Abstract #190

# 190
Intravital imaging of the lactating mammary gland in live mice reveals novel aspects of milk-lipid secretion.
Ian H. Mather*1,2, Andrius Masedunskas2,3, Yun Chen4, Roberto Weigert2,3, 1University of Maryland, College Park, MD, 2National Cancer Institute, NIH, Bethesda, MD, 3National Institute of Craniofacial and Dental Research, NIH, Bethesda, MD, 4Johns Hopkins University, Baltimore, MD.

Milk fat comprises membrane-coated droplets of neutral lipid, which constitute the sole source of all lipids for survival of the suckling neonate. From the perspective of the dairy industry, they are the basis for the manufacture of butter, and essential ingredients in the production of cheese, yogurt and specialty dairy produce. To provide mechanistic insight into the assembly and secretion of lipid droplets during lactation, we developed novel intravital imaging techniques using transgenic mice, which express fluorescently tagged marker proteins. The number 4 mammary glands were surgically prepared under a deep plane of anesthesia and the exposed glands positioned as a skin flap with intact vascular supply on the stage of a laser-scanning confocal microscope. Lipid droplets were stained by prior exposure of the glands to hydrophobic fluorescent BODIPY dyes and their formation and secretion monitored by time-lapse subcellular microscopy over periods of 1–2 h. Droplets arose from basal regions of the cell and were transported to the cell apex by directed (superdiffusive) motion. Transit was relatively slow (0–2 μm/min) and intermittent. Droplets grew by numerous fusion events regardless of size both during transport and in apical regions. On occasion, droplets caught up with and fused with droplets further along the same track or pathway. Lipid expansion was most pronounced in apical nucleation centers and continued as the droplets were budding from the cell enveloped by apical membranes. Surprisingly, droplet secretion was not constitutive but required an injection of oxytocin (i.p.) to induce contraction of the myoepithelium with subsequent release of droplets into luminal spaces. This regulated mechanism may constitute an important means for controlling the size of secreted droplets because they will continue to expand at the apical surface unless they are ejected by oxytocin released from the pituitary gland. Application of intravital imaging techniques should provide novel insights into the secretion of other milk components, including proteins and constituents transported across the mammary epithelium from the systemic circulation.

Key Words: milk lipid globules, oxytocin-mediated lipid secretion, intravital microscopy

Speaker Bio
Ian Mather was born in Cheshire, UK, and graduated from the University of Wales, Bangor, with BSc and PhD degrees in 1966 and 1971, respectively. He came to the United States in 1973 on a postdoctoral fellowship with Professor Tom Keenan at Purdue University, with whom he acquired a life-long interest in the mechanism of milk secretion. He subsequently moved to the University of Maryland, College Park, as an assistant professor in 1975 and rose through the ranks to professor in 1985. Among his other positions, Mather was a visiting professor at the German Cancer Research Center, Heidelberg, in 1982-1983, chair of the Mammary Gland Gordon Research Conference in 1989, and a Fogarty International Fellow at the European Molecular Biology Laboratory in 1989-1990. He is currently a visiting scientist at the US National Cancer Institute and professor emeritus at the University of Maryland. Mather’s research on milk lipids was recognized by the ADSA American Cyanamid and Dean Foods Awards in 1993 and 2001, respectively, and Fellowship of the American Association for the Advancement of Science in 2014.