Dynamic Control of Oligosaccharide Modification in the Mammary Gland: Linking Recombinant Human Erythropoietin

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin α and β are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and α1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable. The data suggest that N-linked sugar chain patterns of recombinant glycoproteins, produced by the mammary gland differ, since GnT-III alters the sugar pattern extensively. In our experiments, rhEPO produced by the transgenic mice contains more tetra-acidic oligosaccharide structures than epoetin α derived from CHO cells, a rhEPO that is widely used therapeutically. Accordingly, we examined milk-derived rhEPO activity, both in vitro and in vivo. The rhEPO protein purified from the milk of mammary glands upregulates the EPO receptor-mediated expression of the STAT5 gene in MCF-7 cells in a dose-dependent manner, similar to the effects of epoetin α. Furthermore, direct injection of rhEPO into the mouse tail vein leads to an increase in the levels of blood components, such as red blood cells and platelets. In light of these findings, we suggest that the mammary glands of transgenic animals provide a sufficient environment to generate rhEPO with post-translational modifications for biopharmaceutical use. These authors are equal contributors to this work.     

Production of human surfactant protein C in milk of transgenic mice

Respiratory distress syndrome (RDS), caused by lack of pulmonary surfactant, affects 65 000 infants annually in the USA. Surfactant replacement therapy reduces the morbidity and mortality associated with RDS. Human surfactant protein C (SP-C) is an important component of pulmonary surfactant. To produce human SP-C, a construct using the rat whey acidic protein (WAP) promoter and 3 untranslated regions to target expression of the human SP-C gene to the mammary gland of transgenic mice was created. WAP/SP-C mRNA expression was detected in all transgenic lines analysed. SP-C was expressed in a copy-number-dependent and integration-site-independent fashion, with levels of expression ranging from 0.01% to 36.0% of the endogenous mouse WAP mRNA, and WAP/SP-C mRNA expresison levels were greater than those of the endogenous mouse lung SP-C mRNA. Expression at the RNA level was specific to the mammary gland and paralleled the endogenous WAP expression pattern during mammary gland development. Expression and secretion of the SP-C protein in the lactating mammary gland was demonstrated by western blots performed on whole milk using an anti-SP-C polyclonal antibody. Immunoreactive proteins of MW 22 and 12–14 kDa appeared only in transgenic milk. The 22 kDa protein represents the proprotein, and the 12–14 kDa is a processed form of SP-C.     

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

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.     

Molecular regulation of milk trace mineral homeostasis

The regulation of milk trace mineral homeostasis requires the temporal integration of three main processes, (A) mineral uptake into the secretory mammary epithelial cell (MEC); followed by (B) mineral secretion from MEC into the alveoli lumen of the mammary gland for sequestration in milk; and then (C) milk release in response to suckling. Trace mineral requirements of term infants are generally met by exclusive breast-feeding through about the first 6 months of life and although milk zinc (Zn), iron (Fe), and copper (Cu) concentrations are relatively refractory to maternal trace mineral status, they normally decline throughout lactation. Recently, Zn-, Fe- and Cu-specific transporters have been identified that regulate trace element uptake and efflux in various cell types; however, there is currently little information available regarding the processes through which the mammary gland regulates milk trace mineral transport. The homology of trace mineral transporters between species permits the utilization of rodent models to examine the regulation of mammary gland mineral transport. Therefore, we have used the lactating rat to determine changes in mammary gland Zn, Fe and Cu transporter expression and localization that occur throughout lactation and in response to maternal trace mineral deficiency in hope of elucidating some of the changes which occur during mammary gland trace element homeostasis and also may be occurring in lactating women.     

Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland

The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na(+), K(+), assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the premature cessation of milk secretion observed in these transgenic goats.     

Recombinant human bile salt-stimulated lipase: an example of defectiveO-glycosylation of a protein produced in milk of transgenic mice

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprisingbssl cDNA, or alternatively genomicbssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomicbssl sequences mediated high levels (0.5–1, mg ml^–1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree ofO-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same, stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavilyO-glycosylated makes species-related restrictions less attractive as an explanation for the reducedO-glycosylation.     

X-Ray Fluorescence Microscopy Reveals Accumulation and Secretion of Discrete Intracellular Zinc Pools in the Lactating Mouse Mammary Gland

Background

The mammary gland is responsible for the transfer of a tremendous amount of zinc (∼1–3 mg zinc/day) from maternal circulation into milk during lactation to support the growth and development of the offspring. When this process is compromised, severe zinc deficiency compromises neuronal development and immune function and increases infant morbidity and/or mortality. It remains unclear as to how the lactating mammary gland dynamically integrates zinc import from maternal circulation with the enormous amount of zinc that is secreted into milk.

Methodology/Principal Findings

Herein we utilized X-ray fluorescence microscopy (XFM) which allowed for the visualization and quantification of the process of zinc transfer through the mammary gland of the lactating mouse. Our data illustrate that a large amount of zinc first accumulates in the mammary gland during lactation. Interestingly, this zinc is not cytosolic, but accumulated in large, discrete sub-cellular compartments. These zinc pools were then redistributed to small intracellular vesicles destined for secretion in a prolactin-responsive manner. Confocal microscopy identified mitochondria and the Golgi apparatus as the sub-cellular compartments which accumulate zinc; however, zinc pools in the Golgi apparatus, but not mitochondria are redistributed to vesicles destined for secretion during lactation.

Conclusions/Significance

Our data directly implicate the Golgi apparatus in providing a large, mobilizable zinc storage pool to assist in providing for the tremendous amount of zinc that is secreted into milk. Interestingly, our study also provides compelling evidence that mitochondrial zinc pools expand in the mammary gland during lactation which we speculate may play a role in regulating mammary gland function.     

Expression of human lactoferrin in milk of transgenic mice

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 g ml^–1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.     

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10???g/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.     

Production of recombinant albumin by a herd of cloned transgenic cattle

Purified plasma derived human albumin has been available as a therapeutic product since World War II. However, cost effective recombinant production of albumin has been challenging due to the amount needed and the complex folding pattern of the protein. In an effort to provide an abundant source of recombinant albumin, a herd of transgenic cows expressing high levels of rhA in their milk was generated. Expression cassettes efficiently targeting the secretion of human albumin to the lactating mammary gland were obtained and tested in transgenic mice. A high expressing transgene was transfected in primary bovine cell lines to produce karyoplasts for use in a somatic cell nuclear transfer program. Founder transgenic cows were produced from four independent cell lines. Expression levels varying from 1–2 g/l to more than 40 g/l of correctly folded albumin were observed. The animals expressing the highest levels of rhA exhibited shortened lactation whereas cows yielding 1–2 g/l had normal milk production. This herd of transgenic cattle is an easily scalable and well characterized source of rhA for biomedical uses.     

Rabbit whey acidic protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.     

Decrease in an Inwardly Rectifying Potassium Conductance in Mouse Mammary Secretory Cells after Forced Weaning

Mammary glands are physiologically active in female mammals only during nursing. Immediately after weaning, most lactation-related genes are downregulated and milk production ceases. In our previous study, we have detected an inwardly rectifying potassium channel (Kir) 2.1-like current in mammary secretory (MS) cells freshly isolated from lactating mice. This current is highly sensitive to external Ba²⁺. The potassium permeability of the Kir channels may contribute to the secretion and/or preservation of ions in milk. We hypothesized that the functions of the Kir channels in MS cells are regulated after weaning. To test this hypothesis, we examined the effect of forced weaning on the Ba²⁺-sensitive Kir current and Kir2.1 expression in the mouse mammary glands. Twenty-four hours after weaning, the lumina of mammary acini were histologically enlarged by milk accumulation. The whole-cell patch-clamp analyses showed that the Ba²⁺-sensitive Kir current in the post-weaning MS cells was smaller than in the lactating MS cells. The inward conductances of the current in the lactating and post-weaning cells were 4.25 ± 0.77 and 0.93 ± 0.34 nS, respectively. Furthermore, real-time PCR and Western blot analyses showed that Kir2.1 mRNA and protein expression decreased in the post-weaning mammary gland (mRNA, 90% reduction; protein, 47% reduction). Moreover, the local milk accumulation caused by teat sealing decreased Kir conductance in MS cells (2.74 ± 0.45 and 0.36 ± 0.27 nS for control and sealed mammary glands, respectively). This was concomitant with the reduction in the Kir2.1 mRNA expression. Our results suggest that milk stasis after weaning immediately decreases the Kir conductance in MS cells. This decrease in the Kir conductance may be partly caused by the reduction in the Kir2.1 mRNA and protein expression. These alterations during the post-weaning period may be involved in the cessation of ion secretion and/or preservation in the milk.     

Transient expression of hepatitis B virus surface antigen (HBsAg) and human erythropoietin (hEPO) genes in milk after direct introduction into ewe

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Origin,concentration and structural features of human mammary gland cells cultured from breast secretions

Summary This study traced the origin of cells observed in human breast secretion samples obtained during lactation and describes the appearance of these cells following prolonged maintenance in vitro. Human milk contains a large number of single vacuolated foam cells and a small proportion of non-vacuolated epithelial cells in clusters. Foam cells are identified by their large size, the polarity of their cytoplasmic organelles, the variation in number and size of lipid vacuoles and the condensed chromatin of their eccentrically located nucleus. Both cell types originate by exfoliation from the mammary gland. This was established by comparing the structural characteristics of cells isolated from milk with those of the cuboidal cell linings of ducts and alveoli in lactating mammary tissue. Relatively pure populations of foam cells could be established from early lactation samples (3–7 days post/partum) while non-vacuolated epithelial cell clusters were more frequently cultured from late lactation specimens (1–10 days postweaning). Foam cells did not divide and lost cytoplasmic organization during prolonged culture. In contrast, non-vacuolated epithelium in clusters proliferated to form colonies of polygonal cells. These results, which imply that foam cells are an active form of the non-vacuolated mammary cells in clusters, call attention to one system for the study of the complex hormonal interactions necessary to induce and maintain lactation.Supported in part by NCI contract NO 1-CB-33898     

Lactation-dependent down regulation of leptin production in mouse mammary gland

Lactation-dependent regulation of leptin expression in mouse mammary gland and parametrial adipose tissue was estimated by RT-PCR analysis for virgin, pregnant, lactating and post-lactating mice, and the serum and milk leptin levels of these mice were also determined by ELISA. Leptin gene expression in mammary gland as well as in adipose tissue was obviously detected before pregnancy, markedly decreased to 30-50% after parturition and kept at the low level during lactation period, and restored to the original level after weaning. The leptin concentration of milk collected just before weaning was about two-fold higher than that of the milk collected at mid-lactating stages. The serum leptin levels of the mid- and late-lactating mice were not significantly higher than those of non-pregnant mice. These results suggested that the lactation-induced down regulation of leptin was associated with autocrine/paracrine action of leptin in mammary and adipose tissues, and that the milk leptin, especially at the latter stages of lactation, was not only ascribed to diffusive transport from maternal blood stream, but also regional production and secretion by mammary epithelial cells. This possible production of leptin by mammary epithelial cells was further supported by the fact that leptin was expressed by cultured cells of mammary epithelial cell line, COMMA-1D, in a manner negatively dependent on the lactogenic hormones.     

Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.