Identification and Quantification of Proteins from Preparative Partition Chromatography and Peptides from Organic Extraction of Fetal Versus Adult Bovine Serum using Nano-Spray-LC-ESI-MS/MS

Blood proteins communicate with many different cells, tissue and organs; perform key functions in the immune system and may be of particular biological complexity. One of the most widely used blood products in the laboratory is fetal bovine serum for cell culture. There are ethical and practical concerns regarding the use of fetal serum from animals and alternative serum-free replacements have been attempted using platelet lysates. Previous biochemical experiments have shown that FBS apparently contained factors such as alpha-feto protein (AFP) and insulin-like growth factors that may support the indefinite cell growth and division of certain cell lines. It is presumed that a set of as yet undefined growth factors transform cells growth resulting in rapid proliferation. Cultured Raw cells 264.7 in adult bovine serum multiplied slowly and differentiated into elongated cells with a dendritic shape, which died after the first few generations. On the contrary, in fetal bovine serum, cultured cells multiplied rapidly and formed many smaller cells with a rounded shape through many cell passages. Three independent batches of fetal bovine serum were tested on Raw cells 264.7 macrophages to confirm that they supported cell growth in culture compared to three independent batches of adult bovine serum. The intact proteins of each serum sample were separated by partition chromatography into 16 fractions with an increasing step gradient of salts over quaternary amine resin (proteomics). The endogenous peptides were precipitated with 90% of acetonitrile and extracted into 10 fractions with a decreasing step gradient of acetonitrile in water (peptidomics). Trypsin digested intact proteins and endogenous peptides were then analyzed on a fresh C18 nano-HPLC column with random and independent sampling by LC-ESI-MS/MS. The fractionated mass spectra were identified with SEQUEST and X!TANDEM algorithms. Redundant use of MS/MS spectra were flirted out with the SQL Server system and the R statistical analysis system was used to perform Chi Square (X2) analysis of frequency counts and ANOVA of the log10 precursor intensity results. Alpha-feto protein, fetal albumin, insulin, insulin like growth factors, platelet derived growth factors and proteins associated with HRAS/AKT growth pathway at the level of ligand, receptors, receptor associated enzyme and nucleic acid binding proteins including transcription factors were observed to be specifically enriched in fetal serum.

Identification and Quantification of Proteins from Preparative Partition Chromatography and Peptides from Organic Extraction of Fetal Versus Adult Bovine Serum using Nano-Spray-LC-ESI-MS/MS

Blood proteins communicate with many different cells, tissue and organs; perform key functions in the immune system and may be of particular biological complexity. One of the most widely used blood products in the laboratory is fetal bovine serum for cell culture. There are ethical and practical concerns regarding the use of fetal serum from animals and alternative serum-free replacements have been attempted using platelet lysates. Previous biochemical experiments have shown that FBS apparently contained factors such as alpha-feto protein (AFP) and insulin-like growth factors that may support the indefinite cell growth and division of certain cell lines. It is presumed that a set of as yet undefined growth factors transform cells growth resulting in rapid proliferation. Cultured Raw cells 264.7 in adult bovine serum multiplied slowly and differentiated into elongated cells with a dendritic shape, which died after the first few generations. On the contrary, in fetal bovine serum, cultured cells multiplied rapidly and formed many smaller cells with a rounded shape through many cell passages. Three independent batches of fetal bovine serum were tested on Raw cells 264.7 macrophages to confirm that they supported cell growth in culture compared to three independent batches of adult bovine serum. The intact proteins of each serum sample were separated by partition chromatography into 16 fractions with an increasing step gradient of salts over quaternary amine resin (proteomics). The endogenous peptides were precipitated with 90% of acetonitrile and extracted into 10 fractions with a decreasing step gradient of acetonitrile in water (peptidomics). Trypsin digested intact proteins and endogenous peptides were then analyzed on a fresh C18 nano-HPLC column with random and independent sampling by LC-ESI-MS/MS. The fractionated mass spectra were identified with SEQUEST and X!TANDEM algorithms. Redundant use of MS/MS spectra were flirted out with the SQL Server system and the R statistical analysis system was used to perform Chi Square (X2) analysis of frequency counts and ANOVA of the log10 precursor intensity results. Alpha-feto protein, fetal albumin, insulin, insulin like growth factors, platelet derived growth factors and proteins associated with HRAS/AKT growth pathway at the level of ligand, receptors, receptor associated enzyme and nucleic acid binding proteins including transcription factors were observed to be specifically enriched in fetal serum.

Dexamethasone induces an imbalanced fetal-placental-maternal bile acid circulation: involvement of placental transporters

Background The use of prenatal dexamethasone remains controversial. Our recent studies found that prenatal dexamethasone exposure can induce maternal intrahepatic cholestasis and have a lasting adverse influence on bile acid (BA) metabolism in the offspring. The purpose of this study was to investigate the effects of dexamethasone on fetal-placental-maternal BA circulation during the intrauterine period, as well as its placental mechanism. Methods Clinical data and human placentas were collected and analyzed. Pregnant Wistar rats were injected subcutaneously with dexamethasone (0.2 mg/kg per day) from gestational day 9 to 20. The metabolomic spectra of BAs in maternal and fetal rat serum were determined by LC-MS. Human and rat placentas were collected for histological and gene expression analysis. BeWo human placental cell line was treated with dexamethasone (20–500 nM). Results Human male neonates born after prenatal dexamethasone treatment showed an increased serum BA level while no significant change was observed in females. Moreover, the expression of organic anion transporter polypeptide-related protein 2B1 (OATP2B1) and breast cancer resistance protein (BCRP) in the male neonates’ placenta was decreased, while multidrug resistance-associated protein 4 (MRP4) was upregulated. In experimental rats, dexamethasone increased male but decreased female fetal serum total bile acid (TBA) level. LC-MS revealed that primary BAs were the major component that increased in both male and female fetal serum, and all kinds of BAs were significantly increased in maternal serum. The expression of Oatp2b1 and Bcrp were reduced, while Mrp4 expression was increased in the dexamethasone-treated rat placentas. Moreover, dexamethasone increased glucocorticoid receptor (GR) expression and decreased farnesoid X receptor (FXR) expression in the rat placenta. In BeWo cells, dexamethasone induced GR translocation into the nucleus; decreased FXR, OATP2B1, and BCRP expression; and increased MRP4 expression. Furthermore, GR was verified to mediate the downregulation of OATP2B1, while FXR mediated dexamethasone-altered expression of BCRP and MRP4. Conclusions By affecting placental BA transporters, dexamethasone induces an imbalanced fetal-placental-maternal BA circulation, as showed by the increase of primary BA levels in the fetal serum. This study provides an important experimental and theoretical basis for elucidating the mechanism of dexamethasone-induced alteration of maternal and fetal BA metabolism and for exploring early prevention and treatment strategies.

Murine Fetal Serum Phosphorus is Set Independent of FGF23 and PTH, Except in the Presence of Maternal Phosphate Loading

Fibroblast growth factor 23 (FGF23) appears to play no role until after birth, given unaltered phosphate and bone metabolism in Fgf23- and Klotho-null fetuses. However, in those studies maternal serum phosphorus was normal. We studied whether maternal phosphate loading alters fetal serum phosphorus and invokes a fetal FGF23 or parathyroid hormone (PTH) response. C57BL/6 wild-type (WT) female mice received low (0.3%), normal (0.7%), or high (1.65%) phosphate diets beginning 1 week prior to mating to WT males. Fgf23+/- female mice received the normal or high-phosphate diets 1 week before mating to Fgf23+/- males. One day before expected birth, we harvested maternal and fetal blood, intact fetuses, placentas, and fetal kidneys. Increasing phosphate intake in WT resulted in progressively higher maternal serum phosphorus and FGF23 during pregnancy, while PTH remained undetectable. Fetal serum phosphorus was independent of the maternal phosphorus and PTH remained low, but FGF23 showed a small nonsignificant increase with high maternal serum phosphorus. There were no differences in fetal ash weight and mineral content, or placental gene expression. High phosphate intake in Fgf23+/- mice also increased maternal serum phosphorus and FGF23, but there was no change in PTH. WT fetuses remained unaffected by maternal high-phosphate intake, while Fgf23-null fetuses became hyperphosphatemic but had no change in PTH, skeletal ash weight or mineral content. In conclusion, fetal phosphate metabolism is generally regulated independently of maternal serum phosphorus and fetal FGF23 or PTH. However, maternal phosphate loading reveals that fetal FGF23 can defend against the development of fetal hyperphosphatemia.

Maternal hypercortisolemia alters placental metabolism: a multiomics view

Previous studies have suggested that increases in maternal cortisol or maternal stress in late pregnancy increase the risk of stillbirth at term. In an ovine model with increased maternal cortisol over the last 0.20 of gestation, we have previously found evidence of disruption of fetal serum and cardiac metabolomics and altered expression of genes related to mitochondrial function and metabolism in biceps femoris, diaphragm, and cardiac muscle. The present studies were designed to test for effects of chronically increased maternal cortisol on gene expression and metabolomics in placentomes near term. We hypothesized that changes in placenta might underlie or contribute to the alterations in fetal serum metabolomics and thereby contribute to changes in striated muscle metabolism. Placentomes were collected from pregnancies in early labor (143 ± 1 days gestation) of control ewes ( n = 7) or ewes treated with cortisol (1 mg·kg−1·day−1 iv; n = 5) starting at day 115 of gestation. Transcriptomics and metabolomics were performed using an ovine gene expression microarray (Agilent 019921) and HR-MAS NMR, respectively. Multiomic analysis indicates that amino acid metabolism, particularly of branched-chain amino acids and glutamate, occur in placenta; changes in amino acid metabolism, degradation, or biosynthesis in placenta were consistent with changes in valine, isoleucine, leucine, and glycine in fetal serum. The analysis also indicates changes in glycerophospholipid metabolism and suggests changes in endoplasmic reticulum stress and antioxidant status in the placenta. These findings suggest that changes in placental function occurring with excess maternal cortisol in late gestation may contribute to metabolic dysfunction at birth.