Porcine endometrial glandular epithelial cells in vitro: transcriptional activities of the pregnancy-associated genes encoding antileukoproteinase and uteroferrin

Abstract

The aim of this investigation was to establish a homologous culture system for study of the transcriptional mechanisms underlying endometrial expression of the pregnancy-associated genes encoding antileukoproteinase (ALP), an elastase/cathepsin G protease inhibitor, and uteroferrin (Uf), a transplacental iron transport protein. Glandular epithelial (GE), Luminal epithelial (LE), and stromal (ST) cells were isolated from pig endometrium at Day 12 of pregnancy by differential enzymatic digestion and sieve filtration. The three cell populations differed with respect to their morphology in culture and with respect to their expression of ALP and Uf. Expression of the ALP gene was much higher in GE than in LE cells and was undetectable in ST cells. Similarly, GE had the highest expression of the Uf gene, and expression in ST was lower but distinct. Western blot analysis of conditioned media (72 h) from GE, LE, and ST, using antiporcine Uf antiserum, detected significant levels of secreted Uf only in GE. The steroid hormone responsiveness of GE cells was monitored by changes in steady-state levels of ALP mRNA after 24-h exposure to estradiol 17 beta (E2; 10 nM) and/or progesterone (P; 10 nM). Glandular epithelial cells treated with E2, P, and E2 + P had increased (p < 0.05) ALP mRNA levels relative to those in control cultures. Glandular epithelial cells were transiently transfected with reporter constructs containing the 5'-flanking genomic regions of each gene. For ALP, the 1266-nucleotide (nt) region of the ALP 5'-flanking genomic DNA, and progressive 5' deletions within this region, were coupled to a luciferase reporter gene (LUCE). The most proximal 119-bp fragment (-119ALP LUCE), which contains the TATAA box (-21 to -26 nt) and a GC-rich sequence (-66 to -74 nt), was sufficient to confer transcriptional activity to the reporter vector. Progressively longer 5'-genomic fragments had promoter activities higher than or similar to those of the 119-nt fragment. Estrogen had no effect on the transcriptional activities of any of the ALP constructs. Uteroferrin 5'-flanking and promoter DNA constructs containing the chloramphenicol acetyl transferase (CAT) reporter gene also exhibited transcriptional activity in GE cells. The presence of multiple interacting cis-regulatory sequences within this region was demonstrated by increased promoter activity, relative to that of the smallest construct (-182 UFCAT-E; basal activity), with the inclusion of sequences between -182 and -484 nf, and drastic reduction to basal activity with the inclusion of sequences between -484 and -831 nt. In summary, primary cultures of GE from early-pregnant porcine endometrium express ALP and Uf, are steroid hormone-responsive, and support the transcriptional activity of endometrial-associated gene promoter and regulatory sequences. The use of primary GE cells thus provides a convenient in vitro system for further study of the endocrine, paracrine, and autocrine factors regulating endometrial gene expression during pregnancy.