Molecular characterization and identification of the E2/P4 response element in the porcine HOXA10 gene

Abstract

Homeobox A10 (HOXA10), a well-known transcriptional factor that can be regulated by estrogen (E(2)) and progesterone (P(4)), is necessary not only for endometrial differentiation but also for establishing the conditions required for implantation. However, little research has focused on the regulation of the porcine HOXA10 gene. In this study, we aimed to (1) characterize the genetic structure of the porcine HOXA10 gene, (2) analyze the tissue expression pattern and differential expression levels in the endometrium of HOXA10 in different developmental stages of Meishan and commercial Yorkshire pigs, and (3) identify the E2/P4 response element in the promoter region of the porcine HOXA10 gene and verify that it induces HOXA10 in human endometrial adenocarcinoma (Ishikawa) cells. The results indicated that the cDNA of porcine HOXA10 was 2,628 bp in length with an open reading frame (ORF) of 1,236 bp encoding a peptide of 411-amino acid residues, which showed 90 and 95 % sequence similarity to that of human and mouse homologs, respectively. Semiquantitative RT-PCR confirmed that the porcine HOXA10 gene is highly expressed in the endometrium, bladder and kidney. Real-time PCR showed that the expression of HOXA10 was significantly higher on day 15 of gestation (gd 15) in comparison to gd 26 (P < 0.01), gd 50 (P < 0.01) and day 15 of the estrous cycle (ed 15) in the endometrium of Meishan pigs. In contrast, the highest expression in the endometrium of Yorkshire pigs was on gd 50. Moreover, the abundance of HOXA10 mRNA was the highest on gd 15 in Meishan pigs than in any other stage tested in the two breeds. Deletion analysis and reporter expression assays identified a promoter region (-1044 bp to +18 bp) which is responsible for E(2)- and P(4)-induced HOXA10 transcription. Moreover, this promoter region enhanced the E2/P4-induced HOXA10 expression in Ishikawa cells. In conclusion, we identified an E(2)/P(4) response element of the porcine HOXA10 gene for the first time. The data from the present study contribute to the mechanism by which the porcine HOXA10 gene regulates embryo implantation and prolificacy traits.