Term decidual tissue consists of terminally differentiated decidual cells, bone marrow-derived cells, and fibroblast cells. Since undifferentiated decidual cells are fibroblast-like cells of the endometrial stroma, the possibility exists that the fibroblast cells in term decidua are undifferentiated decidual cells. To test this hypothesis, a purified population of fibroblast cells was isolated from term decidua and treated under conditions that are known to induce differentiation of endometrial stromal cells. By flow cytometry and immunocytochemistry, the fibroblast cells from term decidua were shown to be free of cells expressing bone marrow-derived cell-surface antigens and the epithelial cell marker cytokeratin. In addition, they tested positive for the cytoskeletal protein vimentin, thus establishing that they were mesenchymal cells. As with endometrial stromal cells, continuous treatment of the decidual fibroblast cells with the progesterone analog medroxyprogesterone acetate and estradiol in combination with either dibutyryl-cAMP or prostaglandin E2 induced cell aggregation and the expression of prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). When cells were plated at an initial cell density of 0.25 x 10(6) cells/well in a 24-well culture dish with medium changes every three days, PRL was first detected on Days 4-6, and the peak of averaged 24 h-PRL release (30 ng/well) occurred on Days 26-28. The mRNA for decidual fibroblast PRL followed a temporal pattern corresponding to that of the released hormone. The size of the PRL mRNA was 1.15 kb, corresponding to the alternately spliced PRL mRNA reported for decidualized endometrial stromal cells and other extrapituitary sources of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

This study demonstrates the synthesis and release of prolactin (PRL) from dermal fibroblasts (>98%) in vitro, suggesting a potential local source of PRL in skin. PRL release was first detected in confluent cultures (0.25 x 10(6) plated cells) on or before day 18 and increased to a maximal level of 2 ng/72 h by day 30. Medroxyprogesterone acetate and estradiol (E2) had no effect on PRL release, but prostaglandin E2 (PGE2) reduced the time required for PRL induction to 6-9 days. The steroids and PGE2 together were synergistic, reaching maximal values of approximately 10 ng/72 h after 2 or more weeks of treatment. Dibutyryl-cyclic AMP, a second messenger in prostaglandin signal transduction, was also synergistic with medroxyprogesterone acetate and E2, but induced significant PRL expression in the absence of the steroids (28 and 12 ng/72 h, respectively). The increase in PRL release was not a result of increased cell proliferation, because the PRL-secreting cultures had 32.2 +/- 8.8% less DNA (N = 3 individuals, 93% confidence limit) than control cultures after 3 weeks of treatment with dibutyryl-cyclic AMP, medroxyprogesterone acetate, and E2. Dermal fibroblast PRL was immunologically and electrophoretically identical to decidual and pituitary PR-Ls, and Northern blot analysis demonstrated a PRL mRNA size of 1.15 kb. Maximal PRL release from fibroblast cells was 32.0 +/- 6.1 ng/72 h (mean +/- SD at 95% confidence limit) for a donor population representing both males (n = 15) and females (n = 7) between the ages of 20-week gestation to 52 years. In contrast to term decidual fibroblast cells that also express PRL, dermal fibroblasts did not co-express insulin-like growth factor-binding protein-1.

When small biological samples are collected by microdissection or other methods, amplification techniques are required to provide sufficient target for hybridization to expression arrays. One such technique is to perform two successive rounds of T7-based in vitro transcription. However the use of random primers, required to regenerate cDNA from the first round of transcription, results in shortened copies of cDNA from which the 5' end is missing. In this paper we describe an experiment designed to compare the quality of data obtained from labeling small RNA samples using the Affymetrix Two-Cycle Eukaryotic. Target Labeling procedure to that of data obtained using the One-Cycle Eukaryotic Target Labeling protocol. We utilized different preprocessing algorithms to compare the data generated using both labeling methods and present a new algorithm that improves upon existing ones in this setting.