Fetal Liver Explants Research Articles

Alterations in one‑carbon metabolism and protein synthesis signals due to methionine supplementation and lipopolysaccharide challenge in Holstein fetal liver explants

One‑carbon metabolism (OCM) fueled by methionine (Met), choline, and folic acid is key for embryo development and fetal growth. We investigated effects of lipopolysaccharide (LPS) to induce inflammation in fetal liver tissue with or without Met on components of OCM and protein synthesis activity. Fetal liver harvested at slaughter from six multiparous pregnant Holstein dairy cows (37 ± 6 kg milk/d, 100 ± 3 d gestation) were incubated (0.2 ± 0.02 g) for 4 h at 37 °C with each of the following: ideal profile of amino acids (control; Lysine:Met 2.9:1), control plus LPS (1 μg/mL), increased Met supply (Met, Lys:Met 2.5:1), and Met+LPS. Data were analyzed as a 2 × 2 factorial (PROC MIXED, SAS 9.4). Ratios of mechanistic target of rapamycin (p-mTOR:mTOR) and eukaryotic elongation factor 2 (p-eEF2:eEF2) protein were lowest (P < 0.0 5) with LPS and highest with Met. Tissue amino acid concentrations were lowest (P < 0.0 5) with Met regardless of LPS suggesting enhanced use via mTOR. The marked increase (P = 0.02) in phosphorylation of S6 ribosomal protein (p-RPS6) with LPS suggested a pro-inflammatory response that was partly alleviated with Met+LPS. No effect (P = 0.4 5) on methionine adenosyl transferase 1 A (MAT1A) protein abundance was detected. Activity of betaine-homocysteine S-methyltransferase (BHMT) was greatest with Met, but Met+LPS dampened this effect (P = 0.0 5). Overall, fetal liver responds to inflammatory challenges and Met supply. The latter can stimulate protein synthesis via mTOR and alter some OCM reactions while having a modest anti-inflammatory effect.

Methylation of inorganic arsenic by murine fetal tissue explants

Although it is generally believed that the developing fetus is principally exposed to inorganic arsenic and the methylated metabolites from the maternal metabolism of arsenic, little is known about whether the developing embryo can autonomously metabolize arsenic. This study investigates inorganic arsenic methylation by murine embryonic organ cultures of the heart, lung, and liver. mRNA for AS3mt, the gene responsible for methylation of arsenic, was detected in all embryonic tissue types studied. In addition, methylated arsenic metabolites were generated by all three tissue types. The fetal liver explants yielded the most methylated arsenic metabolites (∼7% of total arsenic/48 h incubation) while the heart, and lung preparations produced slightly greater than 2% methylated metabolites. With all tissues the methylation proceeded mostly to the dimethylated arsenic species. This has profound implications for understanding arsenic-induced fetal toxicity, particularly if the methylated metabolites are produced autonomously by embryonic tissues.

Visualizing the Effects of Chemicals On Hematopoietic Stem Cell Engraftment in an Endogenous Niche

Abstract 506Hematopoietic stem and progenitor cells (HSPC) self-renew and give rise to all blood cell types throughout adulthood. Definitive HSPC arise from the hemogenic endothelium of the dorsal aorta, are released into circulation, and then seed an intermediate hematopoietic tissue before colonizing the adult marrow. In mammals this intermediate tissue is the fetal liver, and in the zebrafish it is the caudal hematopoietic tissue (CHT), a vascular plexus in the ventral tail of the embryo. We have generated the first highly specific zebrafish transgenic reporter of HSPC, using the previously described mouse Runx1 +23 kb intronic enhancer driving GFP (or mCherry) fluorescent protein. We have demonstrated that these Runx1+23 positive cells are capable of long-term engraftment and multi-lineage contribution. Using time-lapse live imaging in the embryo, we followed HSPC as they migrate to the CHT niche. Together with endothelial (kdrl(flk1):DsRed2) or stromal (cxcl12a(sdf-1a):DsRed2) reporter lines, we could visualize stem cell behavior directly in the endogenous niche. Upon arrival, HSPC underwent a number of distinct steps to engraftment, including: 1) adherence to the vessel wall; 2) extravasation; 3) migration to the abluminal space; 4) triggering of niche formation—endothelial cells actually remodel around a single HSPC to create a niche; 5) cell division decisions. To determine if endothelial niche formation is conserved in mammals during ontogeny, we performed live imaging of mouse fetal liver explants at embryonic day 11.5, the earliest stage of seeding by HSPC. We observed rare c-kit+/Ly6a(Sca1):GFP+ HSPC become centered in a rosette of CD31+/Lyve1+ sinusoidal endothelial cells. This dynamic remodeling of endothelial cells around an HSPC in the niche was strikingly similar to the cellular behaviors we observed in zebrafish. We hypothesized that chemical genetics could reveal the molecular mechanisms and signaling pathways that are associated with the distinct steps of HSPC engraftment. As proof-of-concept, we tested the CXCR4 antagonist AMD3100 because the CXCR4-CXCL12 receptors and ligands are expressed in the CHT, and found that it suppressed CHT hematopoiesis. Next, we performed a chemical genetic screen by applying ∼2400 individual compounds of known action to zebrafish embryos during colonization of the CHT. We found 40 compounds that increased and 107 compounds that decreased CHT hematopoiesis. Applying selected chemical hits in our live imaging assay we found that certain compounds actually modulated distinct steps during engraftment. We identified a role for sphingosine-1-phosphate signaling during extravasation. We observed that regulators of the transcription factor hypoxia inducible factor (HIF)-1α modulated migration into abluminal spaces. The HIF-1α stabilizer dimethyloxalylglycine (DMOG) promoted migration into hypoxic abluminal spaces, while conversely the HIF-1α inhibitor YC-1 promoted migration into normoxic luminal spaces. We found the plant alkaloid Lycorine promoted endothelial niche formation, creating more locations for HSPC and allowing longer residence times in the CHT. The transforming growth factor (TGF)-β receptor inhibitor SB-431542 increased the rate of HSPC division after they had arrived in the niche. Our studies provide the first genetic approach to understanding engraftment, and the chemicals found could be used therapeutically for patients receiving marrow transplantation. Disclosures:Tamplin:Boston Children’s Hospital: Employment, Patents & Royalties. Zon:Fate Therapeutics: Founder Other.

Circadian Rhythms of Fetal Liver Transcription Persist in the Absence of Canonical Circadian Clock Gene Expression Rhythms In Vivo

The cellular circadian clock and systemic cues drive rhythmicity in the transcriptome of adult peripheral tissues. However, the oscillating status of the circadian clocks in fetal tissues, and their response to maternal cues, are less clear. Most clock genes do not cycle in fetal livers from mice and rats, although tissue level rhythms rapidly emerge when fetal mouse liver explants are cultured in vitro. Thus, in the fetal mouse liver, the circadian clock does not oscillate at the cellular level (but is induced to oscillate in culture). To gain a comprehensive overview of the clock status in the fetal liver during late gestation, we performed microarray analyses on fetal liver tissues. In the fetal liver we did not observe circadian rhythms of clock gene expression or many other transcripts known to be rhythmically expressed in the adult liver. Nevertheless, JTK_CYCLE analysis identified some transcripts in the fetal liver that were rhythmically expressed, albeit at low amplitudes. Upon data filtering by coefficient of variation, the expression levels for transcripts related to pancreatic exocrine enzymes and zymogen secretion were found to undergo synchronized daily fluctuations at high amplitudes. These results suggest that maternal cues influence the fetal liver, despite the fact that we did not detect circadian rhythms of canonical clock gene expression in the fetal liver. These results raise important questions on the role of the circadian clock, or lack thereof, during ontogeny.

Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver

Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos. We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1(-/-) embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells.

Macrophage-associated erythropoiesis and lymphocytopoiesis in mouse fetal liver: ultrastructural and ISH analysis.

To elucidate the process of fetal liver hematopoiesis, the relationships between stroma and hematopoietic cells involved in maturation were investigated. Cultured mouse fetal liver explants were established for morphological analysis of the interactions between fetal liver stroma and hematopoietic cells ex vivo. Fetal liver stroma comprised epithelial cells and macrophages, which occupied most of the culture surface. Macrophages proliferated extensively in primary culture, but almost disappeared after 3 passages. Close morphological and functional relationships were established between macrophages and hemopoietic cells, whereas epithelial cells did not interact with blood cells. Scanning electron microscopy revealed that macrophages were in close contact with erythroblasts and formed a three-dimensional network. In each erythroblastic island, 2-3 lymphocytes were also in contact with the macrophages; erythroblasts, lymphocytes and macrophages formed close, firm associations through their cytoplasmic membranes. This cell orientation was confirmed by transmission electron microscopy of fetal liver in vivo. In situ hybridization revealed that the macrophages expressed jagged-1, an important ligand of the Notch signaling system in hematopoiesis.

Gestational age-dependent expression of insulin-like growth factor-binding protein-1 (IGFBP-1) phosphoisoforms in human extraembryonic cavities, maternal serum, and decidua suggests decidua as the primary source of IGFBP-1 in these fluids during early pregnancy.

The insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are important regulators of fetal and maternal tissue development during pregnancy. Posttranslational modification of IGFBP-1 yields up to six IGFBP-1 phosphovariants and a nonphosphorylated form, which in vitro, have some different properties. Nonphospho IGFBP-1 has less affinity for IGFs than the phospho isoforms and also may have IGF-independent actions. Herein, we have investigated the complement of IGFBP-1 phosphoisoforms present in extraembryonic coelomic (EEC) fluid, amniotic fluid (AF), and maternal serum (MS) throughout human gestation. Also, to determine potential tissue source(s) of IGFBP-1 in these fluids, we have quantified IGFBP-1 and examined IGFBP-1 phosphoisoforms in conditioned media (CM) from maternal decidua, fetal liver, and fetal kidney explants throughout gestation. Western immunodetection revealed that IGFBP-1, present in EEC and AF in early pregnancy and in CM from early pregnancy decidua, is primarily in the nonphosphorylated form. MS in this period contains primarily the nonphospho form and, as in nonpregnant adults, the highly phosphorylated form of IGFBP-1. The phosphorylation profile of IGFBP-1 in AF, MS, and decidua CM changes as pregnancy progresses. All the IGFBP-1 phosphoisoforms ultimately are produced by decidua and are present in midgestation MS, and all but the most highly phosphorylated form are present in AF. In late gestation, MS contains primarily the highly phosphorylated form. In contrast, profiles in CM from explants of fetal liver and kidney at different gestational ages remain unchanged. Nonphosphorylated IGFBP-1 is the primary form in fetal kidney CM, whereas fetal liver CM contains all IGFBP-1 phosphoisoforms. Concentrations of IGFBP-1 in fetal liver and kidney CM are significantly lower (482 +/- 146 and 120 +/- 32 ng/mL x 100 mg wet wt tissue, respectively) than in decidua CM (11,417 +/- 2,358 ng/mL x 100 mg wet wt tissue). The data cumulatively suggest that maternal decidua is the primary source of IGFBP-1 in EEC, AF, and MS in early pregnancy and that fetal liver and kidney are not likely significant contributors. The presence of nonphospho IGFBP-1 in AF, EEC, and MS suggests an important role for this isoform during early gestation.

Continuous culture of pig tissue-derived macrophages

Normal tissue macrophages from fetal and adult pig tissues can be continuously cultured by growing simple explant cultures on feeder layers of STO mouse embryonic fibroblasts. Macrophage cultures initiated from fetal and newborn pig testicle and liver explants grew from eight to 30 population doublings. The macrophages grew on top of the STO feeder cells in two forms: either a semi-attached round refractile morphology, or a closely attached ameboid morphology with several extended pseudopods. Cultured macrophages had large lobed nuclei, numerous complex vacuoles, and filopodia by transmission electron microscopic examination. The macrophages rapidly took up and sequestered acetylated-LDL in their vacuoles. They were highly phagocytic and expressed CD14 on their surface. Macrophage cultures were also initiated from tissues of the turkey, rat, mouse, cow, and sheep (data not shown). This simple method of isolating and propagating tissue macrophages could routinely provide macrophages for general research, adoptive immunotherapy, and somatic gene therapy.

Insulin-like growth factor-binding proteins in the human fetus: Tissue-specific protein secretion, immunologic characterization, and gene expression

The objectives of this study were to investigate the profile of insulin-like growth factor-binding proteins secreted by human fetal tissues and their immunologic identification and tissue-specific gene expression. Explants of midgestational fetal tissues from seven fetuses were cultured with and without cycloheximide. Conditioned media were examined for insulin-like growth factor-binding proteins by Western ligand blot analysis, and insulin-like growth factor-binding proteins were identified by immunoprecipitation. Gene expression was analyzed by Northern analysis. Fetal liver and kidney explants secreted insulin-like growth factor-binding protein-1 to insulin-like growth factor-binding protein-4, with insulin-like growth factor-binding protein-1 being the most prominent in liver. Fetal lung secreted insulin-like growth factor-binding protein-2 and insulin-like growth factor-binding protein-4 and lesser amounts of insulin-like growth factor-binding protein-3, whereas white matter explants secreted exclusively insulin-like growth factor-binding protein-2 and insulin-like growth factor-binding protein-4. Cycloheximide inhibited secretion of binding proteins, suggesting de novo synthesis. Northern blot analyses were consistent with the protein studies. These data demonstrate that insulin-like growth factor-binding protein secretion by fetal tissues is tissue specific.

The SCID-ra mouse: rat T-cell differentiation in the severe combined immunodeficient mouse.

The SCID-hu mouse model offers the possibility to study the human thymus in vivo in an isolated xenogeneic environment. Whilst studying the toxicology of the human thymus using the SCID-hu model, a "control" model to test for differences in toxicokinetics due to the different localization of the thymus is desirable when SCID-hu data have to be extrapolated to the normal human situation. For this reason, SCID-ra mice were constructed by implanting rat fetal or postnatal thymus and liver explants under the renal capsule of severe combined immunodeficient (SCID) mice. Implantation of rat fetal thymus in combination with fetal liver resulted in thymus grafts with a histological appearance that was virtually identical to that of normal age-matched rat thymus. T cells of rat origin were found in the circulating blood and lymphoid organs of the recipient mice. After implantation of postnatal thymus and liver, the thymus grafts showed a dysplastic morphology. These grafts were devoid of thymocytes and consisted mainly of thymic microenvironmental components and large numbers of thymic macrophages. Some SCID-ra mice showed signs of graft-versus-host (GVH) reactions in the skin. The incidence of GVH reactions was higher with increasing developmental stage of the donor material used for implantation.

Inhibitors of Glucose Uptake Stimulate the Production of Insulin-Like Growth Factor-Binding Protein (IGFBP-1) by Human Fetal Liver*

Cultured human fetal hepatic explants were used to study the role of hexose uptake in regulating production of the GH-independent insulin-like growth factor-binding protein (IGFBP-1). Cytochalasin B (100 microM), but not cytochalasin E, inhibited 2-deoxyglucose uptake by fetal liver explants and increased production of IGFBP-1 2-fold. Two agents which stimulate cyclic nucleotide-dependent pathways, cholera toxin and theophylline, also inhibited 2-deoxyglucose uptake in this system and both stimulated IGFBP-1 production. When added to a maximally inhibiting concentration of cytochalasin B (250 microM), hexose uptake was not further inhibited by theophylline (5 mM) or cholera toxin (10 micrograms/ml), suggesting that the three substances interact with the same hexose transport mechanism. However, the stimulatory effect of theophylline (5 mM) or cholera toxin (10 micrograms/ml) on IGFBP-1 production was additive to the effect of 250 microM cytochalasin B, suggesting that another pathway, possibly involving cyclic nucleotide accumulation, could further stimulate IGFBP-1 above the effect of blocking glucose transport. Insulin (300 nM) had no effect on hexose uptake by human fetal liver explants but inhibited IGFBP-1 production, both basally and when stimulated by cytochalasin B (100 microM) or cholera toxin (1 microgram/ml). These results are consistent with the conclusion that a cyclic nucleotide-dependent pathway, activated by blocking hexose transport and inhibited by insulin, is involved in the regulation of IGFBP-1 synthesis.

Regulation of Growth Hormone-Independent Insulin Like Growth Factor-Binding Protein (BP-28) in Cultured Human Fetal Liver Explants*

Cultured human fetal liver explants were used to study regulation of the GH-independent insulin-like growth factor-binding protein (BP-28). BP-28 was produced by fetal liver explants at approximately 200 ng/mg total tissue protein.day between days 3 and 6 in culture and was inhibited 60% after 3-day exposure to cycloheximide (10 micrograms/mL). BP-28 production was stimulated over 3-fold by 20 mmol/L theophylline, over 2-fold by 30 mumol/L forskolin, and 90% by 10 micrograms/mL glucagon. The glucose concentration in the medium inversely regulated BP-28 production, which was 68% higher in 2 mmol/L glucose than in 30 mmol/L glucose (P less than 0.001); in contrast, albumin production was 40% lower in 2 mmol/L glucose than in 30 mmol/L (P less than 0.05). BP-28 levels were inhibited 50% by 300 nmol/L insulin or 3 mumol/L dexamethasone. In acid extracts of cultured tissue, BP-28 levels were less than 10% of the amount secreted in 48 h. These results are consistent with the conclusion that intracellular glucose availability is an important regulator of hepatic BP-28 synthesis.

α1-microglobulin (HC protein) in human hepatocellular carcinoma

al -Microglobulin (cx1-m) is a highly glycosylated polypeptide of 182 amino acids associated with a brown chromophore (Ekstr6m et al., 1975; Lopez Otin et al., 1984). The molecule is heterogeneous in charge and was described as HC protein (Tejler & Grubb, 1976) or acl-microglycoprotein (Seon & Pressmann, 1978). It occurs in biological fluids as a 31 kDa monomer and as a 90kDa component covalently associated to one a chain of monomeric immunoglobulin A (IgA) (Grubb et al., 1983; Vincent et al., 1985). The al-m gene is associated with a gene coding for the HI-30 domain of interax-trypsin inhibitor, one of the acute phase proteins (Kaumeyer et al., 1986). The human 31 kDa al-m was shown to be synthesised by fetal liver explants (Tejler et al., 1978) and we recently reported that the molecule was produced in vitro by several human hepatoma cell lines but not by lymphoma or plasmocytoma cell lines (Vincent et al., 1987). For these reasons we have determined serum levels of al-m in a large series of patients suffering from primary hepatocellular carcinoma in order to evaluate the possible use of this molecule as an additional tumour marker. The data show that 31 kDa al-m levels are usually not increased in this disease, whereas the 90kDa component levels are elevated in some cases. Our study involved a hundred South African Blacks with histologically proven hepatocellular carcinoma. They included 11 women and 89 men whose ages ranged from 20 to 73 years (median 39). Seventy-seven healthy South African Black subjects served as controls. All serum samples were sent frozen in dry ice from South Africa and were stored at -40°C until use. The differential quantitation of 31 kDa and IgA-associated 90 kDa ocl-m in unfractionated sera was performed by

Changes in the circulating form of serum somatomedin-C during fetal life.

The predominant form of somatomedin-C (Sm-C) in human postnatal serum elutes from gel chromatographic columns at an approximate molecular weight of 150,000 daltons (150 K). Nine of 10 infants of 30 weeks gestation or more had elution profiles similar to postnatal sera, while in 7 of 9 infants of 27 weeks or less, the immunoreactive Sm-C eluted predominantly at an apparent molecular weight of 40,000 daltons (40 K). Five infants between 26-32 weeks exhibited a transitional pattern. One 43 week gestation anencephalic infant exhibited only 40 K Sm-C, suggesting that the 150 K proteins which bind Sm-C are acquired in response to growth hormone or other pituitary hormones. Even though the 40 K form of Sm-C is the only form found in mid-gestation fetal serum and in media from in vitro fetal mouse liver explants, it probably does not represent the primary gene product. This is suggested by the observation that 40 K Sm-C also binds 125-I-Sm-C and can be dissociated by acid and, therefore, probably is a complex of Sm-C non-covalently bound to other proteins.

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.

Changes in the properties of fetal rat liver during organ culture.

Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight—soluble malate dehydrogenase, glutamate dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine, aminotransferase, and alanine aminotransferase—were unchanged or increased. Glycogen synthetase, aspartate aminotransferase, pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro.

Functional integrity of fetal rat liver explants cultured in a chemically defined medium

A technique is described for the rapid preparation of large numbers of fetal rat liver explants for the study of developmental changes. Uniform pieces (1 mm 3) of liver at 16 and 20 days of gestation were cultured for 3 days in a chemically defined medium in the absence of serum or exogenous proteins. Explants of both gestational ages appeared morphologically normal at the end of 3 days. Net uptakes of glucose remained relatively constant over the culture period. There was an initial loss of protein and DNA which could be largely accounted for by loss of erythropoietic cells. After the first or second day in culture, the DNA content of the explants remained constant. The amount of nonsedimentable protein (nonerythrocyte) released into the medium was 16 to 20 μ per explant per day. Between 40 and 50% of this protein was identified as albumin and α-fetoprotein by immunologic and electrophoretic techniques. A minor protein component was identified as transferrin. These proteins, normally secreted into amniotic fluid, were released in amounts that markedly exceeded their concentrations in the explant. This release was blocked by the presence of cycloheximide in the culture medium. This technique provides a simple method for maintaining viable fetal liver explants in a chemically defined medium, and should be useful for studying metabolic development in antenatal liver.

Production of protein HC by human fetal liver explants

Human fetal lever explants were found to secrete protein HC into the medium in molar amounts comparable to those of albumin, α 1-antitrypsin and orosomucoid. Incorporation of a radioactive amino acid from the medium into the secreted protein HC demonstrated de novo synthesis. The secreted protein HC had the same size and electrophoretic mobility as protein HC of plasma and urine and gave a reaction of immunochemical identity with the protein in these biological fluids.

Tight junctions in fetal rat liver explants grown in vitro

When culturing fetal liver explants in vitro , hepatocytes at the surface of the explants develop numerous interdigitating cytoplasmic processes joined to one another by diminutive tight junctional elements. The formation of such junctions, not present in the liver in situ , indicates the ability of the hepatocyte plasma membrane to establish junctional differentiations not necessarily associated with bile canaliculi.

DECREASE IN HEPATIC LIPOGENESIS IN TERM RAT LIVER EXPLANTS

Hepatic lipogenesis in the fetal rat is highly active until the time of birth when it decreases and remains low throughout the suckling period. High fat intake during suckling is thought to suppress hepatic lipogenesis. By utilizing explants of fetal rat liver we have demonstrated that the decrease in hepatic fatty acid synthesis may occur independent of nutritional influences. Explants obtained from fetal rats at 17 and 22 days gestation were incubated for 48 hours in F12 medium without added hormones or serum. Incorporation of acetate-1-14C into long chain fatty acids during a 6 hour pulse was determined at 0 time and after 48 hours in culture and is shown below. Data are expressed as % incorporation/mg protein/6 hours. Production of 14CO2 from uniformly labeled glucose was examined as an index of viability. There was no significant decrease in glucose oxidation in explants from either 17 or 22 day gestation fetuses during the 48 hours in culture. The results indicate that acetate incorporation into fatty acids in liver explants from 17 day fetuses remained constant between 0 and 48 hours in culture. Explants from 22 day fetuses, however, showed a striking decline in acetate incorporation. These data suggest that the decline in hepatic lipogenesis in the newborn rat may be independent of dietary influences.