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Transit of Rat Uterine Stromal Cells through G1 Phase of the Cell Cycle Requires Temporal and Cell-Specific Hormone-Dependent Changes on Cell Cycle Regulators

Department of Biology, Pittsburg State University, Pittsburg, Kansas 66762

Address all correspondence and requests for reprints to: Dr. Virginia Rider, Pittsburg State University, 1701 South Broadway, Pittsburg, Kansas 66762. E-mail: vrider{at}pittstate.edu.

Progesterone pretreatment increases the number of synchronously proliferating stromal cells in the ovariectomized rat uterus, but estrogen is necessary to stimulate reentry into the cell cycle. To investigate the mechanisms underlying differential hormone actions, sexually mature ovariectomized rats were injected with progesterone (2 mg) for three consecutive days. Estradiol 17-ß (0.6 µg) was administered to initiate cell proliferation. Uterine samples were collected at timed intervals. Cell entry into DNA replication was monitored by injecting 5-bromo-2'-deoxyuridine (1 mg/100 g body weight) 2 h before necropsy. Demicolchicine (400 µg) was injected 30 min before necropsy to assess transit into M phase. Temporal progress through G1 was determined by spatial changes in cyclin D1/D3 proteins. Total cyclin D1/D3 protein and mRNA was measured by Western and Northern blotting. Estrogen increased the number of 5-bromo-2'-deoxyuridine-positive stromal cells (P < 0.05), compared with the number in rats treated with progesterone alone. An increase (P < 0.05) in the number of M-phase cells occurred at 12 h post estrogen. There was no evidence for epithelial cell proliferation in response to steroid treatments. Cyclin D1/D3 mRNA was expressed in the uteri of ovariectomized and hormone treated rats. The D-type cyclin proteins, however, were not evident in stromal cells without estrogen treatment. Progesterone pretreatment inhibited estrogen-dependent epithelial cell proliferation while redirecting D-type cyclin expression to the uterine stroma. Stromal cell transit through G1 required nongenomic steroid-dependent action on signal transduction pathways that control the nuclear localization and cell type-specific expression of the D-type cyclin proteins.

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