Figure 1.
CT analysis demonstrates that a HF-diet leads to increased subcutaneous and abdominal fat.
Fasted rats were anesthetized and subjected to CT scanning. A. Representative CT-scan of a CON-fed dam; B. Representative CT-scan of a HF-fed dam. Adipose tissue is characterized by a lower intensity signal (darker regions on the image). The area occupied by the abdominal fat (AB) and subcutaneous fat (SQ) are indicated; scale bar = 1 cm. C. Adipose tissue absorption in the area between the bottom of the lungs and the top of the sacroiliac joint was calculated with Amira software using attenuation thresholds of −150 to −400 Hounsfield units. The values represent mean ± SEM. * p<0.01; n = 7 in each group. D. The mass of total abdominal adipose tissue during pregnancy (GD15) was also quantified and expressed as a percentage of body weight. The total pool includes gonadal, retroperitoneal and mesenteric fat. Values represent mean ± SEM; *p<0.01, n≥18 in each group.
Table 1.
Pre-pregnancy metabolic parameters of fasted dams.
Figure 2.
Arterial Blood Pressure in elevated in obese dams at GD 15.
Blood pressure was determined at GD15 using tail cuffs. Animals were gently restrained in cages warmed to 37°C using a heating pad. The hatched bars represent CON-fed animals, while the solid black bars represent HF-fed animals. Values are mean ± SEM; *p<0.05, n≥23 in each group. The animals underwent 5 acclimatization cycles and 20 measurement cycles. The reported values are the mean of these 20 cycles.
Table 2.
Obstetrical outcomes of CON and HF fed dams.
Figure 3.
Neonatal health outcomes for pups born to CON or HF-fed dams.
A. The body weight of offspring of CON-fed (hatched bar) or HF-fed (solid black bar) dams on postnatal day 1 (PND1). B. The average number of pups per litter born to CON-fed or HF-fed dams. C. The percentage of CON-fed or HF-fed dams giving birth to at least one stillborn pup. D. The percentage of the total number of live pups in the litter that survived to PND4. E. The fetal/placental weight ratio was calculated for CON-fed and HF-fed dams. F. The average number of resorption sites for CON-fed and HF-fed dams at GD15. Values represent mean ± SEM; *p<0.05; n≥12 dams per group.
Figure 4.
The labyrinth layers of placentas from HF-fed dams exhibit increased expression of endothelial cell markers at GD15.
Representative images, acquired at 100× magnification, of GD15 placenta from CON-fed (A) and HF-fed dams (B) immunostained with CD31 antibody are shown. Scale bar = 50 µm; the labyrinth (L) and junctional zone (JZ) are indicated for reference. Four distinct regions from each histological section were quantified and averaged in determining percent immunopositive area. Images from an individual dam represent a single statistical unit. C. The percentage of area immunopositive for CD31 in the labyrinth of GD15 placenta based on the analysis of cross sections from CON-fed and HF-fed dams sacrificed at GD15. D. The number of blood vessels per field for CON-fed or HF-fed dams at GD15. All values mean ± SEM, *p<0.05; n = 5 dams for each group.
Figure 5.
The labyrinth layers of placentas from HF-fed dams exhibit fewer SMA positive blood vessels at GD15.
Representative images, acquired at 200× magnification, of GD 15 placenta from CON-fed (A) and HF-fed dams (B) immunostained with an antibody to SMA are shown. The SMA positive area within the labyrinth (L) of each group of animals was quantified. C. The average number of vessels per field of view staining positive for smooth muscle actin (SMA) in placentas from CON-fed and HF-fed dams. D. All values mean ± SEM; *p<0.05, n = 5 dams for each group.
Figure 6.
The labyrinth layers of placentas from HF-fed dams exhibit increased levels of carbonic anhydrase staining at GD15.
Representative images, acquired at 200× magnification, of GD 15 placenta from CON-fed (A) and HF-fed dams (B) immunostained with an antibody to carbonic anhydrase are shown. C. The average staining intensity in placentas from CON-fed and HF-fed dams was calculated. All values mean ± SEM, *p<0.05; n = 5 dams for each group.
Figure 7.
8-hydroxy 2-deoxyguanosine, a marker of systemic oxidative damage, is not significantly increased in the HF-dams.
Levels of 8-OH-2-dG were quantified in urine collected from GD15 CON-fed and HF-fed dams at sacrifice using a competitive EIA kit. 8-OH2-dG was normalized to the concentration of creatinine in urine. All values mean ± SEM, n = 15 for CON and n = 10 for HF-fed dams. †p<0.1 by one tailed Students T-test.
Figure 8.
Markers of tissue specific oxidative damage are not significantly increased in placentas from HF-fed dams at GD15.
A. 10 µg of total whole placental homogenate was separated on a 12.5% SDS-PAGE and subjected to Western blot analysis. The average content of 4-HNE was normalized to total protein (using Ponceau-S staining) in CON (hatched bars) or HF-fed (solid black bars) dams. B. Representative lanes containing 10 µg placental homogenates, developed using the 4-HNE monoclonal antibody. C. The relative content of protein carbonyls was quantified using a polyclonal antibody directed towards 2,4-dinitrophenylhydrazine (DNPH). This quantification was carried out using 5 µg of placental homogenate enriched for mitochondria. D. Representative lanes containing 5 µg of protein, enriched for mitochondria, prepared from CON or HF-fed dam placenta were separated on a 12.5% SDS-PAGE and developed using the anti DNPH polyclonal antibody. Values represent mean ± SEM; †p<0.10, n = 6 per group.