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Ovariectomies were done on 6- to 8-week-old mice and 14 days later animals were treated with hormone [E (1 ng/μl) or P (10 ng/μl) or both] dissolved in sesame oil. For protein immunoblot analysis of uterine PR expression and BrdU immunolabeling ovariectomized mice were given daily subcutaneous (s.c.) injections of E (100 ng) alone or E (100 ng) and P (1 mg) for 4 days. Immunoblots were probed with polyclonal rabbit immunoglobulin G (IgG) to PR (1:1000 dilution; Santa Cruz Labs Santa Cruz CA) or polyclonal rabbit IgG to LF as described (21). Uterine sections (5 μm) were probed for immunohistochemistry with rabbit polyclonal IgG to PR (1:100 dilution; DAKO Denmark) as described (17). For BrdU immunolabeling mice were administered intraperitoneal injections of BrdU (30 μg per gram of body weight) 2 hours before being killed. Uteri were fixed in 4% paraformaldehyde and embedded in paraffin and 5-μm sections were prepared. BrdU uptake was detected with the Cell Proliferation kit (Amersham-Pharmacia Biotech Buckinghamshire UK). Image Tool software (UTHSCS San Antonio TX) was used to determine immunoreactive cells per unit area. For each animal four independent sections each containing 1000 to 1500 cells were counted. For protein immunoblot analysis of LF mice were given a single injection of E (100 ng) or E (100 ng) plus P (1 mg). For ribonuclease protection (RPA) analysis uterine RNA was isolated 6 hours (AR) or 14 hours (CT and HDC) after a single dose of P (1 mg). Total RNA (10 μg) was analyzed with 32 P-labeled uridine 5′-triphosphate–antisense RNA probes (MAXI-script; Ambion Austin TX) and the RPA assay kit (RIPA III; Ambion Austin TX). A 400–base pair (bp) CT clone was from EST bank (958362). Clones HDC [350-bp Bam HI–Eco RI fragment in pGEM7ZF (+)] and AR [475-bp Sac I–Dra II fragment in pBS-SKII(+)] were gifts from S. K. Dey (University of Kansas Medical Center Kansas City KS).

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Beeswax pellets containing 20 mg of E and 20 mg of P or no hormones were implanted s.c. on days 1 and 10 and tissues were collected 11 days later. Mammary glands were fixed in 10% formalin and stained with hematoxylin as described (2). For indirect immunofluorescence frozen mammary tissue was sectioned at 50-μm thickness permeabilized with 5% Triton X-100 and fixed in 4% paraformaldehyde. Sections were incubated overnight at 4°C in rabbit polyclonal antiserum to cytokeratin-14 (1:500 dilution; a gift from D. R. Roop Baylor College of Medicine) containing 1% bovine serum albumin and rat monoclonal IgG to E-cadherin (1:500 dilution; Zymed San Francisco CA). Antibodies were visualized with Texas Red–goat antibodies to rabbit IgG (1:200 dilution; Molecular Probe Eugene OR) and fluorescein isothiocyanate–goat antibodies to rat IgG (1:400 dilution; PharMingen San Diego CA). Serial optical sectioning was performed with a confocal laser-scanning microscope (Multiprobe 2001 Molecular Dynamics). Three-dimensional images were reconstructed with Image Space Software (Molecular Dynamics).

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We thank A. Bradley for ES cells pNEOTKLOX and helpful suggestions; S. O'Gorman for pOG231; L. Hadsell M. Gu and A. Ward for technical assistance; and J. S. Richards and D. L. Medina for helpful discussions. Supported by NIH grant HD32007 to O.M.C.