Mammary Gland Morphometric Analysis

Whole mounts mammary glands were viewed and imaged using a Zeiss Axio Imager dissection microscope and Zeiss high-resolution color camera (Carl Zeiss, Oberkochen, Germany). Zeiss ZEN Pro software was used for morphometric analysis using methods developed previously [29 (link), 33 (link)]. Briefly, in females at both ages, specific measurements were quantified including the area subtended by the ducts (ductal area), the growth of the longest duct from the center of the lymph node (ductal extension), the total number of terminal end buds (TEBs, defined as bulb-shaped structures ≥0.03 mm²), and area of TEBs. Ductal density was evaluated using the threshold tool to compare intensity of mammary epithelium within 1.5 cm² areas in the nipple and lateral to the lymph node, or across the entire ductal tree. In males, endpoints that were measured included ductal area and number of branching points. TEBs were never observed in adult males.