(2)血管新生と脱落膜化における性ステロイドホルモンによる調節機構の解明(シンポジウム2「子宮内膜の機能調節とその病態」,第62回日本産科婦人科学会・学術講演会)

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The human endometrium undergoes remarkable cyclic growth and regeneration in response to the female sex steroids estrogen (E) and progesterone. Local autocrine and paracrine molecules that vary periodically during the menstrual cycle have been suggested to play various roles in uterine function. Endometrial growth and repair after menstruation are associated with profound angiogenesis. Several mediators regulate angiogenesis, including, vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR-1) as a VEGF antagonist, and stromal cell-derived factor 1 (SDF-1/CXCL12). Firstly, we determined whether E can regulate VEGF and sVEGFR-1 as a VEGF antagonist, and SDF-1 in human endometrial stromal cells (ESCs) and to determine whether SDF-1 can induce the proliferation mediated by its receptor (CXCR4) in the human endometrium. In human endometrium, local factors that vary periodically during the menstrual cycles have been suggested to play various roles in uterine function. Progesterone is a key factor in establishment and maintenance of pregnancy in the human endometrium. Secondly, differential gene expression in human endometrium was investigated by using a human cDNA expression array system. Proprotein convertase (PC) 6 is suggested to play an important role in the processes of stromal cell decidualization and embryo implantation in the mouse. Thirdly, we investigated the regulation of PC6 mRNA and protein expression in human ESCs during decidualization in vitro. Finally, we have aimed to determine whether infertility in some patients might be attributable to change the expression of local factors regulated by E and P.E significantly induced VEGF mRNA levels, whereas E caused a significant decrease in sVEGFR-1 mRNA levels. E significantly increased the VEGF production levels and attenuated the sVEGFR-1 production compared with control. The decrease in levels of free VEGF was proportionate to the rise in sVEGFR-1 levels in the culture media of ESCs. E stimulates VEGF production and concurrently attenuates sVEGFR-1 production in ESCs. This consequential increase in VEGF/sVEGFR-1 ratio might enhance the biological effects of VEGF on the angiogenic environment in human endometrium. E significantly induced SDF-1 mRNA levels in ESCs compared with that in control cells. E enhanced the SDF-1 production levels in a time- and dose-dependent manner that could be completely abolished by ICI 182,780. P could antagonize the E-stimulated effects. Although SDF-1 was undetectable in Ishikawa cells, CXCR4 mRNA levels in Ishikawa cells were much higher than those in ESCs. SDF-1 induced the proliferation of Ishikawa cells; this could be inhibited by AMD 3100 (CXCR4 antagonist). E induces SDF-1 mRNA and protein production in ESCs; this may play a role in endometrial epithelial cell growth in the paracrine system. By using microarray analysis, we found that the mRNA expression of Interleukin-15 (IL-15) and fibulin-1 were up-regulated by P. In human endometrium, expression of IL-15 mRNA significantly increased during the secretory phase compared with the proliferative phase. The most abundant expression of IL-15 mRNA during the menstrual cycle was observed in the midsecretory phase. Northern blot analyses revealed a significant increase in IL-15 mRNA levels in ESCs treated with P alone or E plus P compared with vehicle. Furthermore, P is a potent inducer of IL-15 mRNA expression in ESCs in a dose-dependent manner. On the other hand, E alone did not increase IL-15 mRNA expression. By ELISA, IL-15 protein secretion was stimulated by P and further enhanced by combined treatment with E and P, whereas E alone was ineffective. It is suggested that IL-15 is deeply involved in the hormonal control of the human endometrium by P and E. By using microarray analysis, we identified that one of the genes upregulated after progesterone treatment was fibulin-1, which codes for an extracellular matrix and plasma glycoprotein. Quantitative analysis with real-time PCR experiments on human endometrial tissues showed significantly higher fibulin-1 mRNA expressions in secretory phase endometria than in proliferative phase. Immunohistochemical studies revealed that the fibulin-1 protein is expressed in the glandular epithelium in proliferative phase endometria, and that expression switched to the stroma in secretory phase endometria. In culture experiments with ESCs, a significant increase of fibulin-1 mRNA expression was observed in cells treated with P.P stimulated the fibulin-1 mRNA expression in a dose-dependent manner, whereas E alone did not increase the fibulin-1 mRNA expression. These results suggest that fibulin-1 is an important molecule that mediates P action in human ESC differentiation towards implantation. Real-time PCR analyses revealed a significant increase in PC6 mRNA levels in ESCs treated with E plus P during decidualization. On the other hand, E alone did not increase PC6 mRNA expression. Using an antisense morpholino approach, PRL production, a typical marker for decidualization, was significantly attenuated in decidualized ESCs following treatment with PC6 morpholino antisense oligonucleotides in comparison to controls. These results suggest that PC6 plays a key role for decidualization in human endometrium. With the use of real-time PCR, we have studied the expression levels of VEGF, SDF-1, IL-15, Fibulin-1, and PC6 mRNA in receptive phase endometria from 31 idiopathic infertile patients. The patients were categorized as follows: Group A had never been pregnant; Group B had become pregnant after intercourse and IUI; Group C had become pregnant after IVF and ICSI. There were no statistically significant differences regarding the expression of VEGF, SDF-1, and PC6 mRNA in all groups. The levels of IL-15 and Fibulin-1 mRNA were significantly decreased in Group B with regard to Group A and C. Significantly decreased levels of these transcripts in human endometria may be associated with implantation failure. Our findings contribute not only on the insight into mechanisms of key reproductive processes such as decidualization and the establishment and maintenance of early pregnancy, but also on future clinical applications for implantation failure.
2010-11-01

(2)血管新生と脱落膜化における性ステロイドホルモンによる調節機構の解明(シンポジウム2「子宮内膜の機能調節とその病態」,第62回日本産科婦人科学会・学術講演会)

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