Fetal Hepatic Tissue Research Articles

Maternal Overconsumption of High Fructose Corn Syrup Causes Hypoxia in Placenta and Fetal Livers of Wistar Rats

BackgroundAmerican maternal overnutrition negatively impacts fetal development. Several epidemiological and experimental studies have demonstrated that overconsumption of diets rich in fats and sugars can have effects spanning three generations. While the negative impact of high fructose corn syrup (HFCS), a component heavily present in many processed American foods, is well‐recognized, its effects during fetal development remain largely underexplored. Thus, this study aims to examine the placental and fetal hepatic effects of HFCS maternal overconsumption.MethodsAdult female Wistar rats were randomized in 2 experimental groups: HFCS Group received a 20% HFCS solution in drinking water for 30 days prior to and during pregnancy. Control Group received regular water. Prior to pregnancy, blood pressure was measured in both experimental groups by using tail‐cuff plethysmography. Fasting glucose readings were measured weekly. Terminal experiments were completed on day 21 of gestation. Placentas and fetal livers were isolated and further processed for analysis of Hypoxia‐Inducible Factor‐α (HIF‐α) expression, a classical transcription factor of hypoxia, using Western Blot. Blood samples were collected for biochemical analysis.ResultsPrior to pregnancy, HFCS group exhibited increased serum triglycerides (441.48 vs. 109.47 mg/dL), systolic (159.03 ± 12.04 vs. 136.29 ± 2.57 mmHg, p<0.05) and diastolic blood pressures (114.66 ± 9.47 vs. 99.29 ± 2.49 mmHg, p<0.05) compared to controls. No differences were found in glucose levels (98.17 ± 1.65 vs. 92.5 ± .24 mg/dL controls). At the terminal experimental protocol, HFCS group displayed increased HIF‐α expression in placenta (2.3 fold‐increase, p<0.05) and fetal liver (3.1 fold increase, p<0.001) compared to controls. Therefore, our results show hypoxic changes in both placental and fetal hepatic tissues. Moreover, oil red staining confirmed increased fat deposition in the maternal livers from HFCS group.ConclusionOur findings show that overconsumption of HFCS prior to and during pregnancy has a negative impact on offspring, as demonstrated by upregulation of hypoxic markers such as HIF‐α. We will further this study to determine whether other tissues undergo hypoxia and the mechanisms by which HFCS leads to hypoxia in maternal and fetal tissues.Support or Funding InformationIn‐house Grant NYITThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of maternal high-fat diet on key components of the placental and hepatic endocannabinoid system.

Maternal obesity in pregnancy has been linked to a spectrum of adverse developmental changes. Involvement of eCBs in obesity is well characterized. However, information regarding eCB physiology in obesity associated with pregnancy is sparse. This study evaluated fetomaternal hepatic, systemic, and placental eCB molecular changes in response to maternal consumption of a HFD. From ≥9 mo before conception, nonpregnant baboons ( Papio spp.) were fed a diet of either 45 (HFD; n = 11) or 12% fat or a control diet (CTR; n = 11), and dietary intervention continued through pregnancy. Maternal and fetal venous plasma samples were evaluated using liquid chromatography-mass spectrometry to quantify AEA and 2-AG. Placental, maternal and fetal hepatic tissues were analyzed using RT-PCR, Western blot, and immunohistochemistry. mRNA and protein expression of endocannabinoid receptors (CB1R and CB2R), FAAH, DAGL, MAGL, and COX-2 were determined. Statistical analyses were performed with the nonparametric Scheirer-Ray-Hare extension of the Kruskal-Wallis test to analyze the effects of diet (HFD vs. CTR), fetal sex (male vs. female), and the diet × sex interaction. Fetal weight was influenced by fetal sex but not by maternal diet. The increase in maternal weight in animals fed the HFD vs. the CTR diet approached significance ( P = 0.055). Maternal circulating 2-AG concentrations increased, and fetal circulating concentrations decreased in the HFD group, independently of fetal sex. CB1R receptor expression was detected in syncytiotrophoblasts (HFD) and the fetal endothelium (CTR and HFD). Placental CB2R protein expression was higher in males and lower in female fetuses in the HFD group. Fetal hepatic CB2R, FAAH, COX-2 (for both fetal sexes), and DAGLα (in male fetuses) protein expression decreased in the HFD group compared with the CTR group. We conclude that consumption of a HFD during pregnancy results in fetal systemic 2-AG and hepatic eCB deficiency.

Cytochrome P450 and flavin-containing monooxygenase families: age-dependent differences in expression and functional activity

Background:Age-dependent differences in pharmacokinetics exist for metabolically cleared medications. Differential contributions in the cytochrome P450 3A (CYP3A), CYP2C, and flavin-containing monooxygenases (FMO) families play an important role in the metabolic clearance of a large number of drugs administered to children.Methods:Unlike previous semi-quantitative characterization of age-dependent changes in the expression of genes and proteins (Western Analysis), this study quantifies both gene and absolute protein expression in the same fetal, pediatric and adult hepatic tissue. Expression was then correlated with the corresponding functional activities in the same samples.Results:CYP3A and FMO families showed a distinct switch from fetal (CYP3A7, FMO1) to adult isoforms (CYP3A4, FMO3) at birth, while CYP2C9 showed a linear maturation from birth into adulthood. In contrast, analysis of CYP2C19 revealed higher expression and catalytic efficiency in pediatric samples compared to fetal and adult samples. Further, CYP3A and FMO enzymes exhibited an unexpected higher functional activity in fetal samples not entirely explained by protein expression.Conclusions:These surprising findings suggest that CYP and FMO enzymes may encounter development-related differences in their micro environments that can influence the enzyme activity in addition to protein expression levels.

MICROSCOPICAL STUDIES ON THE POSSIBLE TOXIC EFFECT OF SODIUM BENZOATE ON THE LIVER OF PREGNANT MICE AND THEIR FETUSES

The present study delt with the possible effects of sodium benzoate (SB), which is commonly used in Egypt as an antimicrobial agent in food industry, on the livers of pregnant mice and their fetuses. The pregnant females were treated with low and high doses of SB (200 & 500mg/kg b.wt) through stomach tube. In this study, 25 pregnant mice were used, which were classified as follow: 5 pregnant mice were used as control, 10 animals in group 1 and in group 2 treated with low and high doses of SB, respectively, during days 8-12 or 12-16 of pregnancy. On day 18 of gestation, the liver of the treated mothers showed some histopathological changes which were more severe in mice given the high dose than those given the low dose. These alterations included highly vacuolated hepatocytes, dilated blood sinusoids, congestion of blood vessels, hypertrophied Kupffer cells and focal necrotic areas. Structureless hepatocytes were also observed with vacuolated, pyknotic and chromatolytic nuclei. On the other hand, the fetal liver maternally treated with SB revealed similar degenerative changes such as degenerated hepatocytes, dilated sinusoids and blood vessels, and large spaces occluded with red blood cells representing severe haemorrhage. The hematopoietec cells showed variable occurrence in the fetal hepatic tissue, probably, due to SB treatment. Ultrastructural alterations were also reported in the liver cells. These changes included large vacuoles of different sizes and forms, lipid droplets and highly degenerated mitochondria with deformed outer and inner mitochondrial membranes. Also, activated Kupffer cells were observed in blood sinusoids with dense elongated nuclei. All these degenerative changes could be related to the direct action of SB on the pregnant mothers and through the placental passage of SB or its metabolites on the developing fetuses.

Particle Size Dependent Teratogenicity of Silver Nanoparticles in Mice

Nanoparticles because of their unique properties have widespread application in biomedicine and many industrial sectors. The present study was undertaken to determine the potential effects of AgNPs of different size on pregnant dams and fetal development after maternal exposure on gestational days (GD) 6-19 in mice. AgNPs, of 20nm and 1300 nm respectively were administered to pregnant mice by oral gavages at concentrations of 0.5 mg/kg/day and 1 mg/kg/day. All dams were subjected to Cesarean section on GD 20. The fetuses were evaluated for signs of embryotoxic and teratogenic effects. AgNPs caused a decrease in Catalase and Reduced Glutathione activities at ≥ 0. 5 mg/kg/day and a reduction in glutathione content at 1 mg/kg/day in maternal liver tissues. However, no treatment-related deaths or clinical signs were observed in any of the animals treated with AgNPs. Fetal liver tissue showed significant decrease in Catalase and Reduced Glutathione activities. Histomorphological alterations in the fetal liver were observed at 1300nm particle group which were exacerbated in 20 nm group. The results show that a repeated oral dose of AgNPs during pregnancy caused oxidative stress in fetal hepatic tissue which is not only dose dependent but also depends on the particle size.

NSG-hu mice generate antigen specific human immune responses after gp96 vaccination (161.9)

Abstract NOD.SCID il-2r γc-/- (NSG) KO mice have no functional adaptive immune system and readily accept human xenografts. We have generated NSG mice with human adaptive immunity (NSG-hu) by transplanting CD34+ hematopoietic stem cells isolated from fetal hepatic tissue. These early progenitors give rise to all members of the human adaptive and innate lineages and can be found localized to immunologically relevant compartments (spleen, thymus, mLN, IEL and LPL). Human engraftment appears stable over time and reaches a high proportion of human CD45+ cells when compared to murine CD45+ cells in peripheral blood (54%±24%). Higher proportions of human CD45+ cells were detected in specific tissues (spleen, PEC, IEL and mLN) (82%±29%). We have also found that by using ACK2, an antagonistic anti-mouse stem cell antibody, to further ablate murine hematopoietic stem cells, human engraftment can be boosted (81%±6%). Previously, our lab has developed a cell secreted heat shock protein gp96 based vaccine expressing HIV gag (293gp96HIVgag). NSG-hu vaccinated with 293gp96HIVgag i.p. generates HIV gag-pentamer positive CD8 T cells in the gut. In addition, an interferon gamma ELIPOST response is also observed in the mucosa when stimulated with relevant peptide but not in spleen. We conclude that NSG-hu mice are able to establish and maintain stable hematopoiesis from CD34+ precursor cells. Furthermore, NSG-hu mice are capable of mounting a human HIV-specific immune response after gp96 vaccination.

Alterations of the Fetal Primate Epigenome due to Maternal High Fat Diet Exposure Are Reversed in Subsequent Offspring with Cessation of High Fat Diet.

The most recent report from the CDC shows that more than one third of American adult women are obese. The Developmental Origins Hypothesis postulates that potentially adverse conditions in utero, such as exposure to a maternal high fat diet and maternal obesity, are associated with fetal alterations, rendering susceptibility to adult metabolic disorders. We have previously reported in a non-human primate model that exposure to a maternal high fat diet results in both pathological and epigenetic changes to the fetal liver. We have shown that high fat diet exposure results in non-alcoholic fatty liver disease, concomitant with covalent modifications to fetal hepatic chromatin structure, specifically increasing histone H3 lysine 14 acetylation (H3K14ac) levels. We now show that cessation of the high fat diet during pregnancy, even in the face of persistent maternal obesity, returns acetylation levels to those of control diet animals (n=6/ group). We also show that this increased acetylation correlates with the reprogramming of several metabolic and circadian regulatory genes. The expression of Npas2, a peripheral circadian transcription factor, is disrupted in fetal hepatic tissue from animals exposed to a maternal high fat diet, returning to those of control diet upon high fat diet cessation. We also report the disruption of other circadian genes regulated by Npas2, such as Per1 and Reverb-alpha in the high fat diet exposed animals. Extensive analysis of the Npas2 promoter region shows that DNA methylation levels do not differ due to maternal diet. However, using chromatin immunoprecipitation of an important regulatory region of the Npas2 promoter, we find enrichment of H3K14ac in animals exposed to a high fat diet compared with those on a control diet. This enrichment is lost from the diet reverted group. We conclude that H3K14ac levels are perturbed by maternal high fat diet exposure, which correlates with altered gene expression. We find that reversion to a control diet, even with persistent maternal obesity, helps to attenuate some epigenomic and circadian perturbations. (platform)

Betaine-homocysteine methyltransferase: Human liver genotype–phenotype correlation

Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity and immunoreactive protein in 268 human hepatic surgical biopsy samples from adult subjects as well as 73 fetal hepatic tissue samples obtained at different gestational ages. BHMT protein levels were correlated significantly ( p < 0.001) with levels of enzyme activity in both fetal and adult tissues, but both were decreased in fetal tissue when compared with levels in the adult hepatic biopsies. To determine possible genotype–phenotype correlations, 12 tag SNPs for BHMT and the closely related BHMT2 gene were selected from SNPs observed during our own gene resequencing studies as well as from HapMap. These SNPs data were used to genotype DNA from the adult hepatic surgical biopsy samples, and genotype–phenotype association analysis was performed. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28 kb upstream, in the 5′-UTR and in intron 1 of BHMT, respectively, were significantly correlated with both BHMT activity ( p = 3.41E-8, 2.55E-9 and 2.46E-10, respectively) and protein levels ( p = 5.78E-5, 1.08E-5 and 6.92E-6, respectively). We also imputed 230 additional SNPs across the BHMT and BHMT2 genes, identifying an additional imputed SNP, rs7700790, that was also highly associated with hepatic BHMT enzyme activity and protein. However, none of the 3 genotyped or one imputed SNPs displayed a “shift” during electrophoretic mobility shift assays. These observations may help us to understand individual variation in the regulation of BHMT in the human liver and its possible relationship to variation in methylation.

Some biological effects of scorpion envenomation in late pregnant rats

Scorpion envenoming is less studied during gestation; however, it may induce various biological disturbances in maternal organism and hypothetical ones on their fetuses. The scope of this report was to elucidate some biological effects of such poisoning in late pregnant rats. Hence, TBARS levels in maternal lung, placental and fetal pulmonary and hepatic tissues and dam's biochemical blood parameters (glucose, creatinine, 17-β estradiol, progesterone, blood nitrogen urea, sodium and potassium maternal plasma concentrations) had been evaluated after saline (G1), and scorpion venom (G2: 30 min and G3: 60 min) injections in 22nd day pregnant rats. Histological microscopic examination of these tissues was also carried out in HE-stained paraffin sections. In addition, the mean arterial blood pressure following the envenomation variations was measured in three rats from the same pool. Our results showed that Buthus occitanus tunetanus crude venom induced significant increase in maternal, placental and fetal tissues lipid peroxidation, concomitant with blood pressure elevation. Maternal plasma creatinine, estradiol and progesterone concentrations levelled up significantly after 30 min or later (60 min) after the venom injection. Except for a probable pronounced oedema and few congestions in maternal lungs and degenerative aspects of trophoblast cells, all examined tissues showed a conserved structure. These results suggest that scorpion envenomation may induce gestation process disturbances and threatens both mother's and fetus’ well-being.

Developmental origins of disease and determinants of chromatin structure: maternal diet modifies the primate fetal epigenome.

Chromatin structure is epigenetically altered via covalent modifications of histones to allow for heritable gene regulation without altering the nucleotide sequence. Multiple lines of evidence from rodents have established a role for epigenetic remodeling in regulating gene transcription in response to an altered gestational milieu. However, to date, it is unknown whether variations in the intrauterine environment in primates similarly induce changes in key determinants of hepatic chromatin structure. We hypothesized that a maternal high-fat diet would alter the epigenomic profile of the developing offspring, which would result in alterations in fetal gene expression. Age- and weight-matched adult female Japanese macaques were placed on control (13% fat) or high-fat (35% fat) breeder diets and mated annually over a 4-year interval. Fetuses in successive years were delivered near term (e130 of 167 days) and underwent necropsy with tissue harvest. Fetal histones were acid extracted for characterization of H3 modification and chromatin immunoprecipitation (ChIP) with differential display PCR; fetal RNA, DNA, and cytoplasmic and nuclear protein extracts were similarly extracted for comparison. Chronic consumption of a maternal high-fat diet results in a threefold increase in fetal liver triglycerides and histologic correlates of non-alcoholic fatty liver disease. These gross changes in the fetal liver are accompanied by a statistically significant hyperacetylation of fetal hepatic tissue at H3K14 (199.85+/-9.64 vs 88.8+/-45.4; P=0.038) with a trend towards the increased acetylation at H3K9 (140.9+/-38.7 vs 46.6+/-6.53; P=0.097) and at H3K18 (69.0+/-3.54 vs 58.0+/-4.04; P=0.096). However, epigenetic modifications on fetal hepatic H3 associated with gene repression were absent or subtle (P>0.05). Subsequent characterization of key epigenetic determinants associated with H3 acetylation marks revealed similar significant alterations in association with a high-fat maternal diet (e.g., relative fetal histone deacetylase 1 (HDAC1) gene expression 0.61+/-0.25; P=0.011). Consistent with our mRNA expression profile, fetal nuclear extracts from offspring of high-fat diet animals were observed to be significantly relatively deplete of HDAC1 protein (36.07+/-6.73 vs 83.18+/-7.51; P=0.006) and in vitro HDAC functional activity (0.252+/-0.03 vs 0.698+/-0.02; P<0.001). We employ these observations in ChIP differential display PCR to attempt to identify potential fetal genes whose expression is reprogramed under conditions of a high-fat maternal diet. We quantitatively confirm a minimum of a 40% alteration in the expression of several genes of interest: glutamic pyruvate transaminase (alanine aminotransferase) 2 (GPT2) (1.59+/-0.23-fold; P=0.08), DNAJA2 (1.36+/-0.21; P=0.09), and Rdh12 (1.88+/-0.15; P=0.01) are appreciably increased in fetal hepatic tissue from maternal caloric-dense diet animals when compared with control while Npas2, a peripheral circadian regulator, was significantly downmodulated in the offspring of high-fat diet animals (0.66+/-0.08; P=0.03). In this study, we show that a current significant in utero exposure (caloric-dense high-fat maternal diet) induces site-specific alterations in fetal hepatic H3 acetylation. Employing ChIP, we extend these observations to link modifications of H3 acetylation with alterations in gene-specific expression. These results suggest that a caloric-dense maternal diet leading to obesity epigenetically alters fetal chromatin structure in primates via covalent modifications of histones and hence lends a molecular basis to the fetal origins of adult disease hypothesis.

Methyl group metabolism in maternal and fetal hepatic tissues is altered by diabetes and retinoic acid treatment

Adequate methyl group supply is vital for numerous biological reactions, especially during development. A lack of methyl groups, B‐vitamins, and changes in relevant enzymes, can result in a host of pathologies, including neural tube defects. If inappropriately activated, hepatic glycine N‐methyltransferase (GNMT) a regulatory enzyme in folate and methyl group metabolism, leads to methyl deficiency. We have shown that both a diabetic state and retinoic acid (RA) treatment result in activation of GNMT. The goal of this pilot study was to determine if induction of GNMT during pregnancy alters methyl group metabolism in the fetus. Non‐diabetic and diabetic (STZ) pregnant rats treated with and without RA, were sacrificed on d 18 of gestation for collection of blood and liver tissue. Two hepatic immunoreactive proteins were clearly detected (32 and ~36 kD) in liver tissue. The lighter form (L‐GNMT) was more responsive in maternal liver, whereas the heavier protein (H‐GNMT) dominated in fetal liver. Rats receiving both RA and STZ exhibited elevated maternal hepatic GNMT activity, L‐GNMT abundance, and DNA hypomethylation, and reduced circulating homocysteine levels. Fetal liver expressed only H‐GNMT which was slightly responsive to treatment; however, L‐GNMT expression was evident in the combined treatment group. Fetal hepatic tissue did not exhibit changes in DNA methylation, due to the lack of L‐GNMT expression.

TERATOID HEPATOBLASTOMA

We present the case of a Middle Eastern child, diagnosed and treated at 8 months of age for a hepatic neuroblastoma. After surgical removal of a 7 cm mass of the left liver lobe at our institution when the child was 15 months of age, the tumor was reclassified as a teratoid hepatoblastoma. The tumor was composed of fetal and embryonal hepatic tissue, undifferentiated tissue, and a teratoid background of loose mesenchymal tissue containing osteoid, squamous, and mucinous epithelium. We speculate on the histogenesis of teratoid hepatoblastoma and discuss its association with chemotherapy.

Maternal Supplementation of L‐carnitine Alters the Maternal and Fetal IGF Axis in Swine

We investigated the influence of supplemental L-carnitine on the uterine and placental insulin-like growth factor (IGF) system, circulating IGF-I in pregnant gilts, circulating fetal IGF-II, and the expression of IGFs and binding proteins in the hepatic tissue and skeletal muscle of fetal pigs at day 40, 55, and 70 of gestation. Maternal IGF-I plasma concentrations decreased (P<0.01) from day 0 to 70 with no influence of supplemental carnitine. Fetal IGF-II levels were lower (P=0.09) and fetuses were heavier (P=0.06) at d 70 in gilts fed L-carnitine. Insulin-like growth factor binding protein-3 (IGFBP-3; P=0.05) and IGFBP-5 mRNA increased (P=0.01) and IGF-I mRNA numerically increased (P=0.16) in the endometrium of gilts supplemented with L-carnitine. From d 40 to 55, IGF-I, IGFBP-3, and IGFBP-5 mRNA decreased (P<0.04) in the myometrium. Placental IGF-I mRNA tended to increase (P=0.08) as gestation length increased. Fetal IGF-I mRNA was lower (P=0.05) in hepatic tissue from dams supplemented L-carnitine. As gestation length increased, IGFBP-3 mRNA increased (P<0.02) in the fetal hepatic tissue and IGF-I mRNA increased (P<0.02) in skeletal muscle. Fetal skeletal muscle IGFBP-3 mRNA decreased (P=0.04) from day 40 to 55, but increased (P=0.02) from day 55 to 70. These data demonstrate that L-carnitine supplementation and gestation length alter the expression of the IGF axis at the fetal-maternal interface in swine. These changes in the IGF axis could play a fundamental role in porcine fetal growth and development.

Shearing ewes at mid-pregnancy is associated with changes in fetal growth and development

This study investigated the effect of mid-pregnancy shearing (at Day 70 of pregnancy, P70) on herbage intake of grazing single- and twin-bearing ewes, lamb birth weight, and cold resistance of new-born lambs. At pregnancy diagnosis on P50, 30 single-bearing and 30 twin-bearing ewes were allocated either to be shorn at P70 (n = 15 for each pregnancy rank) or to remain unshorn to serve as controls (n = 15 for each pregnancy rank). All ewes were mated over a 3-day period with synchronisation of their oestrus. Herbage intake was measured indirectly from in vitro pasture digestibility and faecal output of grazing ewes, with the use of intra-ruminal chromium slow-release capsules, over six 5-day periods from P64 to P105. The weights of placental and fetal tissues were assessed in a subgroup of 16 ewes at P140 and P141. In the remaining sheep, lamb liveweight at birth and during lactation until weaning at 103 days of age was measured, and cold-resistance of new-born lambs was assessed by measuring summit metabolic rate (SMR) by indirect calorimetry. Ewe liveweight (corrected for fleece weight), condition score, and herbage intake during pregnancy were not affected by shearing treatment. Mid-pregnancy shearing did not affect placental weight, but increased the relative weights (i.e. g/kg liveweight) of fetal thyroid gland and lungs and reduced the relative weight of adrenal glands and heart. The ratio of secondary to primary wool follicles in near-term fetal skin was about 10% higher in offspring of shorn than of unshorn ewes. The metabolic rate of fetal hepatic tissue was increased by mid-pregnancy shearing, particularly in twin fetuses, possibly indicative of an increase in placental transport of nutrients to the fetuses. This conclusion is supported by the greater birth weight (average response 0.5 kg) of lambs born to ewes shorn at mid-pregnancy. Mid-pregnancy shearing also increased the SMR of new-born twin lambs by 16%, but decreased the SMR of singleton lambs by 26%. These results indicate that mid-pregnancy shearing can increase lamb birth weight without increasing ewe herbage intake or placental weight. An increase in the efficiency of nutrient uptake by the placenta is implied, and possible effects on the activity of thermogenic tissues are discussed.

Effect of maternal nutrition on brown adipose tissue and its prolactin receptor status in the fetal lamb.

We investigated the influence of maternal nutritional enhancement during the second half of gestation on prolactin receptor (PRLR) abundance in fetal brown adipose tissue (BAT) and liver close to term (i.e. 141-144 d gestation). Ewes were provided with 100% (i.e. control; n = 8) or 150% (i.e. well-fed; n = 7) of their metabolic requirements from 80 to 144 d gestation. Crude plasma membranes were prepared from fetal BAT and hepatic tissue, and individual molecular weight isoforms for the long and short forms of the PRLR were detected by immunoblotting. Mitochondrial preparations were prepared from BAT to measure the amount of the BAT-specific mitochondrial uncoupling protein-1 and its thermogenic activity (i.e. guanosine 5'-diphosphate binding). Fetuses sampled from well-fed ewes were heavier (controls, 3927 +/- 196 g; well-fed, 4783 +/- 219 g; p = 0.01) but possessed less BAT per kilogram body weight (controls, 5.92 +/- 0.43 g/kg; well-fed, 3.85 +/- 0.19 g/kg; p = 0.001), which had a greater uncoupling protein-1 abundance (controls, 56 +/- 5% of reference; well-fed, 78 +/- 9% of reference; p < 0.01) and higher thermogenic activity (controls, 157 +/- 41 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; well-fed, 352 +/- 36 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; p < 0.01) than controls. Multiple isoforms of the long and short forms of the P1LR were detected in all tissues. BAT from well-fed fetuses had a higher abundance of the 15-kD isoform of the long form of the PRLR (controls, 1.6 +/- 0.4 densitometric units; well-fed, 16.3 +/- 2.0 densitometric units; p < 0.001). This isoform was not detected in hepatic tissue. Maternal nutrient intake had no effect on any other isoforms of the PRLR in BAT or liver. In conclusion, increasing the quantity of feed provided in late gestation acts to promote fetal weight and BAT maturation, the combination of which will enhance neonatal viability.

Identification of a novel zinc finger gene, zf5-3, as a potential mediator of neuroblastoma differentiation.

We established a unique parental neuroblastoma cell line, NUB-7, which mimics the bipotentiality of neuroblastoma in vivo along neuronal and Schwann cell lineages following dibutyryl cAMP and retinoic acid treatments, respectively. Differential display identified a putative novel zinc finger gene as a potential differentiation-responsive gene coincident with retinoic acid treatment of NUB-7. This cDNA clone, now designated zf5-3, was mapped to chromosome 19 using somatic cell hybrids, and a larger cDNA clone further localized this gene to band 13.1-13.2 by fluorescent in situ hybridization. zf5-3 possesses 4 characteristic zinc finger DNA-binding motifs as determined by its nucleic acid and proposed amino acid sequence. Expression of zf5-3 is restricted to fetal neuronal, hepatic and renal tissues and their tumor-derived cell lines, including 8/9 neuroblastomas and 2/2 malignant rhabdoid tumors of kidney. The restricted expression in the kidney of zf5-3 to collecting tubules and ureter epithelium is suggestive of an ectodermal histogenesis of malignant rhabdoid tumors of kidney. During development of the fetal human brain, high levels of zf5-3 mRNA are restricted to the mitotically active, undifferentiated neuroblasts. Morphological evidence of overt differentiation was generally accompanied by a marked loss in zf5-3 expression. Therefore, the neuronal tissue expression profile and the down-regulation coincident with retinoic acid-induced neuroblastoma maturation implicate zf5-3 as a potential mediator of their differentiation.

Inhibition of human prenatal biosynthesis of all- trans-retinoic acid by ethanol, ethanol metabolites, and products of lipid peroxidation reactions : A possible role for CYP2E1

Biotransformation of all- trans-retinol (t-ROH) and all- trans-retinal (t-RAL) to all- trans-retinoic acid (t-RA) in human prenatal hepatic tissues (53–84 gestational days) was investigated with HPLC using human adult hepatic tissues as positive controls. Catalysis of the biotransformation of t-ROH by prenatal human cytosolic fractions resulted in accumulation of t-RAL with minimal t-RA. Oxidations of t-ROH catalyzed by prenatal cytosol were supported by both NAD + and NADP +, although NAD + was a much better cofactor. In contrast, catalysis of the oxidation of t-RAL to t-RA appeared to be solely NAD + dependent. Substrate K m values for conversions of t-ROH to t-RAL and of t-RAL to t-RA were 82.4 and 65.8 μM, respectively. At concentrations of 10 and 90 mM, ethanol inhibited the conversion of t-ROH to t-RAL by 25 and 43%, respectively, but did not inhibit the conversion of t-RAL to t-RA significantly. In contrast, acetaldehyde reduced the conversion of t-RAL to t-RA by 25 and 87% at 0.1 and 10 mM respective concentrations. Several alcohols and aldehydes known to be generated from lipid peroxides also exhibited significant inhibition of t-RA biosynthesis in human prenatal hepatic tissues. Among the compounds tested, 4-hydroxy-2-nonenal (4-HNE) was highly effective in inhibiting the conversion of t-RAL to t-RA. A 20% inhibition was observed at a concentration of only 0.001 mM, and nearly complete inhibition was produced at 0.1 mM. Human fetal and embryonic hepatic tissues each exhibited significant CYP2E1 expression as assessed with chlorzoxazone 6-hydroxylation, a highly sensitive western blotting technique, and reverse transcriptase-polymerase chain reaction (PCR) (RT-PCR), suggesting that lipid peroxidation can be initiated via CYP2E1-catalyzed ethanol oxidation in human embryonic hepatic tissues. In summary, these studies suggest that ethanol may affect the biosynthesis of t-RA in human prenatal hepatic tissues directly and indirectly. Ethanol and its major oxidative metabolite, acetaldehyde, both inhibit the generation of t-RA. Concurrently, the CYP2E1-catalyzed oxidation of ethanol can initiate lipid peroxidation via generation of a variety of free radicals. The lipid peroxides thereby generated could then be further converted via CYP2E1-catalyzed reactions to alcohols and aldehydes, including 4-HNE, that act as potent inhibitors of t-RA synthesis.

Expression of Cytochrome P450RAI (CYP26) in Human Fetal Hepatic and Cephalic Tissues

PCR amplifications with two sets of degenerate primers that were targeted to CYP26-specific regions were performed with cDNAs from human fetal liver and brain as templates. PCR products were purified, cloned, sequenced and analyzed with the BLAST program. Our results revealed expression of CYP26 in both human fetal liver and brain. Furthermore, human fetal CYP26 cDNA exhibited 99.2%–100% nucleotide sequence identity to its adult counterpart. Novel isoforms, that would have indicated additional CYP26 genes, were not found. A Northern blot containing poly(A+)RNAs from 43 human adult and 7 human fetal tissues was tested for CYP26 expression. We were able to detect CYP26 message in most tissues but hybridization signals varied in intensity. Highest levels of transcription were in adult liver, heart, pituitary gland, adrenal gland, placenta and regions of the brain. CYP26 expression in fetal tissues was strongest in the brain and comparable with message levels in adult tissues.

Identification of a Unique Form of Protein C in the Ovine Fetus: Developmentally Linked Transition to the Adult Form

To investigate fetal development of protein C, a pregnant ovine model was used. Protein C was isolated from ovine plasma, and a polyclonal antibody was raised. Citrated plasma was obtained from undisturbed chronically catheterized fetal lambs. On Western blot, nonreduced adult ovine protein C had a molecular mass of 70 kD. Fetal ovine protein C was determined to have a molecular mass of 4 to 6 kD larger than the adult molecule. Crossed immunoelectrophoresis demonstrated slightly increased anodal migration of the fetal form. Isoelectric focusing demonstrated a decreased pI of the fetal molecule (4.45 versus 4.6). The ovine protein C molecules were deglycosylated with N-glycanase. Deglycosylated fetal protein C migrated more similarly to the adult form, although a portion of the fetal form persisted. These experiments demonstrate the first example of a unique fetal form of a vitamin K-dependent protein and are compatible with increased glycosylation of fetal ovine protein C. It is speculated that altered posttranslational processing may exist as a general process by which certain coagulation proteins are modified during fetal development. mRNA was isolated from maternal and fetal hepatic tissue and analyzed by Northern hybridization. Fetal plasma concentration and hepatic mRNA for protein C were both 40% of normal maternal values from midgestation onward. At term, protein C mRNA increased to adult range (p < 0.025), although plasma protein C concentration decreased slightly (p < 0.001). A transition from fetal to adult protein C form was found beginning 6 d before term birth, with a doubling time of 24 h. These data are compatible with a gestationally determined maturation of ovine protein C.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of ovine growth hormone (oGH) and ovine placental lactogen (oPL) binding to fetal and adult hepatic tissue in sheep: evidence that oGH and oPL interact with a common receptor

The GH receptor (GHR) plays a key role in postnatal growth regulation. Although plasma concentrations of GH are high during fetal life, its role during fetal development is not well understood. Recent data suggest that GHR are present in fetal hepatic tissue as early as 51 days gestation. However, the levels of GHR expression are markedly lower in fetal hepatic tissue compared to postnatal values, and there are conflicting data suggesting that ovine placental lactogen (oPL) and oGH share a common receptor. Given the uncertainty about whether oPL acts via the oGHR or a distinct receptor, we performed ligand binding and affinity cross-linking studies on hepatic microsomal membranes from adult castrated male, pregnant female, and fetal sheep. Ligand binding assays at a constant concentration of membranes showed that [125I]oPL yielded consistently higher (P < 0.001) specific binding (59.5 +/- 6.4%, 30.5 +/- 5.7%, and 7.6 +/- 2.4% for castrated male, pregnant female, and fetal sheep, respectively) compared to [125I]oGH (17.8 +/- 4.7%, 5.0 +/- 1.6%, and 1.2 +/- 0.4% for castrated male, pregnant female, and fetal sheep, respectively). Cross-reactivity studies showed that unlabeled oPL was consistently more potent than unlabeled oGH in displacing either of the labeled ligands. The dissociation constant (Kd) for oPL binding ranged from 0.16-0.40 nM and was not changed by solubilization with Triton X-100. Equilibrium binding analysis for oGH showed lower affinity for hepatic microsomal membranes (Kd, 1.7-3.2 nM) in each of the three groups of animals. Affinity cross-linking of microsomal membranes from castrated male and pregnant female sheep liver showed four major cross-linked complexes with both [125I]oPL and [125I]oGH, with mol wt of 150, 97, 75, and 60 kilodaltons. All four bands were identified with both ligands. Unlabeled oPL showed markedly higher potency than unlabeled oGH in reducing the signal of the [125I]oPL cross-linked complexes, whereas unlabeled oGH and oPL showed comparable potencies in reducing the signal of the [125I]oGH complexes. Immunoprecipitation of detergent-solubilized hepatic microsomal membranes from pregnant and fetal sheep using a panel of monoclonal antibodies raised against the extracellular region of the rabbit GHR showed potent immunological recognition of the [125I]oPL-receptor complexes. We suggest that oGH and oPL bind to a common or a related receptor protein(s). It is possible that differences in receptor dimerization or association with other membrane proteins are the basis of the differences in affinity and biological actions of the two hormones.