Global gene expression analysis and regulation of the principal genes expressed in bovine placenta in relation to the transcription factor AP-2 family

Abstract

Background: Cell-cell communication is an important factor in feto-maternal units during placentogenesis. The placenta produces pivotal hormones and cytokines for communication between cotyledonary villi and the maternal caruncle. Gene expression in bovine placenta throughout pregnancy was comprehensively screened by a cDNA microarray, and we searched for a common transcription factor in a gene cluster that showed increasing expression throughout gestation in cotyledonary villi and caruncle.

Methods: Placentomal tissues (villi and caruncle) were collected from Day 25 to Day 250 of gestation for microarray analysis. Global gene expression profiles were analyzed using the k-means clustering method. A consensus sequence cis-element that may control up-regulated genes in a characteristic cluster was examined in silico. The quantitative expression and localization of a specific transcription factor were investigated in each tissue using quantitative real-time RT-PCR and in situ hybridization.

Results: The microarray expression profiles were classified into ten clusters. The genes with most markedly increased expression became concentrated in cluster 2 as gestation proceeded. Cluster 2 included placental lactogen (CSH1), pregnancy-associated glycoprotein-1 (PAG1), and sulfotransferase family 1E estrogen-preferring member 1 (SULT1E1), which were mainly detected in giant trophoblast binucleate cells (BNC). Consensus sequence analysis identified transcription factor AP-2 binding sites in some genes in this cluster. Quantitative real-time RT-PCR analysis confirmed that high level expression of transcription factor AP-2 alpha (TFAP2A) was common to cluster 2 genes during gestation. In contrast, the expression level of another AP-2 family gene, transcription factor AP-2 beta (TFAP2B), was extremely low over the same period. Another gene of the family, transcription factor AP-2 gamma (TFAP2C), was expressed at medium level compared with TFAP2A and TFAP2B. In situ hybridization showed that TFAP2A, TFAP2B and TFAP2C mRNAs were localized in trophoblast cells but were expressed by different cells. TFAP2A was expressed in cotyledonary epithelial cells including BNC, TFAP2B was specifically expressed in BNC, and TFAP2C in mononucleate cells.

Conclusion: We detected gestational-stage-specific gene expression profiles in bovine placentomes using a combination of microarray and in silico analysis. In silico analysis indicated that the AP-2 family may be a consensus regulator for the gene cluster that characteristically appears in bovine placenta as gestation progresses. In particular, TFAP2A and TFAP2B may be involved in regulating binucleate cell-specific genes such as CSH1, some PAG or SULT1E1. These results suggest that the AP-2 family is a specific transcription factor for clusters of crucial placental genes. This is the first evidence that TFAP2A may regulate the differentiation and specific functions of BNC in bovine placenta.

Figure 1

K-means clusters of the gene expression pattern from non-pregnant ENDO to Day 250 CAR and Day 25 EEM to Day 250 COT. The 1446 unique genes except for the genes that exhibited low expression intensity were subjected to clustering analysis. The blue line shows to the k-means center of gene expression on ENDO to CAR. The pink line shows to the k-means center of gene expression on EEM to COT. The expression intensity refers log2 value of normalized data.

Figure 2

QPCR analysis and normalized microarray data of ANXA1, CTDSP2, MSX1, HSPA1A, HSPA8, and SULT1E1 mRNA at each stage of bovine tissue (ENDO, CAR, EEM, COT). The gene expressions on Days 25 ENDO (n = 3), 60 CAR (n = 4), 150 CAR (n = 3), 250 CAR (n = 4), 25 EEM (n = 3), 60 CAR (n = 4), 150 CAR (n = 3), and 250 CAR (n = 4) are shown. The QPCR expression of these genes was normalized to the expression of GAPDH measured in the same RNA preparation. The pink bar shows gene expression on fetal side (EEM to COT) by QPCR. The blue bar shows gene expression on maternal side (ENDO to CAR) by QPCR. The yellow line shows normalized value of the microarray. Values are means ± SEM. Values with different letters are significantly different (P < 0.05).

Figure 3

Potential AP-2 binding site in upstream region (-200 to -1) of principal genes in cluster 2, identified by TFBIND software. The AP-2 consensus sequence is "MKCCCSCNGGCG" (M = A/C/G; K = A/G/T; S = G/C; N = A/G/C/T) from TRANSFAC databases. The threshold value exhibits homology with the above consensus sequence. "1" represents perfect coincidence with the consensus sequence.

Figure 4

Localization of SULT1E1 mRNA in a bovine placentome on Day 56 of gestation. SULT1E1 mRNA was detected by in situ hybridization. (A) DIG-labeled anti-sense cRNA probes were used. (B) DIG-labeled sense cRNA probes were used. Seven-micrometer sections of bovine placentome were hybridized with each probe. Scale bar = 20 μm. CaE: caruncular epithelium. CaS: caruncular stroma. CoE: cotyledonary epithelium. BNC: binucleate cell.

Figure 5

QPCR analysis of TFAP2A, TFAP2B, and TFAP2C mRNA at each stage of bovine tissue (ENDO, CAR, EEM, COT). The gene expression on Days 25 ENDO (n = 3), 60 CAR (n = 4), 150 CAR (n = 3), 250 CAR (n = 4), 25 EEM (n = 3), 60 CAR (n = 4), 150 CAR (n = 3), and 250 CAR (n = 4) are shown. The expression of these genes was normalized to the expression of GAPDH measured in the same RNA preparation. The pink bar shows gene expression on fetal side (EEM to COT) by QPCR. The blue bar shows gene expression on maternal side (ENDO to CAR) by QPCR. Values are means ± SEM. Values with different letters are significantly different (P < 0.05).

Figure 6

Localization of TFAP2A, TFAP2B, and TFAP2C mRNA in the bovine placentome on Day 56 of gestation. TFAP2A (A, B), TFAP2B (C, D) and TFAP2C (E, F) mRNA were detected by in situ hybridization. (A, C, E) DIG-labeled anti-sense cRNA probes were used. (B, D, F) DIG-labeled sense cRNA probes were used. Seven-micrometer sections of bovine placentome were hybridized with each probe. Scale bar = 20 μm. CaE: caruncular epithelium. CaS: caruncular stroma. CoE: cotyledonary epithelium. BNC: binucleate cell.