12-15 weeks (n = 5) and 16-19 weeks (n = 5) gestation were studied. OUTCOME MEASURES: Reverse transcription-PCR was performed to simultaneously determine the gene expression of CXCR-1-4 and SDF-1, and immunohistochemical staining of non-hematopoietic tissues was used to determine the specific cellular proteins. RESULTS: CXCR-1-4 and SDF-1 mRNA were present in every tissue examined. The expression of CXCR-3 in kidney, liver, and brain was dependent upon gestational age. CXCR-1-4 protein was expressed in non-hematopoietic cells in the brain, heart, intestine, and kidney. CONCLUSIONS: CXCR-1-4 and SDF-1 genes are widely expressed in the normal human fetus. This suggests that these gene products could influence fetal development.

Effects of enterally administering granulocyte colony-stimulating factor to suckling mice.

Gastrointestinal (GI) tract development is influenced by multiple growth factors, some of which are delivered directly to the GI lumen, as they are swallowed constituents of amniotic fluid, colostrum, and milk. Granulocyte colony-stimulating factor (G-CSF), traditionally known as a granulocytopoietic growth factor, is an example of one such factor. However, it is not clear whether the large amounts of G-CSF that are normally swallowed by the fetus and neonate have systemic effects on circulating neutrophils or local effects in the developing intestine. To assess this, we administered either active or heat-denatured (control) recombinant human G-CSF to 5- to 7-d-old C57BL/6 x 129SvJ mice. Pups received either a low dose (3 ng) that was calculated to approximate the amount of G-CSF swallowed in utero from amniotic fluid or an isovolemic high dose 100 times larger (300 ng). Oral dosing was performed daily for either 3 or 7 d, after which pups were killed and measurements were made on the blood and the GI tract. Absolute blood neutrophil counts and immature to total neutrophil ratios did not differ from controls in any of the test groups. However, intestinal villus area, perimeter, length, crypt depth, and proliferating cell nuclear antigen index increased significantly among those that were treated with active G-CSF. Thus, in suckling mice, enterally administered G-CSF had no effect on the concentration of circulating neutrophils but had trophic effects on the intestine. We speculate that the G-CSF present in amniotic fluid, colostrum, and milk acts as a topical intestinal growth factor and has little or no granulocytopoietic action.

Bioavailability of granulocyte colony-stimulating factor administered enterally to suckling mice.

The developing fetal and neonatal gastrointestinal (GI) tract is influenced by many growth factors, including epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), and erythropoietin (Epo). Granulocyte colony-stimulating factor (G-CSF), typically regarded as a hematopoietic growth factor, might also be included because it exists in high concentrations in amniotic fluid, colostrum, and human milk, and because granulocyte CSF receptors (G-CSF-R) are abundantly expressed on the villous enterocytes of the developing intestine. As a first step toward understanding whether the effects of G-CSF on the GI tract were local or systemic, we sought to determine whether recombinant human G-CSF (rhG-CSF) administered enterally to suckling mice, is absorbed into the circulation, and if so, whether the G-CSF-R is essential for this absorption. We enterally administered rhG-CSF to suckling mice, selecting a daily dose based on the amount of G-CSF normally swallowed by the fetus and neonate (3 ng), or in other mice, a dose of G-CSF 100 times larger (300 ng). Pups were tested at either 5-7 days of age, or at 14-16 days of age. C57BL/6 x 129SvJ mice were used. Some mice had a targeted null mutation in the G-CSF-R gene, producing a non-functional G-CSF-R protein. At intervals following the enteral G-CSF dosing, G-CSF concentrations in plasma were measured by specific ELISA. The bioavailability of G-CSF was invariably <1%, regardless of the dose of rhG-CSF given, the age of the pups, or whether they had a functional G-CSF-R. After enteral administration of rhG-CSF to suckling mice, only minimal quantities of G-CSF are absorbed into the circulation, and the G-CSF-R is not essential for this absorption.

Messenger RNA expression of granulocyte colony-stimulating factor receptor isoforms in the fetus.

Granulocyte colony-stimulating factor (G-CSF) is a growth factor known to support the proliferation, differentiation, and survival of cells of the neutrophil lineage. G-CSF affects these cells after binding to its specific receptor, G-CSF-R, which exists in seven isoforms. While information exists about the distribution of these isoforms in hematopoietic cells and placenta, G-CSF-R isoforms on non-hematopoietic fetal tissues have not been described. Using RT-PCR, we analyzed a variety of human fetal tissues ranging from 6 to 18 weeks gestation. Isoforms I and III were present in all tissues, and the expression of isoform III varied with gestational age. The remaining isoforms were variably expressed in relation to tissue type and gestational age. Thus in the human fetus, G-CSF-R isoform I is the predominant form expressed on non-hematopoietic and hematopoietic tissues.

Circulating concentrations of chemokines in cord blood, neonates, and adults.

Chemokines are critical for the movement of leukocytes. Chemotaxis is deficient in neonates, particularly those delivered prematurely, and this likely contributes to their increased vulnerability to sepsis. The concentrations of circulating chemokines in neonates have not been reported, nor is it known whether low chemokine concentrations contribute to their defective chemotaxis. We hypothesized that serum concentrations of chemokines 1) would be lower in preterm than term neonates, and 2) would be lower in preterm and term neonates than adults. Samples were obtained from preterm and term neonates with normal neutrophil and eosinophil counts, umbilical cord blood samples from pregnancies without clinical evidence of intra-amniotic infection, and healthy adult volunteers. The concentrations of epithelial neutrophil activating peptide-78, growth-related oncogene-alpha, eotaxin, RANTES (regulated upon activation, normal T cell expressed and secreted), and macrophage inflammatory protein-1 alpha were measured using specific ELISA. Serum concentrations from preterm infants were either similar to or higher than those measured in term neonates and adults. We conclude that the chemotactic defect observed in premature neonates is not the result of diminished circulating concentrations of any of the specific chemokines we measured.