Morphological and quantitative assessment of inguinal mammary glands from 12, 14, and 16 month old WT and VDR KO mice.

<p>A. (Left Panels) Glands from VDR KO mice show decreased secondary and tertiary branching, signs of ductal thickening (ectasia), and large lymph nodes as the adipose tissue begins to atrophy with age. In contrast, WT glands show an extensive array of secondary and tertiary branches resting in a vast fat pad of plentiful adipose tissue. (Middle Panels) Glands at higher magnification from VDR KO mice show reduced secondary and tertiary branching and areas of darkly stained lesions along primary ducts (arrows). (Right Panels) Histological sections stained with hematoxylin and eosin Y show clusters of inflammatory cells along the primary ducts within glands of VDR KO mice compared to WT glands. B. Primary, secondary and tertiary branch points were quantitated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016479#s4" target="_blank">materials and methods</a>. Primary branch counts showed a significant decrease between WT and KO glands at 16 months whereas secondary and tertiary branches were significantly decreased in glands from VDR KO mice at 14 and 16 months compared to WT control mice. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05. C. Whole mounts from VDR WT and KO mice were utilized to measure the area of mammary fat pads using AxioVision software. Fat pad area was calculated by tracing around the exterior of the ductal branches of the mammary fat pads. At 14 and 16 months of age, there is a significant reduction in the mammary fat pads of VDR KO mice compared to WT controls. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05. D. Whole mounts from VDR WT and KO mice were utilized to measure the mammary lymph nodes. Using AxioVision software, the exterior of the glandular lymph node was traced to calculate the area. At all time points, lymph nodes within glands from VDR KO mice were significantly enlarged compared to lymph nodes from WT mice. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05.