Evaluation of Mammary Gland Development and Function in Mouse Models

Summary

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Abstract

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts¹. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane². At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones^3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy². At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli^6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups ⁸. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium^9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation^12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

Protocol

1. Mammary gland dissection Euthanize the mouse using CO2 inhalation. Avoid cervical dislocation if possible since it may damage major blood vessels in the neck and result in accumulation of blood around mammary glands, rendering the dissection more difficult. However, if other criteria are to be evaluated in the same mouse, such as blood levels of oxytocin, alternative euthanasia methods may be necessary, though this must be evaluated by your IACUC. On a polystyrene foam wrapped in foil,…

Discussion

Mammary gland development and function are tightly orchestrated. Mutant mice may harbor gene mutations that can impair mammary gland development and function highlighting the need to evaluate gland architecture^13,12. Whole mounting can be performed as described in this article at all stages of mammary gland development. Typically, development of the epithelium is assessed at the beginning of puberty ( ˜4 weeks old), in the middle of puberty ( ˜6 weeks old) and after puberty ( ˜ 10-12 weeks ol…

Materials

Reagent	Company	Catalogue number
Carmine	Sigma-Aldrich	C1022
Aluminum potassium sulfate	Sigma-Aldrich	A6435
Thymol	Sigma-Aldrich	T0501
Methyl salicylate	Sigma-Aldrich	M6752
Oxytocin	Sigma-Aldrich	O3251