-Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17 β (E 2 ) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α ), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-α increased the percentage of BrdU-labeled cells in a dose-dependent manner. Administration of the combination of E 2 (10 -9 M) and P (10 -7 M) for 2 days increased the percentage of BrdU-labeled cells 2.3-fold. The stimulatory effect of EGF, TGF-α and the combination of E 2 and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10 -5 M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-α -, and EGF-mRNA was carried out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-α , both produced in the stromal cells, acting for DNA synthesis through EGF receptors.

-Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17 β (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.

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Runx3 is a transcription factor that belongs to the Runx family. We studied the localization of Runx3 mRNA in the mouse uterus, and its function in the mouse endometrium using Runx3 knockout (Runx3(-/-)) mice. Runx3 mRNA was detected in the endometrial luminal epithelial cells, glandular epithelial cells and stromal cells below the epithelial cell layer on the luminal side. The uteri of Runx3(-/-) mice were smaller than those of wt mice. The endometrial layer and uterine glands of Runx3(-/-) mice were less developed than those of wild-type mice, and the endometrial stromal layer was thinner. Transforming growth factor β1 and β3 (TGFβ1 and β3) mRNA levels in endometrial stromal cells of Runx3(-/-) mice were low compared with those of wild-type mice. Estradiol-17β (E2) increased Tgfb2 mRNA levels in endometrial stromal cells of Runx3(-/-) mice, but not in those of wild-type mice. E2 increased epidermal growth factor (EGF) mRNA levels in endometrial stromal cells of wild-type mice, but did not increase those of Runx3(-/-) mice. The diminished Tgfb1 and Tgfb3 mRNA expressions may lead to the reduced proliferation of endometrial stromal cells. Alterations of E2-associated expressions of Tgfb2 and Egf mRNA in endometrial stromal cells of Runx3(-/-) mice may be associated with suppression of E2-dependent endometrial epithelial cell proliferation in Runx3(-/-) mice. Thus, Runx3 is likely to be a regulatory factor responsible for endometrial growth.

Transforming growth factor-α (TGFα) is thought to be involved in the regulation of endometrial cells. We investigated Tgfa mRNA expression, and the effects of TGFα on DNA-synthesis and gene expression of insulin-like growth factor 1 (IGF1), IGF binding protein-3 (IGFBP3) and IGF1 receptor in the mouse endometrial cells, because IGF1 is involved in estrogen-induced growth of endometrial cells. We also investigated the role of TGFα on matrix metalloproteinase (MMP) expression, as MMPs are involved both in tissue remodeling during cell proliferation and in enhancement of IGF1 signaling through the degradation of IGFBP3. Tgfa mRNA expression was detected in endometrial luminal and glandular epithelial cells, and stromal cells. Tgfa mRNA signals did not appear to change in endometrial luminal epithelial cells, but signals in glandular epithelial cells were higher at diestrus 1, 2 and proestrus, and the number of stromal cells showing strong signals appeared to increase at diestrus 1 and 2. Endometrial epithelial and stromal cells were treated with estradiol-17β (E2) or progesterone (P4). E2 or P4 stimulated Tgfa mRNA expression in stromal cells. TGFα stimulated DNA synthesis in endometrial epithelial and stromal cells, while E2 and P4 stimulated DNA synthesis in stromal cells. In stromal cells, TGFα, at as low as 1 ng/ml, decreased Igfbp3 and Mmp9 mRNA levels, while high dose (10 ng/ml) of TGFα decreased Igf1 mRNA level and increased Mmp3 mRNA level. These results imply that TGFα stimulates proliferation of endometrial stromal cells through multiple mechanisms, including its regulation of Igfbp3 and Mmp3 transcription.