Detection of differentially expressed genes between Erhualian and Large White placentas on day 75 and 90 of gestation

Abstract

Background: Placental efficiency is strongly associated with litter size, fetal weight and prenatal mortality. Together with its rapid growth during late gestation, the Large White pig breed shows a significant increase in placental size and weight, but this does not occur in the highly prolific Chinese pig breeds. To understand the molecular basis of placental development during late gestation in Chinese indigenous and Western breeds with different placental efficiency, female placental samples were collected from six pregnant Erhualian gilts at gestation day 75 (E75) and day 90 (E90) and from six pregnant Large White gilts at gestation day 75 (L75) and day 90 (L90). Two female placentas from one sow were used to extract RNA and then pooled in equal volumes. Twelve pooled samples were hybridized to the porcine Affymetrix GeneChip.

Results: A total of 226 and 577 transcripts were detected that were differentially expressed between E75 and L75 and between E90 and L90 (p < 0.01, q < 0.2), respectively. Gene Ontology (GO) analysis revealed that these genes belong to the class of genes that participate in angiogenesis and development. Real-time RT-PCR confirmed the differential expression of eight selected genes. Significant differential expression of five genes in the VEGF pathway was also detected between the breeds. A search of chromosomal location revealed that 44 differentially expressed genes located to QTL regions related to reproduction. Differential expression of six candidate imprinted genes was also confirmed. Three of the six genes (PLAGL1, DIRAS3, and SLC38A4) showed monoallelic expression in the porcine placenta.

Conclusion: Our study detected many genes that showed differential expression between placentas of two divergent breed of pigs, and confirmed the imprinting of three genes. These findings help to elucidate the genetic control of placental efficiency and improve the understanding of placental development.

Figure 1

Gene Ontology (GO) molecular function classification of all genes expressed in placenta and differentially expressed genes. (A) All expressed genes in placenta. (B) Differentially expressed genes in E75 vs L75. (C) Differentially expressed genes in E90 vs L90. The x-axis shows the gene percentage of each GO category with regard to the placenta transcripts or the declared differentially expressed genes and the y-axis represents each GO category.

Figure 2

Real time RT-PCR results of 13 genes. (A) Validation of the Microarray data by real-time RT-PCR analysis of eight representative genes. Expression levels of eight genes were detected in E75, L75, E90 and L90 by real-time RT-PCR (red) and microarray (blue). R represents the Pearson correlation coefficient. (B) real-time RT-PCR results of genes related to vascular development and vascular permeability. The x-axis represents the genes and the y-axis shows the fold change in expression. Different superscript alphanumeric characters indicate a statistically significant difference at p < 0.05.

Figure 3

Hierarchical cluster of differentially expressed genes. We have performed a data adjustment (median center and normalization) in the cluster analysis. The color codes of red, white, black and dark green represented expression levels of high, average, low and absent respectively. A detailed view of the genes expression levels in clustering patterns is shown in the plot areas from A to E.

Figure 4

Imprinting status of PLAGL1, SLC38A4 and DIRAS3 genes in porcine placentas. The results indicated that PLAGL1 and SLC38A4 genes are paternal expression in porcine placenta. DIRAS3 gene is monoallelic expression in porcine placenta.