Ovine Fetal Liver Research Articles

Upregulation of hepatic nuclear receptors in extremely preterm ovine fetuses undergoing artificial placenta therapy

Objective Extremely preterm infants have low Nuclear Receptor (NR) expression in their developing hepatobiliary systems, as they rely on the placenta and maternal liver for compensation. NRs play a crucial role in detoxification and the elimination of both endogenous and xenobiotic substances by regulating key genes encoding specific proteins. In this study, we utilized an Artificial Placenta Therapy (APT) platform to examine the liver tissue expression of NRs of extremely preterm ovine fetuses. This fetal model, resembling a “knockout placenta,” lacks placental and maternal support, while maintaining a healthy extrauterine survival. Methods Six ovine fetuses at 95 ± 1 d gestational age (GA; term = ∼150 d)/∼600 g delivery weight were maintained on an APT platform for a period of 120 h (APT Group). Six age-matched, in utero control fetuses were delivered at 99–100 d GA (Control Group). Fetal liver tissue samples and blood samples were collected at delivery from both groups and assessed mRNA expression of NRs and target transporters involved in the hepatobiliary transport system using quantitative PCR. Data were tested for group differences with ANOVA (p < .05 deemed significant). Results mRNA expression of NRs was identified in both the placenta and the extremely preterm ovine fetal liver. The expression of HNF4α, LRH1, LXR, ESR1, PXR, CAR, and PPARα/γ were significantly elevated in the liver of the APT Group compared to the Control Group. Moreover, target transporters NTCP, OATP1B3, BSEP, and MRP4 were upregulated, whereas MRP2 and MRP3 were unchanged. Although there was no evidence of liver necrosis or apoptotic changes histologically, there was an impact in the fetal liver of the ATP group at the tissue level with a significant increase in TNFα mRNA, a cytokine involved in liver inflammation, and blood elevation of transaminases. Conclusion A number of NRs in the fetal liver were significantly upregulated after loss of placental-maternal support. However, the expression of target transporter genes appeared to be insufficient to compensate role of the placenta and maternal liver and avoid fetal liver damage, potentially due to insufficient excretion of organic anions.

Dietary rumen-protected L-arginine or N-carbamylglutamate attenuated fetal hepatic inflammation in undernourished ewes suffering from intrauterine growth restriction.

This study aimed to explore whether dietary rumen-protected L-arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation to feed-restricted pregnant ewes counteracts fetal hepatic inflammation and innate immune dysfunction associated with intrauterine growth retardation (IUGR) in ovine fetuses. On d 35 of pregnancy, twin-bearing Hu ewes (n = 32) were randomly assigned to 4 treatment groups (8 ewes and 16 fetuses per group) and fed diets containing 100% of the NRC requirements (CON), 50% of the NRC requirements (RES), RES + RP-Arg (20 g/d) (RESA), or RES + NCG (5 g/d) (RESN). At 08:00 on d 110 of gestation, fetal blood and liver tissue samples were collected. The levels of triglyceride, free fatty acid, cholesterol and β-hydroxybutyrate in the fetal blood of RESA and RESN groups were lower (P < 0.05) than those of the RES group, but were higher (P < 0.05) than those of the CON group. The interleukin (IL)-6 and IL-1 levels in fetal blood and liver tissue as well as the myeloid differentiation primary response 88 (MyD88), transforming growth factor β (TGFβ), and nuclear factor kappa B (NF-κB) mRNA levels in the fetal liver were decreased (P < 0.05) by the NCG or RP-Arg supplementation compared to the RES treatment. Similarly, the toll-like receptor (TLR)-4, MyD88, TGFβ, and p-c-Jun N-terminal kinase (JNK) protein levels in the fetal liver were reduced (P < 0.05) in the NCG and RP-Arg -supplemented groups compared to the RES group. These results showed that dietary supplementation of RP-Arg or NCG to underfed pregnant ewes could protect against IUGR fetal hepatic inflammation via improving lipid metabolism, down-regulating the TLR-4 and the inflammatory JNK and NF-κB signaling pathways, and decreasing cytokine production in ovine fetal blood and liver tissue.

Effects of intrauterine growth restriction during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver

This study investigated the effect of intrauterine growth restriction (IUGR) during late pregnancy on the cell apoptosis and related gene expression in ovine fetal liver. Eighteen time-mated Mongolian ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.18 MJ ME kg BW−0.75 d−1, n = 6), Restricted Group 2 (RG2, 0.33 MJ ME kg BW−0.75 d−1, n = 6) and a Control Group (CG, ad libitum, 0.67 MJ ME kg BW −0.75 d −1, n = 6). Fetuses were recovered at slaughter on d 140. Fetal liver weight, DNA content and protein/DNA ratio, proliferation index, cytochrome c, activities of Caspase-3, 8, and 9 were examined, along with relative expression of genes related to apoptosis. Fetuses in both restricted groups exhibited decreased BW, hepatic weight, DNA content, and protein/DNA ratio when compared to CG (P < 0.05), as well as reduced proliferation index (P < 0.05). However, the increased numbers of apoptotic cells in fetal liver were observed in both restricted groups (P < 0.05). Fetuses with severe IUGR (RG1) exhibited increased (P < 0.05) activities of Caspase-3, 8, 9, as higher levels of mitochondrial cytochrome c in fetal liver; intermediate changes were found in RG2 fetuses, but the difference were not significant (P > 0.05). Hepatic expression of gene related to apoptosis showed reduced protein 21 (P21), B-cell lymphoma 2 (Bcl-2) and apoptosis antigen 1 ligand (FasL) expression in RG1 and RG2 (P < 0.05). In contrast, the increased hepatic expression of protein 53 (P53), Bcl-2 associated X protein (Bax) and apoptosis antigen 1 (Fas) in both IUGR fetuses were found (P < 0.05). These results indicate that the fetal hepatocyte proliferation were arrested in G1 cell cycle, and the fetal hepatocyte apoptosis was sensitive to the IUGR resulted from maternal undernutrition. The cell apoptosis in IUGR fetal liver were the potential mechanisms for its retarded proliferation and impaired development.

Conjugation and deconjugation reactions within the fetoplacental compartment in a sheep model: a key factor determining bisphenol A fetal exposure.

The widespread human exposure to bisphenol A (BPA), an endocrine disruptor targeting developmental processes, underlines the need to better understand the mechanisms of fetal exposure. Animal studies have shown that at a late stage of pregnancy BPA is efficiently conjugated by the fetoplacental unit, mainly into BPA-glucuronide (BPA-G), which remains trapped within the fetoplacental unit. Fetal exposure to BPA-G might in turn contribute to in situ exposure to bioactive BPA, following its deconjugation into parent BPA at the level of fetal sensitive tissues. The objectives of our study were 1) to characterize the BPA glucurono- and sulfoconjugation capabilities of the ovine fetal liver at different developmental stages, 2) to compare hepatic conjugation activities in human and sheep, and 3) to evaluate the extent of BPA conjugation and deconjugation processes in placenta and fetal gonads. At an early stage of pregnancy, and despite functional sulfoconjugation activity, ovine fetuses expressed low hepatic BPA conjugation capabilities, suggesting that this stage of development represents a critical window in terms of BPA exposure. Conversely, the late ovine fetus expressed an efficient detoxification system that metabolized BPA into BPA-G. Hepatic glucuronidation activities were quantitatively similar in adult sheep and humans. In placenta, BPA conjugation and BPA-G deconjugation activities were relatively balanced, whereas BPA-G hydrolysis was systematically higher than BPA conjugation in gonads. The possible reactivation of BPA-G into BPA could contribute to an increased exposure of fetal sensitive tissues to bioactive BPA in situ.

Effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver

This study investigated the effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Eighteen ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.175MJMEkgBW−0.75d−1, n=6), Restricted Group 2 (RG2, 0.33MJMEkgBW−0.75d−1, n=6) and a Control Group (CG, ad libitum, 0.67MJMEkgBW−0.75d−1, n=6). Fetuses were recovered at slaughter on d 140. Fetuses in the RG1 group exhibited decreased (P<0.05) liver weight, total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD), cholinesterase (CHE), total protein (TP), globulin (GLB), and alanine transaminase (ALT). In addition, intermediate changes were found in the RG2 fetuses, including decreased liver weight, T-AOC and CHE (P<0.05). In contrast, increases in fetal hepatic collagen fibers and reticular fibers, glutathione peroxidase (GSH-Px), malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOs), monoamine oxidase (MAO), albumin (ALB)/GLB, aspartate transaminase (AST), and AST/ALT were found in the RG1 fetuses (P<0.05). The RG2 fetuses had increased fetal hepatic collagen fibers, NOs and MAO (P<0.05) relative to the control fetuses. These results indicate that impaired fetal hepatic growth, fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition.

Alcohol exposure during late ovine gestation alters fetal liver iron homeostasis without apparent dysmorphology

High levels of alcohol (ethanol) exposure during fetal life can affect liver development and can increase susceptibility to infection after birth. Our aim was to determine the effects of a moderate level of ethanol exposure in late gestation on the morphology, iron status, and inflammatory status of the ovine fetal liver. Pregnant ewes were chronically catheterized at 91 days of gestation (DG; term ~145 DG) for daily intravenous infusion of ethanol (0.75 g/kg maternal body wt; n = 8) or saline (n = 7) over 1 h from 95 to 133 DG. At necropsy (134 DG), fetal livers were collected for analysis. Liver weight, general liver morphology, hepatic cell proliferation and apoptosis, perivascular collagen deposition, and interleukin (IL)-1β, IL-6, or IL-8 mRNA levels were not different between groups. However, ethanol exposure led to significant decreases in hepatic content of ferric iron and gene expression of the iron-regulating hormone hepcidin and tumor necrosis factor (TNF)-α (all P < 0.05). In the placenta, there was no difference in transferrin receptor, divalent metal transporter 1, and ferritin mRNA levels; however, ferroportin mRNA levels were increased in ethanol-exposed animals (P < 0.05), and ferroportin protein tended to be increased (P = 0.054). Plasma iron concentration was not different between control and ethanol-exposed groups; control fetuses had significantly higher iron concentrations than their mothers, whereas maternal and fetal iron concentrations were similar in ethanol-exposed animals. We conclude that daily ethanol exposure during the third-trimester-equivalent in sheep does not alter fetal liver morphology; however, decreased fetal liver ferric iron content and altered hepcidin and ferroportin gene expression indicate that iron homeostasis is altered.

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans.

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good long-term engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

Morphologic Alterations in Ovine Placenta and Fetal Liver Following Induced Severe Placental Insufficiency

Umbilical-placental embolization with microspheres has been used as a model of placental insufficiency and intrauterine growth restriction (IUGR). However, the effects of embolization on placental structure and organ morphology of the resulting IUGR fetus are relatively unexplored. In this study using ovine fetuses, we determined the location and distribution of microspheres within the placenta and explored the extent of placental and fetal organ morphologic changes induced by placental embolization. We hypothesized that microspheres administered into the umbilical circulation over 4 days would cause placental damage without significant morphologic alterations in fetal kidney or liver. Eleven pregnant sheep at 118 +/- 1 (SE) days' gestation were studied. In six fetuses, embolization was induced by injections of 15-microm diameter microspheres on 4 successive days into the fetal descending aorta proximal to the umbilical arteries. Five fetuses served as time controls. In embolized fetuses, microspheres were detected in the placenta embedded in the fetal cytotrophoblastic layer or maternal parenchyma adjacent to villous cytotrophoblasts. Fetal cytotrophoblasts appeared normal except for loss of distinct separation between fetal and maternal cell layers. Microspheres were also detected in the fetal membranes within capillaries. The body weights of embolized fetuses were lower than controls, as were the body weight-normalized liver but not kidney weights. In the liver of the embolized fetuses, the number of hematopoietic cell clusters was markedly reduced, whereas the fetal kidneys appeared normal. We conclude that after 4 days of umbilical-placental embolization, microspheres were concentrated at the fetal villi proximal to the apical maternal-fetal interface and in the fetal membranes. There were noticeable morphologic changes in the embolized placentas, with no apparent gross damage to the placenta. The reduction in fetal liver weight and liver extramedullary hematopoietic cell abundance associated with embolization may predispose the fetus to alterations in liver function that could persist after birth.

Induction of glutamate dehydrogenase in the ovine fetal liver by dexamethasone infusion during late gestation.

Glucocorticoids near term are known to upregulate many important enzyme systems prior to birth. Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes both the reversible conversion of ammonium nitrogen into organic nitrogen (glutamate production) and the oxidative deamination of glutamate resulting in 2-oxoglutarate. The activity of this enzyme is considered to be of major importance in the development of catabolic conditions leading to gluconeogenesis prior to birth. Ovine hepatic GDH mRNA expression and activity were determined in near-term (130 days of gestation, term 147 +/- 4 days) control and acutely dexamethasone-treated (0.07 mg(-1) hr(-1) for 26 hr) fetuses. Dexamethasone infusion had no effect on placental or fetal liver weights. Dexamethasone infusion for 26 hr significantly increased hepatic GDH mRNA expression. This increased GDH mRNA expression was accompanied by an increase in hepatic mitochondrial GDH activity, from 30.0 +/- 7.4 to 58.2 +/- 8.1 U GDH/U CS (citrate synthase), and there was a significant correlation between GDH mRNA expression and GDH activity. The generated ovine GDH sequence displayed significant similarity with published human, rat, and murine GDH sequence. These data are consistent with the in vivo studies that have shown a redirection of glutamine carbon away from net hepatic glutamate release and into the citric acid cycle through the forward reaction catalyzed by GDH, i.e., glutamate to oxoglutarate.

Regulation of erythropoietin gene expression in fetal sheep by glucocorticoids.

Erythropoietin (Epo) gene expression in ovine fetal liver and kidneys was measured by competitive RT-PCR in situations in which fetal glucocorticoid status was altered. Bilateral adrenalectomy at 120 +/- 0.3 days gestation (term is 145-150 days) caused a significant (P < 0.05) 5-fold increase in renal Epo messenger RNA (mRNA) levels at 145 +/- 1 days compared to those in age-matched controls. With cortisol replacement in adrenalectomized fetuses, renal Epo mRNA levels dropped to 17% of this values (P < 0.05). Cortisol infusion (230 micrograms/h for 48 h) at 108 +/- 1 day decreased renal Epo gene expression significantly (P < 0.01) to 23% of the control value; dexamethasone treatment of the ewe at midgestation (0.76 mg/h for 48 h) also decreased fetal, but not adult, renal Epo mRNA levels (to 12% of control value; P < 0.01). Fetal and maternal liver Epo mRNA levels were unaffected by glucocorticoid status at any stage of pregnancy. Thus, glucocorticoids can influence fetal renal, but not maternal, Epo gene expression. In the presence of high concentrations of fetal glucocorticoids, plasma Epo values were consistently 4-5 mU/ml, close to the sensitivity of the assay, whereas in seven adrenalectomized fetuses, the plasma Epo value was 9.1 +/- 1.4 mU/ml.

Differential expression of 11 beta-hydroxysteroid dehydrogenase 1 and 2 in the developing ovine fetal liver and kidney.

In adult mammals, liver and kidney are the two major sites of biosynthesis for 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) 1 and 2 respectively. In the present study, the expression of these two isozymes in the developing ovine fetal liver and kidney was characterized. Livers and kidneys were obtained from fetal sheep at days 85, 100-120 and 140-143 of gestation (term = 145 days). Tissue levels of 11 beta-HSD2 mRNA were assessed by Northern blot analysis. 11 beta-HSD dehydrogenase and reductase activities in tissue homogenates were determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates respectively. The unidirectional 11 beta-HSD2 dehydrogenase activity was identified by its distinct cofactor preference (NAD), and by its unique ability to metabolize dexamethasone (Dex). In the liver, 11 beta-HSD1 dehydrogenase and reductase activities were present by day 85, and their levels did not change between days 85 and 100-120 but increased more than twofold at days 140-143. This was consistent with changes we reported previously in the fetal hepatic 11 beta-HSD1 mRNA. 11 beta-HSD1 reductase activity was always higher than the dehydrogenase activity. 11 beta-HSD2 mRNA and activity were undetectable in the fetal liver at all three ages. By contrast, 11 beta-HSD2 mRNA was present in the fetal kidney by day 85, and its abundance increased progressively thereafter. There was a parallel increase in the renal 11 beta-HSD2 activity. Dex was also converted to 11-dehydro-Dex by the fetal kidney. In keeping with the absence of the full-length 11 beta-HSD1 mRNA, 11 beta-HSD1 activity was undetectable in the kidney. These results indicate that (1) 11 beta-HSD1 and 2 genes are differentially expressed and regulated in the fetal liver and kidney during development, (2) since the hepatic 11 beta-HSD1 reductase activity is always higher than the dehydrogenase activity, the fetal liver may be a potential extra-adrenal source of cortisol, and (3) 11 beta-HSD2 in the kidney may play a very important role in protecting the fetus from elevated levels of bioactive glucocorticoids.

Ontogeny of a specific high-affinity binding site for ovine placental lactogen in fetal and postnatal liver

Ovine placental lactogen (oPL) exerts actions in sheep and rodent fetal tissues that growth hormone (GH) does not. However, in postnatal tissues, both oPL and GH possess these activities. Although a high-affinity binding site for oPL in ovine fetal liver has been reported, some investigators believe this to be the GH receptor. It was our objective to discriminate between oPL and GH binding to fetal liver microsomes using competitive saturation analyses. Microsomal membranes from fetal liver (Days 60, 90, 105, 120, and 135 of gestation) and postnatal liver (1 wk of age) were incubated with increasing amounts of [ 125I]oPL in the absence or presence of a 100-fold molar excess of unlabeled oPL. Saturable binding of [ 125I]oPL was observed with fetal liver and postnatal liver microsomes. The K d of the oPL-binding site in fetal liver was 122.1 ± 8.2 pM (mean ± standard error), and receptor concentrations remained relatively constant (9.8 ± 1.1 fmol/mg of membrane protein) across gestation. The highest concentration of oPL binding was detected in 1-wk postnatal liver microsomes (53.0 fmol/mg of membrane protein). Saturation analyses using [ 125I]GH and [ 125I] prolactin (PRL) were also conducted with fetal liver membrane preparations. Although specific binding for these two radiolabeled ligands was observed in control tissues, no specific binding was observed in fetal liver. These data are in agreement with earlier reports that a high-affinity binding site for oPL exists in fetal tissues. The fact that saturable binding could not be demonstrated for either GH or PRL with fetal liver microsomes contradicts recent suggestions that oPL is binding the GH receptor.

Propranolol elimination by right and left ovine fetal liver: Studies in the intact isolated perfused organ: [formula omitted]. Depts. of Medicine and Surgery, Austin & Repatriation Medical Centre, Melbourne 3081, and ∗Victorian College of Pharmacy, Melbourne 3052, Australia

Propranolol elimination by right and left ovine fetal liver: Studies in the intact isolated perfused organ: [formula omitted]. Depts. of Medicine and Surgery, Austin & Repatriation Medical Centre, Melbourne 3081, and ∗Victorian College of Pharmacy, Melbourne 3052, Australia

The growth hormone receptor messenger ribonucleic acid present in ovine fetal liver is a variant form.

Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

The growth hormone receptor messenger ribonucleic acid present in ovine fetal liver is a variant form

Specific binding of ovine GH (oGH) to microsomal membranes isolated from fetal sheep liver is slight to nonexistent. The complementary DNA sequence encoding the oGH receptor (oGHR) has been reported, and Northern blot analysis has indicated that oGHR messenger RNA (mRNA) is present in fetal liver and skeletal muscle from mid- to late gestation. In human tissues, the GHR mRNA exists in multiple forms, including the deletion of exon 3 and variable 5'-untranslated regions. In rodents, the GHR mRNA exists in two forms, one encoding the membrane-bound receptor and the other encoding the soluble GH-binding protein. To further characterize the oGHR mRNA transcript present in ovine fetal liver during gestation, we designed a series of primers to be used in reverse transcriptase-polymerase chain reactions (RT-PCR), which generate products that span from the 5'-untranslated region through the coding region of the oGHR mRNA. Nucleotide sequences of the resulting complementary DNAs revealed that an oGHR mRNA is present from mid- to late gestation (days 60-135) which contains the region analogous to exon 3 of the human GHR gene. However, the 5'-untranslated region previously reported in adult tissues was not present until day 135 of gestation in fetal liver, nor was it present in day 100 fetal skeletal muscle. Northern hybridization analysis indicates that the major oGHR transcript in day 105 fetal liver is 5.8 kilobases (kb) in size, with minor transcripts observed at 4.7 kb and three transcripts greater than 6.5 kb. By day 135 of gestation, the transcript size is the same as that observed in day 100 pregnant ewe liver (5.5 kb). We conclude that the oGHR mRNA present in midgestation fetal liver differs structurally from the transcript present in late gestation fetal liver and adult liver, and this difference may explain the lack of specific GH binding to ovine fetal liver membranes. Furthermore, our results suggest that there is a developmental switch in the structure of oGHR mRNA that occurs shortly before term, potentially preparing the fetus to respond to GH postnatally.

Two forms of the prolactin receptor messenger ribonucleic acid are Present in ovine fetal liver and adult ovary

Previous binding studies indicated that there is little to no specific prolactin binding in ovine fetal liver and adult ovary. Therefore, we sought to determine if ovine prolactin receptor (PRLR) mRNA is present in those tissues. Primers were designed from the bovine PRLR cDNA sequence for use in reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR analysis of ovine fetal liver total cellular RNA (tcRNA) isolated from days 60, 90, 105, 120 and 135 of gestation, and luteal tcRNA isolated from days 3, 7, 10, 13 and 16 of the estrous cycle revealed that PRLR mRNA was present in these tissues. However, two RT-PCR products were generated from both tissues. The two RT-PCR products did not differ between the two tissue sources in sequence, and were designated oPRLR-1 and oPRLR-2. Ovine PRLR-1 is 513 bp in length and is 96.4% identical to the bovine cDNA. Ovine PRLR-2 is identical to oPRLR-1 until nucleotide (nt) 420 at which point a 39 bp insertion occurs. This insertion occurs between Homology Boxes 1 and 2 within the cytoplasmic domain of the receptor, resulting in an 11 amino acid divergent sequence, followed by two stop codons. Ribonuclease-protection assay revealed that oPRLR-1 mRNA is the most abundant in these tissues. Our data indicate that two forms of oPRLR mRNA are Present in fetal liver and adult ovary, and that one form (oPRLR-2) is predicted to encode a truncated PRLR.

Development of primary culture of ovine fetal hepatocytes for studies of amino acid metabolism and insulinlike growth factors

We report the development and characterization of a system of primary culture of ovine fetal hepatocytes to aid in the understanding of the cellular regulation of fetal growth and metabolism with emphasis on amino acid metabolism and insulinlike growth factor gene expression and to allow comparison to in vivo studies. Hepatocytes were isolated from late gestation fetal lambs by in situ perfusion and collagenase digestion utilizing occlusion of the ductus venosus to limit intrahepatic shunting. Hepatocytes were cultured in media modified to mimic fetal concentrations of glucose, lactate, and amino acids. Ovine fetal hepatocytes in primary culture maintain the pattern of fetal amino acid production and utilization seen across the fetal liver in vivo. Specifically, there is a net production of serine and a net utilization of glycine. Cultured ovine fetal hepatocytes specifically increase tritiated thymidine incorporation in response to insulin and insulinlike growth factor II (IGF-II). IGF-II mRNA abundance is high and IGF-I mRNA is low in cultured ovine fetal hepatocytes as in the fetal sheep liver in vivo. These data demonstrate the successful isolation of ovine fetal hepatocytes that retain some of the characteristics of the ovine fetal liver while maintained in short-term culture.

Induction of cytosolic phosphoenolpyruvate carboxykinase in the ovine fetal liver by chronic fetal hypoglycemia and hypoinsulinemia.

Fetal glucose production has been observed in the chronically hypoglycemic, hypoinsulinemic (HH) fetal lamb. The purpose of this study was to test the hypothesis that induction of hepatic gluconeogenic enzymes occurs in this condition. The activities of both mitochondrial and cytosolic phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase, and glucose-6-phosphatase, three key enzymes of gluconeogenesis, were determined in fetal sheep liver from HH lambs and controls (CONT). Pregnant ewes were maintained chronically hypoglycemic by continuous hyperinsulinemic clamps from approximately 80 d of gestational age (53% of gestation) for 6 wk. Fetuses (gestational age: HH = 136 +/- 2.6, CONT = 133 +/- 3.7 d) were maintained chronically hypoglycemic [HH = 0.51 +/- 0.05 versus CONT = 1.22 +/- 0.11 mmol/L (9.2 +/- 1.0 versus 21.9 +/- 11.9 mg/dL)] and hypoinsulinemic (HH = 3.3 +/- 0.6 versus CONT = 12.0 +/- 2.2 microU/mL) and delivered by cesarean section after measurement of fetal glucose production rate. Hepatic cytosolic PEPCK was 6.0 +/- 1.4 nmol/min/mg protein in CONT and 19.7 +/- 2.5 in HH lamb (p < 0.05), whereas mitochondrial PEPCK was not different between the two groups. Neither fructose-1,6-diphosphatase or glucose-6-phosphatase activities nor plasma glucagon levels were different between groups. These results suggest that chronic fetal hypoglycemia and hypoinsulinemia prematurely induce hepatic cytosolic PEPCK in the fetal lamb. The observed fetal glucose production in the HH fetal lamb may be due to gluconeogenesis.

Purification and structural characterization of ovine placental lactogen

Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4.2 mg of oPL, with an approximately 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0.18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4.1 nmol/l) and oPRL (ED50 = 1.1 mumol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a lambda ZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides).(ABSTRACT TRUNCATED AT 250 WORDS)

Nutritional Regulation of the Placental Lactogen Receptor in Fetal Liver: Implications for Fetal Metabolism and Growth*

We have recently identified and purified from fetal liver a distinct receptor that mediates the effects of placental lactogen (PL) on amino acid transport, glycogen synthesis, and somatomedin production in fetal tissues. At present, the factors that regulate the number and affinity of PL receptors in the fetus are unknown. Since maternal nutrition plays a critical role in fetal metabolism and growth, we have examined the role of nutrition in the regulation of the PL receptor in fetal lambs. Pregnant ewes at 123-126 days gestation were fed ad libitum (FED), fasted for 3 days (FASTED), or fasted for 3 days and then refed for an additional 3 days (REFED). The ewes were then killed, and the binding of [125I]ovine (o) PL to hepatic microsomes from the fetal lambs was examined. Maternal fasting caused a 60-75% reduction in the specific binding of oPL to fetal liver; the effect of fasting was reversed in part by refeeding [specific binding per mg protein: FED, 11.8 +/- 2.2% (n = 8); FASTED, 2.8 +/- 0.4% (n = 7); REFED, 7.2 +/- 2.6% (n = 3)]. The decrease in oPL binding resulted from an 80% reduction in the number of fetal oPL-binding sites (Scatchard analysis); there were no changes in the affinity of the oPL receptor (Kd, 0.6 nM), the subunit structure of the receptor, or the degree of occupancy of the receptor in vivo by endogenous fetal hormones. The specific bindings of GH (0.6%), PRL (0.3%), and insulin (35%) to fetal liver were not affected by maternal fasting, indicating that caloric restriction exerted a specific effect on oPL binding in the fetus. The number of fetal oPL-binding sites was positively correlated with the fetal liver glycogen content (r = 0.69; P less than 0.01) and the fetal plasma concentrations of glucose (r = 0.68; P less than 0.01) and insulin-like growth factor-I (r = 0.74; P less than 0.001), suggesting a role for the PL receptor in the regulation of fetal carbohydrate metabolism and growth. The number of fetal PL receptors was inversely correlated with the fetal plasma oPL concentration (r = 0.47; P less than 0.05). These studies demonstrate that fasting of the pregnant ewe reduces the number of PL receptors in ovine fetal liver. The reduction in fetal hepatic PL receptors may contribute to the mobilization and depletion of fetal liver glycogen stores and may play a role in the pathogenesis of the fetal growth retardation that accompanies maternal caloric deprivation.