The role of ZnT4 in mammary gland development and lactation

The production of breast milk of optimal quality and quantity is critical for providing the nursing infant with optimum nutrition during the critical neonatal period. The World Health Organization and American Pediatrics Association recommend 6 months of exclusive breastfeeding in order to confer optimal health benefits to both the mother and nursing infant. Exclusive breastfeeding provides numerous benefits for the developing infant including increased growth, optimal immune function, and improved cognitive function compared to infants who don't breastfeed.;Numerous factors serve a comprehensive role in regulating mammary gland development and function. One such factor is Zn, which has been shown to regulate mammary gland development and function from a cellular, physiological, and dietary perspective. Zn is required for numerous mammary gland proteins that are critical to mammary gland function and milk synthesis such as the lactose enzyme complex, which is responsible for the synthesis of lactose, the most abundant carbohydrate in milk.;The Zn transporter, ZnT4 (slc30a4) has been implicated as a critical regulator of milk production and quality. The "lethal milk" mouse (C57bl/6Jlm/lm) contains a spontaneous mutation in slc30a4, which results in a non-functional, prematurely truncated protein of greatly reduced abundance. The C57bl/6J lm/lm mouse displays numerous signs of suboptimal lactation including decreased mammary gland weight, milk volume, and milk Zn concentration which result in the death of pups within one week of birth. The primary goal of my dissertation is to understand the role of ZnT4 in mammary gland development and function during lactation.;Physiological Zn requirements are increased during lactation, especially in the mammary gland where a significant amount of Zn is imported into the mammary gland and transported by ZnT4 to supply Zn to Zn-dependent proteins that are critical for mammary gland expansion and milk synthesis. Increased demand is partially met through increased absorption of Zn from the diet. Moreover, it is estimated that 60 to 80% of women of reproductive age are at risk for Zn deficiency due to low intake of bioavailable Zn and increased demands during pregnancy and lactation. How Zn is redistributed within the body to meet the demands of lactation and whether Zn redistribution is altered during Zn deficiency remains unknown.;In study 2, we explored the sub-cellular location of Zn accumulation in the mammary gland and mammary epithelial cell during lactation. We utilized x-ray fluorescence microscopy and identified the accumulation of large, discrete, labile Zn pools in the lactating mammary gland. We identified the GA as the sub-cellular location of these Zn pools, which was subsequently redistributed in response to PRL stimulation to vesicles destined for secretion from the mammary gland. These studies demonstrate the fundamental role of Zn accumulation in the GA as a critical component of mammary gland Zn homeostasis and function during lactation.;Previous studies noted the presence of ZnT4 proximal to the luminal membrane in breast biopsies from lactating women. Additionally, we found that ZnT4 was localized to an intracellular compartment and at the luminal membrane of the lactating mammary gland in rodents, and furthermore, expression is dramatically enhanced during lactation. Therefore, in study 3 we delineated the role of ZnT4 in cultured mammary epithelial cells (HC11) and showed that it transports Zn into the trans-GA for Zn-dependent proteins that are critical for mammary gland function and optimal milk synthesis including 1,4-beta- galactosyltransferase and carbonic anhydrase VI.;In study 4, we used ZnT4-null mice (C57bl/6Jllm/lm) to investigate the role of ZnT4 in mammary gland development and lactation. Histological analysis revealed significantly impaired ductal expansion as measured by decreased alveolar abundance in nulliparous C57bl/6J lm/lm mammary glands. Furthermore, C57bl/6Jlm/lm mammary glands had decreased MMP-9 activity suggesting that ZnT4 plays a critical role in mediating Zn-dependent proteins that are critical in expansion of the ductal tree during mammary gland development. Additionally, lactating C57bl/6Jlm/lm mammary glands display a sparser ductal tree structure and fewer alveoli compared to wild-type littermates, indicating that the defects observed in nulliparous C57bl/6J mice carry over to lactation.;In summary, results supported a central role for ZnT4 in mammary gland development and function; specifically in its ability to transport Zn to proteins that are critical to optimal lactation and to maintain intracellular Zn homeostasis. Overall, these studies show that ZnT4 is critical for mammary gland ductal growth and expansion during puberty, and understanding the function of ZnT4 will have major implications towards improving our understanding of how the mammary gland is able to properly develop and produce milk of optimal quality and quantity, thereby improving maternal and infant health. (Abstract shortened by UMI.).