Figure 4

Progesterone-induced RANKL expression contributes to the proliferative response in the pubertal mammary gland. Pubertal 4-week-old BALB/c mice were OVX, allowed to recover for 3 weeks, and then treated for 5 days with vehicle control (C), E2, P, or E2+P, as described in the Materials and Methods section. (A) Immunofluorescent detection of RANKL (green) in P and E2+P-treated mammary glands. (B) Dual immunofluorescent detection of RANKL (green) and PR (red) expression in representative images of an E2+P-treated duct and end bud. RANKL was most strongly expressed in ducts. Nuclei (A, B) were counterstained with DAPI (blue). Scale bars, 25 μm. (C) Effect of 5d P versus 5-day P+RANK-Fc on EB formation; note lack of significant EB regression with RANK-Fc treatment. Scale bar, 1 mm. (D) End-bud quantitation. Values represent the mean ± SEM number of end buds in the thoracic mammary gland (n = 3 mice). (E) The percentage of proliferating cells in ducts and EBs was determined by immunofluorescent detection of BrdU incorporation after C, P, and P+RANK-Fc treatment. P+RANK-Fc treatment had fewer proliferating cells in ducts than in P treatment (*P < 0.05). P+RANK-Fc treatment had fewer proliferating cells in EB than in P treatment (#P < 0.05).