Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation*

Background: ZnT2 is expressed in non-secreting and secreting mammary epithelium; however, the physiological role is not understood. Results: ZnT2-null mice have impaired mammary expansion and compromised mammary differentiation and milk secretion during lactation. Conclusion: ZnT2-mediated zinc transport is critical for mammary development and function during lactation. Significance: This study identifies novel consequences of ZnT2 function in the mammary gland. The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.

lactin, leading to N-terminal (Lys-4/Lys-6) ubiquitination, which retargets ZnT2 to secretory vesicles to facilitate zinc accumulation, traffics ZnT2-containing vesicles to the cell membrane for zinc efflux into milk, and then activates proteasomal degradation to abrogate zinc secretion (10,14). ZnT2 function in the mammary gland is not limited to lactation as it is also detected in the non-lactating mammary gland and nonsecreting MECs (13,15). In addition, ZnT2 imports zinc into mitochondria, which constricts ATP generation and activates apoptosis (15). This implicates ZnT2-mediated zinc transport in a plethora of cellular processes in the mammary gland; however, these roles are not well understood. The importance in understanding ZnT2 function and regulation reflects the fact that 5 missense mutations in SLC30A2 have been identified (H54R, G87R, W152R, S296L, R340C) that reduce milk zinc concentration by 50 -90% in breastfeeding women, causing severe neonatal zinc deficiency (11, 16 -18), which clinically presents as alopecia and dermatitis, impaired neuronal and behavioral development, impaired immune function, and increased morbidity and mortality (19,20). Therefore, understanding the role of ZnT2 in mammary gland biology has major implications for both infant and maternal health.
To better understand the role of ZnT2-mediated zinc transport in mammary gland biology, we used ZnT2-null mice and characterized the consequences of loss of ZnT2 function in both the nulliparous and lactating mammary gland. Although ZnT2-null mice were not zinc-deficient, they exhibited profound morphological defects in the mammary gland and MEC, indicating impairments in mammary gland development, lactational differentiation, and severe dysregulation in secretion pathways. Consistent with observations made in the mammary gland, ZnT2-attenuated MECs failed to activate differentiation and had impaired secretion of milk components. Moreover, although ZnT2-null dams had reduced milk zinc levels, they also had profound alterations in milk composition, including reduced protein, fat, and lactose concentrations compromising offspring survival. Our studies underscore the importance of ZnT2 in mammary gland biology and suggest that women with genetic variation in ZnT2 may have critical defects in mammary gland function and milk composition and secretion.

Experimental Procedures
Generation of ZnT2-null Mice-All animal protocols were approved by the Institutional Animal Care and Use Committee at the Pennsylvania State University, which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Heterozygous ZnT2 transgenic mice were generated by Dr. Keigo Nishida (Suzuka University of Medical Sciences, Suzuka, Japan) with the following targeting strategy; a neomycin cassette (Neo) was inserted into the SLC30A2 (ZnT2) gene at position exons 5 and 6. Male and female heterozygous mice were mated to generate wild-type (wt) and ZnT2-null (ZnT2ko) mice. Offspring were genotyped by isolating DNA from ear snips using the Extract-N-Amp Tissue PCR kit (Sigma) followed by PCR amplification with primers (primer 1 (P1), 5Ј-CATTGCCCGCTTACCCTGAG-3Ј; Primer 2 (P2), 5Ј-GACT-GATGGAGGGCCAACCCCATTC-3Ј; primer 3 (P3), 5Ј-CAGCAGCCTCTGTTCCACATACACTTCAT-3Ј). PCR products generated from the P1/P2 primer set (256 bp) for the wt allele and the P1/P3 primer set (176 bp) for the ZnT2ko allele were run on a 1% agarose gel containing 0.1% ethidium bromide and visualized using UV light. Mice were housed in polycarbonate ventilated cages and fed ad libitum on a standard commercial rodent diet (LabDiet, Quakertown, PA). They were maintained on a 12-h light/dark cycle under controlled temperature and humidity.
Nulliparous and Lactating Mice Experiments-To study the nulliparous mammary gland, we used post-pubertal mice at 10 weeks of age during the proestrus/estrus phase to control for the morphological and cellular changes seen in the mammary gland throughout the estrous cycle (21). To study lactating mammary gland, wt and ZnT2ko female mice were mated with a wt male (2:1 ratio) for 2 weeks and allowed to deliver naturally and nurse their litters up to lactation day (LD) 10. The number of offspring was counted, litter size was normalized to 6 -7 pups/dam on LD2, and the number of dams still nursing a litter and their litter size at LD10 was recorded to calculate the percent of dams maintaining litters. Nulliparous and lactating mice were euthanized by CO 2 asphyxiation, and tissues were immediately excised and mounted on slides, fixed in 4% paraformaldehyde, frozen in isopentane, or stored at Ϫ80°C until analysis.
Histological Analysis-For histological examination, mammary glands were fixed in 4% paraformaldehyde in phosphatebuffered saline overnight and embedded in paraffin, and sections (5 m) were stained with hematoxylin and eosin (H&E) or Masson's trichrome as previously described (3). The adipocyte size in three 10ϫ fields of view per section from 5 mice/genotype was measured, and data were expressed as adipocyte area (m 2 ) ϮS.D. A TACS-XL Basic In Situ Apoptosis (TUNEL) detection kit was used to identify apoptotic cells according to the manufacturer's instruction (Trevigen, Inc., Gaithersburg, MD). The number of cells stained positive for TUNEL in five 40ϫ fields of view per section (n ϭ 3 mice/genotype) was counted, and data were expressed as a mean apoptotic cell number ϮS.D. The following antibodies were used for immunofluorescent staining: anti-ZnT2 (1 g/ml; Ref. 15), anti-F4/80 (macrophage marker, 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX), and anti-Ki-67 (proliferation marker, 1:300; Vector Laboratories, Burlingame, CA). Antibodies were visualized with Alexa Fluor 488 or Alexa Fluor 568 (Life Technologies, Carlsbad, CA) and counterstained with DAPI nuclear stain (Life Technologies). Stained sections were examined using the Leica DM IL LED microscope equipped with a digital Leica DFC425 camera (Leica Microsystems, Buffalo Grove, IL) or the Leica Inverted Confocal Microscope SP8 (Leica Microsystems). The number of cells stained positive for Ki-67 in three 10ϫ fields of view per section (n ϭ 5 mice/genotype) was counted, and data were expressed as a mean % Ki-67 ϩ cells ϮS.D.
Whole Mount Analysis-Mammary glands were fixed in Carnoy's solution overnight and stained with carmine alum, as previously described (3). Stained whole mounts were examined using Olympus microscope (BX6), and 4ϫ objective images were automatically stitched on cellSens Dimension software. To quantify ductal length (mm), the distance between the cen-ter of the lymph node and the distal end of the longest duct was measured using Adobe Photoshop CS3. The ductal length was measured in mammary glands (n ϭ 5 mice/genotype), and data were expressed as mean ductal length (mm) ϮS.D. The number of lateral branch points was measured in mammary glands (n ϭ 5 mice/genotype), and data were expressed as mean number of branch points ϮS.D. The ratio of glandular tissue to stromal was calculated by the area of glands over the area of adipose stroma in whole mount images (n ϭ 5 mice/genotype), and data were expressed as mean glandular over stromal ratio ϮS.D.
Transmission Electron Microscopy-Excised mammary glands were immediately harvested in Karnovsy's fixative (4% paraformaldehyde, 1 M sodium hydrozide, 25% glutaraldehyde, 0.2 M sodium cacodylate, pH 7.3) for 24 h and stored in 0.1 mM sodium cacodylate until processed and imaged with a JEOL JEM1400 Digital Capture transmission electron microscope at the Penn State Hershey College of Medicine Imaging Facility.
Matrix Metalloproteinase (MMP) Activity-MMP-2 and MMP-9 activity was assessed by gel zymography as previously described (22). Relative MMP activity was assessed by measuring gelatin lysis area in pixels using Adobe Photoshop CS3 and normalized to the amount of MMP protein detected by immunoblotting.
Milk Secretion and Milk Composition Analysis-Milk secretion was carefully assessed by weigh-suckle-weigh as previously described (5) and was calculated as the difference between the final and initial litter weights. Collected milk samples (n ϭ 5 dams/genotype) were immediately used to measure the percentage of milk fat by creamatocrit as previously described (5). Frozen milk samples were thawed on ice and centrifuged at 2000 ϫ g for 15 min 4°C to skim. Milk lactose was measured in skimmed milk samples using a lactose assay kit according to manufacturer's instructions (Abcam). Milk ␤-casein was measured in skimmed milk by immunoblotting as described above and normalized to total milk protein concentration measured by a modified Bradford assay (5).
Zinc Analysis-Plasma, tissue, and milk were digested in nitric acid, and zinc concentrations were determined by atomic absorption spectrometry as previously described (23).
siRNA Transfection of Mammary Epithelial Cells and Secretion Assay-Mouse MECs (HC11) were a gift from Dr. Jeffery Rosen (Baylor College of Medicine, Houston, TX) and used with the permission of Dr. Bernd Groner (Institute for Biomedical Research, Frankford, Germany). Cells were maintained in growth medium (RPMI 1640 supplemented with 10% fetal bovine serum, 5 g/ml insulin, 10 ng/ml epidermal growth factor, and gentamycin). Cells were transfected with ZnT2 siRNA (5Ј-CCAUCUGCCUGGUGU UCAU-3; Sigma) using Lipofectamine 2000 (Life Technologies) as previously described (14) to attenuate ZnT2 (ZnT2KD). Transfected cells were stimulated into a secreting cell with prolactin (1 g/ml) and cortisol (2 M) for 24 h. Cell lysates and conditioned medium (CM) were collected and immunoblotted for p-Stat5 or ␤-casein as described above. The percentage of ␤-casein secreted was calculated using the following formula: % secreted ϭ (BSI of ␤-casein in CM)/(BSI of ␤-casein in CMϩBSI of ␤-casein in lysate) ϫ 100.
Statistical Analysis-Results are presented as the mean ϮS.D. Statistical comparisons were performed using Student's t test or two-way analysis of variance where appropriate (Prism GraphPad, Berkeley, CA), and a significant difference was demonstrated at p Ͻ 0.05.

Results
To understand the role of ZnT2 in mammary gland biology, we used nulliparous adult (10 -13 weeks of age) and lactating (LD 10) ZnT2-null (ZnT2ko) mice and their wild-type (wt) littermates. Briefly, ZnT2ko mice were generated by insertion of a neomycin cassette into exon 5 and exon 6 in the SLC30A2 gene (Fig. 1A). The colony was maintained through ZnT2 Ϫ/ϩ breeding, and progeny were genotyped by PCR followed by DNA agarose gel electrophoresis (Fig. 1B). We confirmed successful deletion of ZnT2 in the mammary gland by the absence of ZnT2 protein expression using immunoblotting (Fig. 1C) and the absence of ZnT2 staining using immunofluorescence (Fig. 1D).

Nulliparous ZnT2-null Mice Have Reduced Mammary Ductal Invasion and Expansion into the Fat Pad, Impairing Mammary Growth and Development-To first determine where
ZnT2 was expressed in the nulliparous mammary gland, we used immunofluorescence and stained for ZnT2 (Fig. 1D). In addition to expression in non-secreting MECs as we have previously shown (13), here we report for the first time that ZnT2 is expressed in macrophages and to a much lesser extent in adipocytes of the stroma in the nulliparous mammary gland. To confirm that ZnT2 was expressed in macrophages, we used immunofluorescence to detect the macrophage marker F4/80 and observed distinct co-localization between ZnT2 and F4/80 (Fig. 1E). Interestingly, these macrophages were abnormally activated in ZnT2ko mice as indicated by the significant increase in MMP-9 expression in the mammary gland ( Fig. 1F; Ref. 25). Importantly, there was no significant effect on plasma (wt, 12.13 mol/liter Ϯ 1.4; ZnT2ko, 11.14 mol/liter Ϯ 1.9), liver (wt, 29.33 g Zn/g of tissue Ϯ 3.2; ZnT2ko, 28.07 g Zn/g of tissue Ϯ 3.1) or mammary gland (wt, 11.44 g Zn/g of tissue Ϯ 1.3; ZnT2ko, 11.15 g Zn/g of tissue Ϯ 1.8) zinc concentrations.
To assess the effects of a ZnT2-null phenotype on the nulliparous mammary gland, we examined the morphology of the mammary glands taken from nulliparous mice by whole-mount analysis and histology. Mammary glands from nulliparous ZnT2ko mice showed no gross defects in alveolar bud formation; however, both primary and secondary ducts had reduced invasion and expansion into the surrounding mammary fat pad, indicated by the reduction in ductal length and lateral branching, respectively (Fig. 2, A-C). As a result, the relative ratio of glandular tissue to stromal area was significantly lower in the mammary glands of ZnT2ko mice (0.67 Ϯ 0.03) compared with wt littermates (0.80 Ϯ 0.07; p Ͻ 0.05). Consistent with a lower glandular/stromal area, images of H&E-stained mammary gland sections (Fig. 2D) documented significantly fewer ducts in ZnT2ko mice (10 ducts/field of view Ϯ 2) compared with wt littermates (22 ducts/field of view Ϯ 1; p Ͻ 0.005), and these ducts were generally collapsed. Also noted in the H&E images was the expansion of adipocyte size in ZnT2ko mice (discussed below). To determine if these effects were associated with FIGURE 1. Verification of ZnT2-null genotype and localization of ZnT2 in the mammary gland. A, targeting strategy used to generate the ZnT2-null mice. A neomycin cassette (Neo) was inserted into the SLC30A2 (ZnT2) gene to eliminate exons 5 and 6; insertion resulted in an Exon5/6 knock-out genotype (ZnT2ko). B, agarose gel of ZnT2 DNA isolated from ear snips for verification of mouse genotype: wt was identified by a product resulting from P1/P2, ZnT2ko was identified by a product resulting from P1/P3, and heterozygotes (Het) were identified by products resulting from both P1/P2 and P1/P3. P1, primer 1; P2, primer 2; P3, primer 3. C, representative immunoblot of ZnT2 expression (green) in total membrane fractions of mammary glands prepared from ZnT2ko mice and their wild-type (wt) littermates. ␤-Actin (red) served as a loading control. Note the complete absence of ZnT2 in ZnT2ko mice. D, representative images of ZnT2 (red) in the nulliparous (top) and lactating (bottom) mammary glands of wild type (wt) and ZnT2-null (ZnT2ko) mice. Nuclei were counterstained with DAPI (blue). Note the complete absence of ZnT2 detected in mammary glands from nulliparous and lactating ZnT2ko mice. Note the presence of ZnT2 in the MEC of the ducts and alveoli buds and also in the stromal cells in the nulliparous mammary glands. Images of lactating mammary glands, which are naturally depleted of adipocytes, illustrate the substantial amount of ZnT2 expressed in MECs. Magnification, 40ϫ. Scale bar, 100 m. E, representative images of ZnT2 (red) and F4/80 (green, macrophage marker) in mammary glands of wild-type (wt) mice. Boxed areas in the merged image are enlarged in a, b, and c. Note co-localization (yellow) of ZnT2 and F4/80 around epithelial cells of ducts and alveoli buds. F, representative immunoblot of MMP-9 expression (green) in mammary glands from wt and ZnT2ko mice. ␤-Actin (red) served as a loading control. MAY 22, 2015 • VOLUME 290 • NUMBER 21 reduced cell proliferation, we counted the number of nuclei that were stained positive for Ki-67 and found that the number of Ki-67-positive cells in the stroma of ZnT2ko mice was significantly lower compared with wt mice (Fig. 2, E and F). Moreover, apoptotic cells were identified using the TUNEL assay and were frequently detected around the ductal cells in the mammary glands of ZnT2ko mice (Fig. 2, G and H). Collectively, this indicates that loss of ZnT2 function resulted in a hypoplastic mammary gland phenotype in nulliparous adult mice.

ZnT2 Regulates Mammary Gland Development and Function
To explore mechanisms through which the loss of ZnT2 impaired ductal expansion, we first measured the activity and expression MMP-2, a zinc-dependent enzyme that is activated in vesicles (26,27) and is known to be critical for ductal cell proliferation and the process of ductal elongation (22,28). Con-sistent with defects in ductal elongation, the mammary glands of ZnT2-null mice had reduced MMP-2 activity without changes in MMP-2 protein abundance (Fig. 3, A and B; Ref. 22), suggesting that the loss of ZnT2 reduced zinc availability for MMP-2 activation. Reduced MMP-2 activity was consistent with the substantial increase in collagen deposition around collapsed ducts in the mammary glands of ZnT2ko mice ( Fig. 3C; Ref. 29). Alternatively, alterations in the stromal environment can affect ductal expansion (30). Consistent with a potential effect of the stromal environment, an interesting finding we noted was that the adipocytes in the mammary glands of ZnT2ko mice were ϳ4ϫ larger (7070 m 2 Ϯ4624) compared with the adipocytes in wt littermates (1770 m 2 Ϯ 33; p Ͻ 0.05) with no effect on the overall size of the mammary gland (data

ZnT2 Regulates Mammary Gland Development and Function
not shown). Collectively, these data indicate that the loss of ZnT2 impaired ductal invasion and expansion into the mammary fat pad.
Lactating ZnT2-null Mice Accumulate Zinc and Have Profound Defects in Morphology and Impaired MEC Differentiation-Consistent with results in nulliparous ZnT2ko mice and previous reports in breastfeeding women with heterozygous mutations in ZnT2 (11,17), overall zinc status as measured by plasma (wt, 13.08 mol/liter Ϯ 1.4; ZnT2ko, 13.95 mol/liter Ϯ 1.3) and liver (wt, 32.38 g Zn/g of tissue Ϯ 2.6; ZnT2ko, 31.78 g Zn/g of tissue Ϯ 4.5) zinc concentration was not altered in the lactating ZnT2ko mice. Additionally, similar to previous reports in rats and mice (31,32), ZnT2 was localized intracellularly and associated with the apical membrane of MECs in the mammary gland during lactation (Fig. 1D). As observed previously (32,33), the zinc concentration in the mammary gland was higher during lactation compared with the non-lactating state, and this relationship was not affected by loss of ZnT2 (Fig. 4A). However, we noted that during lactation, ZnT2ko mice accumulated ϳ30% more zinc in the mammary gland compared with wt littermates. Because zinc accumulation is cytotoxic and is buffered by either increasing expression of ZnT2 for sequestration into vesicles or increasing expression of metallothionein (MT) for sequestration in the cytoplasm (9, 34), we measured expression of MT as a proxy for cytoplasmic zinc accumulation. We found that without the ability to sequester zinc via ZnT2 in ZnT2ko mice, there was a ϳ7-fold increase in MT expression in the mammary gland (Fig. 4B), suggesting that loss of ZnT2 results in zinc accumulation in the cytoplasm. To verify this, we performed a histological zinc stain using Zinpyr-1 (ZP-1), which is a cell-permeable fluorescent probe selective for labile zinc, and confirmed that the loss of ZnT2 impairs zinc vesicularization and accumulates zinc in the cytoplasm of the mammary epithelium (Fig. 4C). To determine if the increase in zinc accumulation in the mammary glands of ZnT2null mice was specific to loss of ZnT2 in MECs, we transfected HC11 cells with ZnT2 siRNA (ZnT2KD) and loaded with FluoZin-3, a zinc-specific fluorophore that fluoresces upon zinc binding. Again, the loss of ZnT2 in MECs impaired zinc vesicularization and increased cytoplasmic zinc (Fig. 4D).
We next examined the mammary glands of lactating ZnT2null mice using whole-mount analysis. Images of the mammary gland from lactating wt mice illustrated a fully differentiated mammary gland with the expected depletion of adipocytes and development of mature alveoli (Fig. 5A). In contrast, the mammary glands of lactating ZnT2ko mice had markedly less lobuloalveolar expansion (Fig. 5B). Consistent with these defects, H&E-stained sections revealed a profound reduction in alveoli with abnormal preservation of adipose in the stroma of ZnT2ko mice, which was absent in the mammary glands of wt mice (Fig.  5, C and D). Under higher magnification, defects in the alveoli of ZnT2ko mice were more clearly observed. wt mice had well expanded alveoli surrounded by flat MECs and lumens filled with lipid droplets, hallmarks of a well differentiated and actively secreting mammary gland ( Fig. 5E; Refs. 35 and 36). In contrast, ZnT2ko mice had constricted alveoli, and the MECs lining the alveoli were swollen with an accumulation of lipidladen and proteinaceous material and an absence of active lipid droplet fusion at the apical membrane as seen in the wt mice (Fig. 5F). The irregular shape and narrow distention of the alveolar lumen, determined by the significant reduction of alveolar circularity in lactating ZnT2ko mice (Fig. 5, G and H), suggested impaired alveologenesis (37).
To determine if mammary gland defects during lactation were associated with enhanced cell turnover, we analyzed apoptosis and cell proliferation by TUNEL and Ki-67 staining, respectively. Apoptotic cells were profoundly evident in the mammary epithelium and alveolar lumen of mammary glands from ZnT2ko mice (Fig. 6, A and B), which were not present in the mammary glands from wt littermates. Interestingly, mammary glands from lactating ZnT2ko mice also had a significantly greater number of proliferating MECs compared with wt mice (Fig. 6, C and D), consistent with an increase in cell turnover. Because the mammary gland undergoes profound cell turnover at the time of weaning or involution (38,39), we next determined if increased cell turnover in response to the loss of ZnT2 function was a consequence of premature involution by measuring the abundance of phosphorylated Stat3 (p-Stat3) (40). We found no expression of p-Stat3 in the lactating mammary glands of either ZnT2ko or wt mice (Fig. 6E), excluding the possibility that loss of ZnT2 function spontaneously activates premature involution.
Next, we measured the abundance of phosphorylated Stat5 (p-Stat5) to determine if the loss of ZnT2 impaired mammary gland differentiation (41). Strikingly, we found a significant loss of p-Stat5 expression with no change in total Stat5 expression in the mammary glands of lactating ZnT2ko mice (Fig. 6F), suggesting that loss of ZnT2 reduced Stat5 activation (Fig. 6G) and mammary gland differentiation (42). To determine if the loss of differentiation observed in the mammary glands of lactating

ZnT2 Regulates Mammary Gland Development and Function
ZnT2-null mice was specific to loss of ZnT2 in MECs, we transfected HC11 cells with ZnT2 siRNA (ZnT2KD) and differentiated them with PRL to activate p-Stat5 signaling (43). These data showed that differentiation was impaired in ZnT2KD MECs as both p-Stat5 (Fig. 7A) and ␤-casein expression and secretion (Fig. 7, B-D) were significantly reduced in ZnT2-attenuated MECs. Thus, consistent with our observations in lactating mice, the loss of ZnT2 in MECs impaired differentiation and secretion, implicating ZnT2 as a critical regulator of mammary gland function during lactation.
ZnT2-null Mice Have Defects in Subcellular Organization-In light of the observation that loss of ZnT2 function compromised differentiation, we next visualized the lactating mammary glands using transmission electron microscopy to further characterize the consequence of a ZnT2-null phenotype at the subcellular level. Transmission electron microscope images revealed that in lactating wt mice, the alveoli were lined with polarized MECs that had a defined basal membrane (Fig. 8A) and an apical membrane with extended microvilli and tight junctions connecting the cells (Fig. 8C). These MECs were normally filled with endoplasmic reticulum and contained well organized Golgi apparatus and mitochondria that were oriented around a spherical nucleus (Fig. 8, A, D, and E; Ref. 44), which together regulate the secretion of milk constituents across the apical membrane. Moreover, secretory vesicles containing casein micelles and fused lipid droplets were directed toward the apical membrane, indicating active milk secretion (Fig. 8, F and G). In stark contrast, the MECS of ZnT2-null mice

ZnT2 Regulates Mammary Gland Development and Function
were severely disorganized, lacking a distinct cell-cell junctions and presented with numerous pseudolumina (Fig. 8B), similar to the morphology seen in Stat5-null mice with impaired MEC differentiation (45). Most notable were the disturbed apical membrane (Fig. 8H), swollen mitochondria (Fig. 8I) and fragmented endoplasmic reticulum (Fig. 8J) that were scattered throughout the cell. Frequently, we detected secretory vesicles that did not properly package casein micelles, which were dispersed throughout the cytosol (Fig. 8K). Moreover, lipid droplets failed to fuse for secretion into the lumen as milk fat globules and instead accumulated inside the cell (Fig. 8L). Collectively, these observations confirm impaired MEC differentiation in the ZnT2ko mice and provide evidence that the loss of ZnT2 function in MECs disrupts secretory activation and cellular secretion processes during lactation.
Milk Secretion Is Impaired in ZnT2-null Dams, Altering Milk Composition and Affecting Pup Survival-Consistent with reduced milk zinc secretion in women who have mutations in ZnT2 (11, 16 -18), ZnT2-null mice had an ϳ30% reduction in milk zinc concentration (Fig. 9A). Because women with mutations in ZnT2 have a much greater (50 -90%) reduction in milk zinc concentration (10,11,13,16,17), we expected to see a greater loss of zinc transfer into milk in ZnT2ko mice. Given this unexpected finding, we tested the hypothesis that ZnT4 expression was augmented to compensate for the lack of ZnT2, as we and others (24,46) have shown that ZnT4 is important for zinc secretion into milk. However, we found that ZnT4 abundance was actually lower in the mammary glands from ZnT2ko mice (data not shown), eliminating a compensatory response as an alternative explanation. Intriguingly, we found that alterations in milk composition extended well beyond reduced zinc concentration in ZnT2ko mice. We found that the concentrations of major milk components, ␤-casein, fat, and lactose, were also significantly reduced by ϳ30 -40% in the milk collected from ZnT2-null dams (Fig. 9, B-D). Moreover, milk secretion was severely impaired in lactating ZnT2-null dams during both early (ϳ25% less) and peak (ϳ30% less) lactation periods (Fig.  9E). As a result, ϳ50% of ZnT2-null dams failed to maintain MECs. Lactating mammary glands (Lact. mg) served as a positive control, and ␤-actin (red) served as a loading control. C, quantification of ␤-casein expression in cell lysates of mock and ZnT2KD MECs. Data represent mean protein abundance normalized to total protein (band intensity/g of protein) ϮS.D., n ϭ 3 samples/group; **, p Ͻ 0.01. D, percentage of ␤-casein that was secreted and calculated using the following formula: % secreted ϭ (BSI of ␤-casein in CM)/(BSI of ␤-casein in CMϩBSI of ␤-casein in lysate). Data represent the mean secreted ␤-casein (%) ϮS.D., n ϭ 3 samples/group; **, p Ͻ 0.01. their litters up to LD10 (Fig. 9F) and had a significantly lower litter survival rate (ZnT2ko, 53.9% Ϯ 45.7 versus wt, 92.9% Ϯ14.1; p Ͻ 0.05). These data demonstrate that the ZnT2 is a critical regulator of mammary gland differentiation and milk secretion during lactation, which is crucial for sustaining infant health.

Discussion
ZnT2 is expressed in the mammary gland (47), and previous studies have focused on its role in importing zinc into vesicles (10,13) for secretion into milk during lactation (11,16,17). ZnT2 is also found in mitochondria and vesicles in non-lactating mammary glands; however, the physiological role of ZnT2mediated zinc transport beyond that of zinc secretion into milk is almost entirely unknown. Here, we found that ZnT2 is critical for normal mammary gland development and function during lactation. Using nulliparous and lactating ZnT2-null mice, we demonstrate that the loss of ZnT2 1) impaired mammary expansion during development, 2) reduced mammary gland differentiation and impaired secretion during lactation, and 3) compromised milk composition and offspring survival.
The Role of ZnT2-mediated Zinc Transport in Mammary Expansion-Non-lactating ZnT2-null mice had profound defects in mammary development and ductal expansion. During puberty, the mammary gland undergoes key developmental phases including terminal end bud formation, ductal elonga-tion, and ductal branching that collectively establish a branching network of parenchymal ducts surrounded by a supporting adipose and stroma (48). One explanation for impaired mammary development may be alterations in zinc-dependent functions. A key observation from our study is that MMP-2 activity was decreased, suggesting that the inability to vesicularize zinc resulted in cytoplasmic zinc accumulation and buffering by MT, perhaps withholding zinc from activating MMP-2 within vesicles for ductal invasion into the stromal tissue (49 -51). An alternative explanation may reflect mitochondrial dysfunction as ZnT2 transports zinc across the inner mitochondrial membrane and regulates apoptosis (15), a key mechanism critical for ductal expansion in the mammary gland (52). Consistent with this postulate, we noted greater apoptotic activity in MECs and significantly fewer mammary ducts in ZnT2-null mice. This suggests that either mitochondrial zinc depletion or cytoplasmic zinc accumulation may have perturbed closure of the mitochondrial permeability transition pore (53) and, thereby, induced release of apoptotic-inducing factors that disrupt ductal expansion. Further studies on mitochondrial function are required to understand these defects.
In addition to MECs, the stroma, which includes adipocytes and macrophages, is crucial for mammary gland development by facilitating epithelial-mesenchymal interactions for branching morphogenesis and ductal outgrowth (30,54). An unex-

ZnT2 Regulates Mammary Gland Development and Function
pected finding from our study was that ZnT2 was expressed in macrophages and adipocytes. Consequently, the loss of ZnT2 in these cells may also affect optimal development of the ductal tree. Adipocytes dictate the extent of which the glandular tissue develops into a functional branching network in the mammary gland (55) by secreting leptin and other endocrine factors that contribute to paracrine interactions with ductal epithelial cells (56); thus, enhanced adipocyte mass may affect these interactions. Moreover, zinc has insulin-like effects in adipocytes, stimulating lipogenesis and increasing fatty acid accumulation (57,58). Consistent with effects of adipocyte zinc accumulation, we observed adipocyte hypertrophy in ZnT2-null mammary glands, which could indicate increased lipogenesis (59). Therefore, the loss of ZnT2 in adipocytes may mirror insulinlike effects on lipogenesis that stunt ductal growth (60) or may affect the synthesis or release of paracrine factors, such as adiponectin and collagen VI (61) from adipocytes that initiate epithelial/mesenchymal interaction and facilitate ductal expansion (55). Finally, macrophages are also important for mammary gland development, as they are recruited to the epithelial cells to support proliferation for ductal outgrowth (30,62) by supplying growth factors, cytokines, and chemokines (63) or by engulfing apoptotic cells that interfere with epithelial proliferation (64). The lack of cell proliferation in ZnT2-null mice suggests that loss of ZnT2 in macrophages may not be able to support normal epithelial and fibroblast proliferation. Because MMP-9 expression was greater in ZnT2-null mice, one explanation may be that the loss of ZnT2 hyperactivates macrophages (25), which can stimulate tumor growth factor-␤ signal-ing pathway and impair ductal outgrowth (65,66). Additionally, adipocyte hypertrophy combined with abnormal macrophage activation may be indicative of an inflammatory mammary gland that in turn interrupts optimal mammary development (67,68). Further studies are required to understand the role of ZnT2-mediated zinc transport in the stromal cells in the mammary gland.
ZnT2 Is Critical for Mammary Differentiation during Lactation-It is possible that defects in mammary gland development persist through pregnancy, affecting morphological development and differentiation. However, we chose to focus on the loss of ZnT2 function on the lactating mammary gland as previous studies clearly show that ZnT2 imports zinc into secretory vesicles, thus playing a vital role in zinc secretion into milk during lactation (10,13,16,17). Although milk zinc concentration was significantly reduced in ZnT2-null mice, much more profound was the accumulation of zinc and the morphological defects observed in the lactating mammary glands that indicate impaired mammary differentiation. Mammary gland differentiation, milk production, and active secretory mechanisms during lactation (69) are primarily activated by prolactininduced Stat5 signaling. Here, we noted that loss of ZnT2 reduced p-Stat5 in the mammary gland and MECs in vitro, indicating that differentiation was dramatically impaired (41,70). The loss of ZnT2-mediated zinc transport into vesicles may activate zinc-dependent dephosphorylation enzymes in the cytoplasm, such as Shp-2 (71), or inhibit prolactin signaling through aberrant stimulation or suppression of factors that regulate Stat5 activity, such as ErbB4, Pak1, caveolin-1, Socs2, Id2, FIGURE 9. Compromised milk secretion in lactating ZnT2-null dams affect offspring survival. A, quantification of zinc concentration in milk from lactating wild-type (wt) and ZnT2-null (ZnT2ko) mice. Data represent the mean milk zinc concentration (mol/liter) ϮS.D., n ϭ 5 mice/genotype; *, p Ͻ 0.05. B, quantification of ␤-casein levels in milk from lactating wt and ZnT2ko mice. Data represent the mean ␤-casein abundance normalized to total protein level (BSI/g of protein) ϮS.D., n ϭ 5 mice/genotype; *, p Ͻ 0.05. C, quantification of fat levels in milk from lactating wt and ZnT2ko mice. Data represent mean fat (%) ϮS.D., n ϭ 5/genotype; **, p Ͻ 0.01. D, quantification of lactose concentration in milk from wt and ZnT2ko mice. Data represent mean milk lactose concentration (mg/ml) ϮS.D., wt n ϭ 10 mice and ZnT2ko n ϭ 5 mice; *, p Ͻ 0.05. E, milk volume calculated as the difference between litter weight before and after suckling on LD5 and LD10 in wild-type (wt) and ZnT2-null (ZnT2ko) mice. Data represent mean milk yield (g) ϮS.D. wt n ϭ 10 litters, and ZnT2ko n ϭ 5 litters); *, p Ͻ 0.05. F, pie graph of the percentage of dams maintaining the indicated litter size at LD10: no survivor, 1-3 pups, 4 or 5 pups and 6ϩ pups. Note the large percentage (ϳ50%) of ZnT2ko dams failing to maintain a litter up to LD10. and Elf5 (70). Notably, functional and morphological defects in ZnT2-null mice were consistent with compromised MEC polarity, tight junction formation, and cellular secretion pathway (72), reinforcing the notion that the direct loss of ZnT2 in the MEC impairs the maintenance of a secreting and differentiated epithelium.
Furthermore, we noted abnormal preservation of adipocytes in the mammary gland of ZnT2-null mice, and thus, the inability to deplete adipocytes may have profound implications on developing and/or maintaining a secreting mammary epithelium. Adipocytes secrete numerous factors that can impact mammary gland function, such as leptin and estrogen (73). The persistence of these factors may have adverse consequences on lactation. For example, high levels of leptin suppress milk ejection by inhibiting oxytocin-induced contractions (74,75). Moreover, prolactin stimulates leptin secretion from mammary adipose, which up-regulates expression of estrogen receptor ␣ and can negatively impact the maintenance of the secreting epithelium through inhibition of Stat signaling in MECs during lactation (76). Because we detected ZnT2 in adipocytes, it is possible that ZnT2-mediated zinc transport plays a role in lactation-induced lipolysis via lipoprotein lipase (55). The inability to clear triglycerides from adipocyte depots may partially underlie the lower level of milk fat observed in ZnT2-null mice. Collectively, our data demonstrate that ZnT2-mediated zinc transport is required for mammary differentiation and the maintenance of a secreting epithelium during lactation.
Loss of ZnT2 Impairs Milk Secretion-Consistent with reports in women with mutations in ZnT2, loss of ZnT2 function in ZnT2-null mice reduced milk zinc levels by ϳ30%. Although milk zinc levels were lower, we do not know if their offspring suffer from zinc deficiency or would survive if we allowed them to age past 10 days. However, in addition to zinc, milk protein, lactose, and fat is significantly lower as well. Thus the impact on offspring survival likely resulted from a general malnutrition. In response to increased prolactin levels at parturition, the differentiated epithelium finely coordinates the synthesis and transport of various milk constituents that are ultimately secreted into the alveolar lumen to provide adequate milk to the developing infant (77). Because milk lipid is a major constituent of mouse milk, the active secretion of lipid droplets from MECs into the alveolar lumen is indicative of the secretory capacity of the mammary gland. Surprisingly, we found a significant accumulation of lipid droplets in the MECs in ZnT2null mice, suggesting a profound defect in secretory activation (36). Impaired secretion was further verified by reduced milk protein, lactose, and total volume in ZnT2-null mice, resulting in overall suboptimal milk delivery. In addition to the macronutrients described above, milk is a complex biological fluid, and MECs secrete a plethora of bioactive factors, anti-infectious and anti-inflammatory agents, growth factors, and prebiotics, all of which are essential for infant growth and immune development (78). Here, we only report reductions in zinc and macronutrients, but it is probable that dysregulation of numerous cellular secretion pathways suppress the secretion of other micronutrients and non-nutritive factors or may even augment the efflux of unwanted components, thereby producing suboptimal milk and negatively affecting neonatal health.
In summary, our study shows that ZnT2-mediated zinc transport is critical for mammary gland expansion and differentiation and milk secretion during lactation. Collectively, the loss of ZnT2 resulted in mammary gland hypoplasia, the underlying cause of failed lactogenesis II where the mother is unable to produce an adequate milk volume (79). The etiology of breast hypoplasia is unknown, but our observations along with previous studies regarding zinc deficiency and the mammary gland (3)(4)(5) suggest that tight regulation of zinc metabolism plays an important role in regulating mammary gland biology.