Fig 1.
The layers of the human placenta at term and resident lipid droplets.
(A) TEM of human placenta at term. MAT, maternal blood space; MVM, microvillous membrane; SCT, syncytiotrophoblast; CTB, cytotrophoblast; END, endothelium; FET CAP, fetal capillary; RBC, red blood cell; BL, basal lamina. Scale Bar: 1μm. (B-E) Lipid droplets (LD, arrow heads) in the human placenta can be detected by multiple staining methodologies. (B) Oil-Red O (C) Nile Red (D-E) Structured illumination microscopy (SIM, a super-resolution technique). Only the syncytiotrophoblast is imaged in (E). (F) The LD volume distribution in freshly delivered placenta is not different (One-way ANOVA) between cytotrophoblast (CTB) and syncytiotrophoblast (SCT) layers, as measured by SIM. (Data are mean ± SEM, n = 7, unpaired t-test). Scale Bar (B-E): 5μm.
Table 1.
Maternal Characteristics.
Table 2.
Primer sequences utilized for quantitative PCR.
Fig 2.
BODIPY-C12 fatty acid uptake in human term placental explants (30 min exposure).
Dashed line represents the syncytiotrophoblast-cytotrophoblast interface. Co-localized BODIPY-C12 and LipidTOX forms yellow LD, indicating esterified BODIPY-C12. (A) BODIPY-C12 (green) is localized to punctate structures (arrow heads) and diffuse areas in syncytiotrophoblasts (SCT) and cytotrophoblasts (CTB). Dark “holes” are unstained nuclei. (B) Pre-existing neutral lipids (LipidTOX, red) are found in punctate structures which are lipid droplets (LD) (arrow head); (C) LipidTOX co-localizes with BODIPY-C12 (double arrowhead) predominantly in CTB, providing evidence that BODIPY-C12 is esterified and localized within LD. Conversely, in SCT, LD (arrowhead) contains little BODIPY-C12 (corresponding region 2A open circle). (D-F) images of explants after fixation showing BODIPY-C12 (green) and LipidTOX (red). (D) LD are present in both SCT and CTB, but BODIPY-C12 seen in (A) has mostly washed out in processing but some remain in sequestered organelles. (E) Hoechst (blue) marks nuclei and HAI-1 (magenta) marks CTB, (F) BODIPY-C12 (green) is found primarily in CTB (magenta) but not in SCT (see double arrow head). (G) BODIPY-C12 containing lipid droplets (arrow heads) are found extensively in the CTB layer (e-cadherin, red) but are sparse in the SCT layer. (H) phloretin (a blocker of protein mediated transport), and (I) triacsin C (a blocker of long-chain fatty acyl-CoA synthetases) reduces the population of lipid droplet containing BODIPY-C12 in CTB. These experiments suggest that transporters and acyl-CoA synthetases are required for production of LD. (J) Quantification of lipid droplets synthesized in explants illustrating the effects of chemical inhibitors and shows under control conditions BODIPY-C12 LD accumulation is higher in 30 min in the CTB vs. SCT; (I): Data are mean ± SEM; n = 6 control, n = 5 phloretin, and n = 4 triacsin C. 2-way ANOVA, *** = p<0.001 vs CTB vehicle; VS, villous stroma; FET CAP, fetal capillary; Scale Bar: 5μm.
Fig 3.
BODIPY-C12 incorporation into lipid droplets in primary cytotrophoblasts incubated for 15 or 30 min.
Cytotrophoblasts (CTB) were in two states of maturation after being in culture for 4hr(A,C-E) or 72hr which leads to syncytialization (SCT) (B,F-H). (A,B) Analyses of BODIPY- C12 incorporation into lipid subclasses via thin-layer chromatography in CTB and SCT in vitro (A) Time course of HepG2 cells (positive control) and primary cytotrophoblast incubated continuously with BODIPY-C12. BODIPY-C12 is incorporated into neutral lipids (NL) and phospholipids (PL) within 30 minutes indicating esterification of this long-chain fatty acid analogue. NEFA, non-esterified fatty acid. (B) CTB incorporate more BODIPY-C12 into esterified lipid subclasses than SCT in vitro within 30 minutes. (D) Cultures (D,E and G,H) were stained with Nile Red to visualize neutral lipid droplets (LD). Images (C,F) shows that BODIPY-C12 incorporation is greater among CTB than among SCT. (E) In CTB, BODIPY-C12 merges with Nile-Red in LD. (H) When CTB have undergone syncytialization, they incorporate very little BODIPY-C12 in LD. (I) Quantification of lipid droplets shows that cultured (4 hours) cells accumulate more BODIPY-C12 LD than those that have syncytialized (72hrs). Phloretin and triacsin C significantly reduce BODIPY-C12 LD accumulation in cytotrophoblasts but not in syncytialized cells. (J) The uptake of BODIPY-C12 using a 96-well plate-reader indicates that uptake is higher in cytotrophoblast before syncytialization (K) The log-dose response curve of CB-2, a FATP2 inhibitor, supports that both cytotrophoblast and syncytialized cells are equally sensitive to inhibition of BODIPY-C12 uptake by CB-2. The efficacy of this inhibition of BODIPY-C12 uptake suggests that FATP2 largely mediates long-chain fatty acid uptake in these cells. TLC plates are representative of 3 technical replicates. (C-H): Scale Bar: 5μm, LD (arrow heads), co-localization (double arrowhead). Image intensity ranges are the same in all panels (I-K): Data are mean ± SEM; (I) n = 6 for each group. 2-way ANOVA, (J,K) n = 4. Paired t-test. * = p<0.05, ** = p<0.01 vs CTB vehicle.
Fig 4.
BODIPY-C12 incorporates into Perilipin-2 and Perilipin-3 labeled cytotrophoblast lipid droplets.
Cytotrophoblast plated for 4hr onto glass coverslips were incubated with 2μM BODIPY-C12 for 30 minutes and fixed as described in the methods section. Cells were immunolabeled with Perilipin-2 or Perilipin-3. Nuclei are labeled using Hoechst dye. All BODIPY-C12 cytotrophoblast lipid droplets contain some degree of Perilipin-2 (A-C) or Perilipin-3 (D-F) on the surface. In addition to the presence of Perilipin-3 on lipid droplets, Perilipin-3 is also distributed on the plasma membrane of cytotrophoblast (E). n = 5. Scale Bar: 1μm.
Fig 5.
mRNA expression of key lipid processing genes before and after cytotrophoblast syncytialization.
(A) In 72hr syncytia, the expression of BHCG is significantly higher compared to cytotrophoblast at 4hrs, indicating differentiation. (B-I) By 72 hours, there are significant changes in expression of several fatty acid uptake and processing genes. (B) SLC27A2 (FATP2, the most highly expressed fatty acid transport protein in placenta). (C) FABP4 (fatty acid binding protein). (D) GPAT3 (glycerol-3-phosphate acyltransferase). (E) ACSL5 (Acyl-CoA synthetase, long-chain). (F) LPCAT3 (lysophosphatidylcholine acyl transferase). (G) SLC27A4 (FATP4, fatty acid transport protein). (H) CD36 (FAT, fatty acid translocase). (I) SLC27A1 (FATP1, fatty acid transport protein). Data are mean ± SEM, n = 6 for all groups. Expression is relative to GAPDH, which is not significantly different between groups. Paired t-test * = p<0.05, ** = p<0.01, *** = p<0.001. (J) Immunofluorescence of FATP2 (green), nuclei are blue (Hoechst). (K) Merged immunofluorescence illustrating cytotrophoblast (E-cadherin, red), syncytiotrophoblast (BHCG, magenta) with FATP2/SLC27A2 (green). FATP2 can be found in both cytotrophoblast and syncytiotrophoblast, but appears to be more strongly expressed in cytotrophoblast which fits gene expression data in panel (B). Dashed line represents the syncytiotrophoblast-cytotrophoblast interface. Scale Bar: 5μm.
Fig 6.
Hypothetical simplified model of placental transport of fatty acids.