Temporal spatial and cell type specific gene expression and immunolocalization of proteins involved in implantation

ANAHÍ FRANCHI; XIAOMEI ZHANG; SILVINA BOCCA; SERGIO OEHNINGER

Barcelona, Spain.

Congreso; 24th Annual Meeting of the European Society of Human Reproduction and Embryology; 2008

European Society of Human Reproduction and Embryology

Introduction: We previously established endometrial gene expression profiles during the early and mid-secretory (window of implantation-WOI) phases of the menstrual cycle using genomic microarrays. We identified three genes with functional significance based upon the presence of putative estrogen receptor elements and up-regulation between 7?35-fold comparing the receptive and pre-receptive endometrium. Here, we further characterized the temporal gene expression of decay accelerating factor (DAF), interleukin 15 (Il-15) and osteopontin (OPN), and localized their products and ligands/receptors (complement component 3 or C3, IL-15Ra and alphavbeta3 integrin), at the opening and closure of the WOI. In addition we used laser capture microdissection (LCM) to analyze the cell type-specific gene expression in epithelial versus stromal cells. Materials and methods: Endometrial tissue was obtained from regularly cycling, ovulatory women at 3, 8 or 11 days after the LH surge, corresponding to cycle day 16 (pre-receptive, n=3), 21 and 24 (opening, n=3 and closure, n=3 of the WOI). Real time RT-PCR was used to determine the expression of DAF, OPN and IL-15, normalized to the housekeeping gene 18S, in LH+3, +8 and +11 samples and in laser microdissected epithelial and stromal cells. The protein products of these genes and their known ligands/receptors were localized by immunohistochemistry. Results: Gene expression levels for all three genes during the WOI were significantly higher than on day 16 (P , 0.03). Relative mRNA levels showed increased expression for all genes: 17- and 46-fold change for DAF, 3.1- and 5.3-fold change for IL-15, and 9.1- and 19.9-fold change for OPN, for day 21 and day 24 respectively, compared with day 16. By immunohistochemistry DAF was found mainly in the epithelial cells with marked increase on day 21, which was sustained on day 24. C3, the ligand of DAF, was found in both the epithelium and stroma at all time points. IL-15 and its specific receptor IL-15Rá, were present in all days in the secretory phase. For IL-15, the immunostaining intensity in the stroma was strong throughout time, while in the epithelial compartment was higher by days 21 and 24. IL-15Rá was localized in the nuclear region; both stromal and epithelial cells showed stronger staining by day 21. A decreased expression was seen by day 24, with patched immunoreactions in the stroma. OPN was localized in the glands and surface epithelium, with stronger staining in the apical region of the cells, and an increased expression observed during the WOI. Nearly identical temporal and spatial patterns were found for its receptor, alphavbeta3 integrin, whose expression was markedly increased on days 21 and 24. The cell type-specific pattern of gene expression was analyzed by real time PCR in epithelial and stromal areas obtained by LCM. The relative expression of DAF was 4.8-fold increased in epithelium versus stroma, whereas for OPN there was a 2-fold change. For IL-15, the expression in stroma was 8.7-fold higher than in epithelial cells. In addition, progesterone and estrogen receptors were analyzed, showing 3.2-fold and 1.5-fold higher expression, respectively, in stromal versus epithelial cells. Conclusions: This study demonstrated that three immunomodulatory genes, DAF, IL-15 and OPN, are up-regulated at the onset of the WOI and that a progressive increase in gene expression occurs throughout the putative closure of the window. Furthermore, their specific proteins products, as well as their ligands/receptors, demonstrated similar temporal variations suggesting that these proteins are increased at the time of receptiveness. We showed a gene expression pattern within specific endometrial compartments, which is the first step to improve our understanding on the endocrine and paracrine modulation of these proteins playing an important role during the establishment of the WOI in the human.