Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta

doi:10.1016/0005-2736(90)90157-J

Biochimica et Biophysica Acta (BBA) - Biomembranes

Volume 1029, Issue 2, 16 November 1990, Pages 218-226

https://doi.org/10.1016/0005-2736(90)90157-J Get rights and content

Abstract

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg²⁺-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg²⁺-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 ± 4% right-side out for MVM and 73 ± 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that ‘paired’ MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.

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Cited by (128)

Sex-specific responses in placental fatty acid oxidation, esterification and transfer capacity to maternal obesity
2021, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Fatty acid metabolism and oxidation capacity in the placenta, which likely affects the rate and composition of lipid delivered to the fetus remains poorly understood. Long chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are critical for fetal growth and brain development. We determined the impact of maternal obesity on placental fatty acid oxidation, esterification and transport capacity by measuring PhosphatidylCholine (PC) and LysoPhosphatidylCholine (LPC) containing DHA by mass spectrometry in mother-placenta-baby triads as well as placental free carnitine and acylcarnitine metabolites in women with normal and obese pre-pregnancy BMI. Placental protein expression of enzymes involved in beta-oxidation and esterification pathways, MFSD2a (lysophosphatidylcholine transporter) and OCTN2 (carnitine transporter) expression in syncytiotrophoblast microvillous (MVM) and basal (BM) membranes were determined by Western Blot. Maternal obesity was associated with decreased umbilical cord plasma DHA in LPC and PC fractions in male, but not female, fetuses. Basal membrane MFSD2a protein expression was increased in placenta of males of obese mothers. In female placentas, despite an increased MVM OCTN2 expression, maternal obesity was associated with a reduced MUFA-carnitine levels and increased esterification enzymes. We speculate that lower DHA-PL in fetal circulation of male offspring of obese mothers, despite a significant increase in transporter expression for LPC-DHA, may lead to low DHA needed for brain development contributing to neurological consequences that are more prevalent in male children. Female placentas likely have reduced beta-oxidation capacity and appear to store FA through greater placental esterification, suggesting impaired placenta function and lipid transfer in female placentas of obese mothers.
Decreased placental folate transporter expression and activity in first and second trimester in obese mothers
2020, Journal of Nutritional Biochemistry
Obese women have an approximately twofold higher risk to deliver an infant with neural tube defects (NTDs) despite folate supplementation. Placental transfer of folate is mediated by folate receptor alpha (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC). Decreased placental transport may contribute to NTDs in obese women. Serum folate levels were measured and placental tissue was collected from 13 women with normal BMI (21.9±1.9) and 11 obese women (BMI 33.1±2.8) undergoing elective termination at 8–22 weeks of gestation. The syncytiotrophoblast microvillous plasma membranes (MVM) were isolated using homogenization, magnesium precipitation, and differential centrifugation. MVM expression of FR-α, PCFT and RFC was determined by western blot. Folate transport capacity was assessed using radiolabeled methyl-tetrahydrofolate and rapid filtration techniques. Differences in expression and transport capacity were adjusted for gestational age and maternal age in multivariable regression models. P<.05 was considered statistically significant. Serum folate levels were not significantly different between groups. Placental MVM folate transporter expression did not change with gestational age. MVM RFC (−19%) and FR-α (−17%) expression was significantly reduced in placentas from obese women (P<.05). MVM folate transporter activity was reduced by−52% (P<.05) in obese women. These differences remained after adjustment for gestational age. There was no difference in mTOR signaling between groups. In conclusion, RFC and FR alpha expression and transporter activity in the placental MVM are significantly reduced in obese women in early pregnancy. These results may explain the higher incidence of NTDs in infants of obese women with adequate serum folate.
A potential role for lysophosphatidylcholine in the delivery of long chain polyunsaturated fatty acids to the fetal circulation
2019, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
The mechanisms of placental transfer of long chain polyunsaturated fatty acids are poorly understood, however fatty acid transporters in the syncytiotrophoblast microvillous (MVM) and basal plasma membrane (BM) are believed to be involved. Using LC-MS/MS and samples from normal term pregnant women, we found that the concentration of lysophosphatidylcholine-docosahexaenoyl (DHA-LPC) was 4-fold higher in umbilical vein plasma compared to maternal venous concentrations while conversely phosphatidylcholine (PC) containing DHA or arachidonic acid were higher in maternal circulation. Using primary human trophoblast cells incubated with DHA we show that DHA was highly incorporated into PC and LPC species in the placenta. Protein expression of Phosphatidylcholine Transfer Protein (PCTP) was 14-fold higher in MVM and the expression of MFSD2a, an LPC transporter, was 5-fold higher in BM than in MVM. Interestingly, BM MFSD2a expression was positively correlated with DHA-LPC in the umbilical vein. These findings suggest that syncytiotrophoblast takes up PC from the maternal circulation, as well as preferentially incorporating DHA into PC. Placental PC are converted to LPC forms, which are transported across the BM mediated by MFSD2a, with a strong selectivity for DHA.
Role of the efflux transporters BCRP and MRP1 in human placental bio-disposition of pravastatin
2018, Biochemical Pharmacology
The expression and activity of human placental transporters during pregnancy could be altered by several factors including pathological changes associated with preeclampsia. The aims of this study were to identify the placental efflux transporters involved in the bio-disposition of pravastatin, determine the protein expression of these transporters and their encoding genes as well as the activity of pravastatin uptake in placentas obtained from patients with preeclampsia. ATP-dependent uptake of [3H]-pravastatin by trophoblast tissue apical and basal membrane vesicles exhibited sigmoidal kinetics. The curved shapes of Eadie-Hofstee plots indicate that more than one placental transporter are involved in the uptake of pravastatin. ATP-dependent uptake of [3H]-pravastatin into vesicles expressing MRP1-5, BCRP, and P-gp, as well as the results of inhibition studies suggest that BCRP and MRP1 are the major placental efflux transporters responsible for the in vitro uptake of pravastatin. Compared to placentas from healthy pregnancies, preeclamptic placentas had increased number of syncytial knots with increased expression of BCRP in their apical membrane and increased expression of MRP1 in the cytoplasm of the syncytiotrophoblast and in cytoplasm of syncytial knots. There was a concomitant increase in ABCC1 but not in ABCG2 gene expressions in preeclamptic placentas. ATP-dependent uptake of [3H]-pravastatin by vesicles prepared from apical membranes of preeclamptic placentas was similar to the uptake by vesicles prepared from placentas obtained after uncomplicated pregnancies (13.9 ± 6.5 vs 14.1 ± 5.8 pmol·mg protein⁻¹ min⁻¹). The transporter-specific changes in the expression of BCRP and MRP1 in preeclamptic placentas did not affect the efflux activity of transporters localized on the apical membrane of the syncytiotrophoblast.
Down-regulation of placental folate transporters in intrauterine growth restriction
2018, Journal of Nutritional Biochemistry
Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th–75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (−34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (−38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR.
Glyburide treatment in gestational diabetes is associated with increased placental glucose transporter 1 expression and higher birth weight
2017, Placenta
Citation Excerpt :
Placental homogenates were frozen in liquid nitrogen and stored at −80 °C until further processing. Isolation of syncytiotrophoblast MVM and BM was performed according to established protocol [35]. Briefly, after initial separation of cellular debris, the MVM was purified by MgCl2-precipitation and BM was purified by sucrose gradient centrifugation.
Use of glyburide in gestational diabetes (GDM) has raised concerns about fetal and neonatal side effects, including increased birth weight. Placental nutrient transport is a key determinant of fetal growth, however the effect of glyburide on placental nutrient transporters is largely unknown. We hypothesized that glyburide treatment in GDM pregnancies is associated with increased expression of nutrient transporters in the syncytiotrophoblast plasma membranes.
We collected placentas from GDM pregnancies who delivered at term and were treated with either diet modification (n = 15) or glyburide (n = 8). Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated and expression of glucose (glucose transporter 1; GLUT1), amino acid (sodium-coupled neutral amino acid transporter 2; SNAT2 and L-type amino acid transporter 1; LAT1) and fatty acid (fatty acid translocase; FAT/CD36, fatty acid transporter 2 and 4; FATP2, FATP4) transporters was determined by Western blot. Additionally, we determined GLUT1 expression by confocal microscopy in cultured primary human trophoblasts (PHT) after exposure to glyburide.
Birth weight was higher in the glyburide-treated group as compared to diet-treated GDM women (3764 ± 126 g vs. 3386 ± 75 g; p < 0.05). GLUT1 expression was increased in both MVM (+50%; p < 0.01) and BM (+75%; p < 0.01). In contrast, MVM FAT/CD36 (−65%; p = 0.01) and FATP2 (−65%; p = 0.02) protein expression was reduced in mothers treated with glyburide. Glyburide increased membrane expression of GLUT1 in a dose-dependent manner in cultured PHT.
This data is the first to show that glyburide increases GLUT1 expression in syncytiotrophoblast MVM and BM in GDM pregnancies, and may promote transplacental glucose delivery contributing to fetal overgrowth.

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Regular paperSimultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta

Abstract

Placenta

Biochim. Biophys. Acta

Mol. Cell. Endocrinol.

Anal. Biochem.

Biochim. Biophys. Acta

Anal. Biochem.

Am. J. Obstet. Gynecol.

Am. J. Obstet. Gynecol.

J. Reprod. Immunol.

J. Immunol. Methods

Exp. Cell Res.

Am. J. Obstet. Gynecol.

Nature

Regular paper
Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta