PORCINE ENDOMETRIAL GLANDULAR EPITHELIAL-CELLS IN-VITRO - TRANSCRIPTIONAL ACTIVITIES OF THE PREGNANCY-ASSOCIATED GENES ENCODING ANTILEUKOPROTEINASE AND UTEROFERRIN

The aim of this investigation was to establish a homologous culture sy stem for study of the transcriptional mechanisms underlying endometria l expression of the pregnancy-associated genes encoding antileukoprote inase (ALP), an elastase/cathepsin G protease inhibitor, and uteroferr in (Uf), a transplacental iron transport protein. Glandular epithelial (GE), luminal epithelial (LE), and stromal (ST) cells were isolated f rom pig endometrium at Day 12 of pregnancy by differential enzymatic d igestion and sieve filtration. The three cell populations differed wit h respect to their morphology in culture and with respect to their exp ression of ALP and Uf. Expression of the ALP gene was much higher in G E 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 l evels of secreted Uf only in GE. The steroid hormone responsiveness of GF cells was monitored by changes in steady-state levels of ALP mRNA after 24-h exposure to estradiol 17 beta (E(2); 10 nM) and/or progeste rone (P; 10 nM). Glandular epithelial cells treated with E(2), P, and E(2) + P had increased (p < 0.05) ALP mRNA levels relative to those in control cultures. Glandular epithelial cells were transiently transfe cted with reporter constructs containing the 5'-flanking genomic regio ns of each gene. For ALP, the 1266-nucleotide (nt) region of the ALP 5 '-flanking genomic DNA, and progressive 5' deletions within this regio n, were coupled to a luciferase reporter gene (LUCE). The most proxima l 119-bp fragment (-119ALP LUCE), which contains the TATAA box (-21 to -26 nt) and a CC-rich sequence (-66 to -74 nt), was sufficient to con fer transcriptional activity to the reporter vector. Progressively lon ger 5'-genomic fragments had promoter activities higher than or simila r to those of the 119-nt fragment. Estrogen had no effect on the trans criptional activities of any of the ALP constructs. Uteroferrin 5'-fla nking and promoter DNA constructs containing the chloramphenicol acety l transferase (CAT) reporter gene also exhibited transcriptional activ ity in GE cells. The presence of multiple interacting cis-regulatory s equences within this region was demonstrated by increased promoter act ivity, relative to that of the smallest construct (-182 UFCAT-E; basal activity), with the inclusion of sequences between -182 and -484 nt, and drastic reduction to basal activity with the inclusion of sequence s between -484 and -831 nt. In summary, primary cultures of CE from ea rly-pregnant porcine endometrium express ALP and Uf, are steroid hormo ne-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 endomet rial gene expression during pregnancy.