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Vitamin D₃ receptor ablation alters mammary gland morphogenesis

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA

View larger version (20K): [in a new window]   Fig. 1. Fertility and growth of wild-type and Vdr knockout mice. Homozygous wild-type and Vdr knockout mice maintained on a high Ca2+ diet were mated at 8 weeks of age. (A-C) Litter size (A), pup survival to weaning (B) and growth after weaning (C) were monitored for both male and female offspring. (D) Serum estradiol was measured by radioimmunoassay in virgin females. Results are mean±s.e.m. (n=7-8); no statistically significant differences were observed between the genotypes.

View larger version (125K): [in a new window]   Fig. 2. Representative wholemounts of mammary glands from wild-type and Vdr knockout mice. (A,B) Inguinal mammary glands from 6-week-old virgin female wild-type (A) and Vdr knockout (B) mice maintained on the high Ca2+ diet were fixed in Carnoy’s and stained with Carmine Alum to visualize ductal development. Arrows indicate terminal end buds. Note the greater ductal extension and density and more numerous end buds in the gland from the Vdr knockout mouse compared with the wild-type mouse. The darkly stained round object within the ducts is the inguinal lymph node. Scale bar: 2 mm.

View larger version (24K): [in a new window]   Fig. 3. Quantitative analysis of mammary gland morphogenesis in virgin wild-type and Vdr knockout mice. (A) Fresh weights of surgically dissected mammary gland fat pads from virgin wild-type and Vdr knockout mice (mean±s.e.m., n=10-12 per group), expressed as organ weight to body weight ratio (mg/g). (B) The number of terminal end buds per mammary gland was counted in wholemounts of inguinal glands removed from weight-matched wild-type and Vdr knockout females from 4-10 weeks of age. (C) Ductal extension (defined as the distance from the center of the lymph node to the leading edge of the gland) was measured on inguinal wholemounts from 6-week-old wild-type and Vdr knockout virgin females. (D) The number of primary, secondary and tertiary branch points was quantitated on inguinal wholemounts from 6-week-old wild-type and Vdr knockout virgin females. Data in B-D represent mean±s.e.m. of at least six animals per genotype per time point. *Statistically significant, wild-type versus Vdr knockout (P<0.05).

View larger version (127K): [in a new window]   Fig. 4. Morphology of mammary glands from wild-type and Vdr knockout mice. Formalin fixed inguinal mammary glands from 6-week-old virgin wild-type (A) and Vdr knockout (B) mice were sectioned and stained with Hematoxylin and Eosin. Longitudinal sections through two terminal end buds are shown in each panel. The terminal end bud in B is undergoing bifurcation, which will result in a branch point. Scale bar: 100 µm.

View larger version (75K): [in a new window]   Fig. 5. Expression of the VDR in mammary gland terminal end bud of wild-type mice. Formalin fixed sections of mammary gland from a 6-week-old virgin wild-type mouse were subjected to immunohistochemistry with a monoclonal antibody directed against VDR. VDR positive cells appear brown against the blue Hematoxylin counterstain. (A) VDR was localized to the nuclei of both stromal and epithelial cells in the terminal end bud. VDR expression was low in the leading edge (box B) compared to the trailing edge (box C). (B) Higher magnification of boxed area B (leading edge of terminal end bud) from A, showing weak VDR staining in highly proliferative cap cells (arrowheads) and epithelial body cells (arrows). (C) Higher magnification of boxed area C (trailing edge of terminal end bud) from A, showing strong nuclear VDR positivity in the luminal and myo-epithelial cells (arrows) and weaker but detectable staining in stromal cells (arrowheads). Scale bar, 50 µm.

View larger version (151K): [in a new window]   Fig. 6. Expression of the VDR in mature ducts and lymph node of wild-type mice. Formalin fixed sections of mammary glands (A-E) and lymph node (F) from virgin wild-type mice were subjected to immunohistochemistry with a monoclonal antibody directed against VDR. VDR-positive cells appear brown against the blue Hematoxylin counterstain. The number of cells positive for nuclear VDR staining was highest in ducts of mice examined at 5 (A) and 6 (B) weeks of age. Both the number of VDR-positive cells and the intensity of VDR staining declined in mice at 7 (C), 8 (D) and 10 (E) weeks of age. VDR expression was also detected in a subset of lymphocytes within the central lymph node (F), but this did not change with age. Scale bars: 50 µm.

View larger version (92K): [in a new window]   Fig. 7. DNA synthesis and apoptosis in terminal end buds of wild-type and Vdr knockout mice. Immunohistochemistry was performed on mammary gland sections from 6-week-old wild-type (A,C) and Vdr knockout (B,D) mice to detect BrdU incorporation (A,B) and TUNEL (C,D). For both BrdU and TUNEL, positive cells appear brown against the blue Hematoxylin counterstain. Arrows in C,D indicate TUNEL-positive cells with characteristic apoptotic morphology. No differences in either proliferation or apoptosis were noted to be due to genotype. Scale bars: 50 µm.

View larger version (171K): [in a new window]   Fig. 8. Response of mammary glands from wild-type and Vdr knockout to exogenous hormones. Representative wholemounts of mammary glands removed from wild-type (A,C,E) and Vdr knockout (B,D,F) mice before and after estrogen plus progesterone supplementation. Wholemounts shown in A (wild-type) and B (Vdr knockout) are representative of glands removed from 5-week-old animals that were not supplemented with estrogen and progesterone. Wholemounts shown in C (wild-type) and D (Vdr knockout) represent glands removed at 5 weeks of age after 15 days in vivo supplementation with estrogen and progesterone via subcutaneous pellets. Glands shown in E (wild-type) and F (Vdr knockout) are the contralateral glands to those shown in C (wild-type) and D (Vdr knockout), which were removed after 15 days in vivo supplementation with estrogen and progesterone and placed in organ culture in the presence of estrogen, progesterone and lactogenic hormones for 10 additional days. Arrows indicate alveolar growth and side branching in D and undeveloped ducts in C. Images are representative of 10 glands per genotype examined. Scale bar: 500 µm.

View larger version (148K): [in a new window]   Fig. 9. Effect of 1,25-dihydroxyvitamin D3 on in vitro growth of mammary glands from wild-type and Vdr knockout mice. Inguinal mammary gland pairs were removed from wild-type (A,C) and Vdr knockout (B,D) mice pre-treated with estrogen and progesterone for 15 days and placed in organ culture with estrogen, progesterone and lactogenic hormones for 14 days. One gland from each pair was exposed to ethanol vehicle (A,B), while the contralateral gland was exposed to 100 nM 1,25-dihydroxyvitamin D3 (C,D). Glands were removed from culture, stained and wholemounted after 14 days in vitro growth. Arrows indicate alveolar growth and side branching in A and undeveloped ducts in C. Images are representative of 10 gland pairs per genotype examined. Scale bar: 300 µm.

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