Antenatal dexamethasone treatment leads to changes in gene expression in a murine late placenta

Abstract

Antenatal steroids like dexamethasone (DEX) are used to augment fetal lung maturity and there is a major concern that they impair fetal growth. If delivery is delayed after using antenatal DEX, placental function and hence fetal growth may be compromised even further. To investigate the effects of DEX on placental function, we treated 9 pregnant C57/BL6 mice with DEX and 9 pregnant mice were injected with saline to serve as controls. Placental gene expression was studied using microarrays in 3 pairs and other 6 pairs were used to confirm microarray results by semi-quantitative RT-PCR, real-time PCR, in situ hybridization, western blot analysis and Oligo ApopTaq assay. DEX-treated placentas were hydropic, friable, pale, and weighed less (80.0+/-15.1mg compared to 85.6.8+/-7.6mg, p=0.05) (n=62 placentas). Fetal weight was significantly reduced after DEX use (940+/-32mg compared to 1162+/-79mg, p=0.001) (n=62 fetuses). There was >99% similarity within and between the three gene chip data sets. DEX led to down-regulation of 1212 genes and up-regulation of 1382 genes. RT-PCR studies showed that DEX caused a decrease in expression of genes involved in cell division such as cyclins A2, B1, D2, cdk 2, cdk 4 and M-phase protein kinase along with growth-promoting genes such as EGF-R, BMP4 and IGFBP3. Oligo ApopTaq assay and western blot studies showed that DEX-treatment increased apoptosis of trophoblast cells. DEX-treatment led to up-regulation of aquaporin 5 and tryptophan hydroxylase genes as confirmed by real-time PCR, and in situ hybridization studies. Thus antenatal DEX treatment led to a reduction in placental and fetal weight, and this effect was associated with a decreased expression of several growth-promoting genes and increased apoptosis of trophoblast cells.

Figure 1. Gross and microscopic appearance of saline and DEX treated placentas and Oligo-ApopTag assay showing trophoblast apoptosis

Panel A shows gross appearance of saline and DEX treated placentas. Panels B and C are haematoxylene and eosine stained sections of placentas, control placenta in panel C has a well defined layers of junctional trophoblast cells (small arrow) and labyrinthine trophoblast cells (large arrow) in contrast DEX treated placenta in panel B show loss of trophoblast cells mainly in the junctional zone as outlined by small arrowheads. Bar represents 100 μm. Panel D and E represents Oligo-ApopTaq assay to study trophoblast apoptosis. There is marked increase in apoptotic nuclei (large arrows) in DEX-exposed placenta (panel D) compared to saline-exposed control placenta (panel E). Bar represents 50 μm.

Figure 2. Semi-quantitative RT-PCR

There was down-regulation of cyclin B1, D2, F1 along with MC protein kinase, cdk2, cdk4 and epithelial growth factor receptor (EGF-R), and bone morphogenic protein-4 (BMP-4) after DEX-treatment. Expression of HPRT was used to normalise data from each group and * represents statistically significant change in expression.

Figure 3. Representative western blot from two control and two DEX-exposed placentas

There was increased expression of caspases 1 p10 and caspase 3 p11, along with pJNK1, pERK1 but no change in expression of serine/threonine kinase Akt or pAkt. β-actin served as internal control.

Figure 4. Quantitative real-time RT-PCR for aquaporin 5 and tryptophan hydroxylase

DEX-treatment led to up-regulation of aquaporin 5 gene by 23 folds and tryptophan hydroxylase gene by 12 folds (n=6 in each group). * represents statistically significant change in expression.

Figure 5. In situ hybridization study to show expression pattern of aquaporin 5

Panel A and C are control placentas, panel A is hybridized with a sense probe and panel C with antisense probe. Panels B and D are DEX-exposed placenta hybridized with a sense and antisense probes respectively. Large arrows indicate junctional trophoblast cell layer and arrow heads delineate areas of loss of trophoblast cells in panels B and D of DEX-treated placentas. Bar represents 50 μm.