Over-expression of human lipoprotein lipase in mouse mammary glands leads to reduction of milk triglyceride and delayed growth of suckling pups

Background

The mammary gland is a conserved site of lipoprotein lipase expression across species and lipoprotein lipase attachment to the luminal surface of mammary gland vascular endothelial cells has been implicated in the direction of circulating triglycerides into milk synthesis during lactation.

Principal Findings

Here we report generation of transgenic mice harboring a human lipoprotein lipase gene driven by a mammary gland-specific promoter. Lipoprotein lipase levels in transgenic milk was raised to 0.16 mg/ml, corresponding to an activity of 8772.95 mU/ml. High lipoprotein lipase activity led to a significant reduction of triglyceride concentration in milk, but other components were largely unchanged. Normal pups fed with transgenic milk showed inferior growth performances compared to those fed with normal milk.

Conclusion

Our study suggests a possibility to reduce the triglyceride content of cow milk using transgenic technology.     

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.     

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.     

Mammary growth patterns in guinea pigs during puberty, pregnancy and lactation

Mammary gland growth patterns were studied in 110 guinea pigs during the growth phase, pregnancy and lactation. Body weight changes were studied and, in addition, mammary indices were wet weight, dry fat-free tissue (DFFT), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Statistical analyses were mathematical regression models to best fit the actual data. These included linear, quadratic, cubic, and several forms of exponential regression models. Data were separated into growth phase (60 guinea pigs in 10 age groups), pregnancy (20 guinea pigs in 4 groups), and lactation (30 guinea pigs in 6 groups). Data during pregnancy and the first 5 days of lactation were pooled and analyzed also because mammary growth continued beyond pregnancy to Day 5 of lactation. Mammary wet weight increased according to a cubic expression in the growth phase, while mammary DFFT, DNA and RNA were rectilinear through 200 days of age. During pregnancy and the first 5 days of lactation, mammary growth parameters followed the pattern of an exponential equation. Daily rates of increase for mammary DFFT and DNA were twice the rate for mammary wet weight. During lactation, mammary gland indices increased to Day 5 and then decreased gradually from Day 10 to Day 20. The best mathematical models for these change were those which are used to describe lactation curves, but all mammary gland indices decreased later and more gradually than milk production. Comparisons in growth rates of guinea pig mammary glands were made with those published for dairy goats and dairy cows. Rates of mammary DNA changed inversely to lengths of gestation in these 3 species.     

The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.     

Mammary Gland Specific Knockdown of the Physiological Surge in Cx26 during Lactation Retains Normal Mammary Gland Development and Function

Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26^fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function.     

The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production

Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.     

Distribution and source of lipoprotein lipase in mouse mammary gland

During lactation lipoprotein lipase (LPL) is elevated in mammary tissue and depressed in adipose tissue to redirect lipids for incorporation into milk fat. The cellular origin of lipoprotein lipase in mammary tissue is thought to be the mammary epithelial cell which is the predominant cell type noticeable in the lactating gland; however, mammary adipocytes are also present. If lipoprotein lipase is produced by adipocytes in other sites of the body, then the question remains as to why mammary adipocytes have not been shown to produce lipoprotein lipase. In this study we present several lines of evidence that indicate that the mammary adipocyte is a source of LPL in the lactating mammary gland of mice. This evidence includes the absence of extracellular and intracellular lipoprotein lipase activity in two types of primary mammary epithelial cell cultures and a similarity in the changes of lipoprotein lipase activity in genital adipose tissue from nonpregnant mice and lactating mammary tissue to the nutritional state of the animal. Other evidence presented here includes strong localization of lipoprotein lipase protein and messenger RNA by fluorescence immunohistochemistry and in situ hybridization, respectively, to interstitial cells located between epithelial structures. We postulate that these interstitial cells are regressed, lipid-deleted mammary adipocytes.     

Suppression of lactation by pregnancy-dependent mammary tumors in GR/A mice

Pregnancy-dependent mammary tumors (PDMT) in GR/A mice appear during pregnancy, disappear soon after parturition, and appear again during subsequent pregnancies. The retardation of pup growth, an indication of the level of milk production, was also observed with the advance of lactation numbers in this strain. This study was performed to elucidate the relationship between PDMT and lactational performance. At the end of the second pregnancy, mice were divided into two groups according to the presence of PDMT [PDMT(-) and PDMT(+) groups]. Although all PDMT disappeared within a day after parturition, the weight and growth of pups on Day 12 of lactation were significantly less in the PDMT(+) group than in the PDMT(-) group. Associated with this, the DNA and RNA contents of the mammary glands were apparently lower in the former than in the latter, although the differences were not statistically significant. There was little difference in mammary RNA/DNA ratio between groups. No difference was also observed between groups in endocrine organ weights, mother body weights, morphology of the mammary glands, adrenals and ovaries and plasma prolactin and progesterone levels. These results suggest that PDMT suppression of lactation is principally due to the retardation of mammary gland growth. Furthermore, no significant correlations were obtained between the size of PDMT and the parameters for mammary gland function. The data suggest that the development of PDMT per se is important for the retarded mammary gland growth.     

Overexpression of des(1-3) insulin-like growth factor 1 in the mammary glands of transgenic mice delays the loss of milk production with prolonged lactation

During prolonged lactation, the mammary gland gradually loses the capacity to produce milk. In agricultural species, this decline can be slowed by administration of exogenous growth hormone (GH), which is believed to act through insulin-like growth factor 1 (IGF1). Our previous work demonstrated delayed natural mammary gland involution in des(1-3)IGF1-overexpressing transgenic mice (Tg[Wap-des{1-3}IGF1]8266 Jmr), hereafter referred to as WAP-DES mice. The present study tested the hypothesis that overexpressed des(1-3)IGF1 would delay the loss of milk production during prolonged lactation. Accordingly, we examined lactational performance in WAP-DES mice by artificially prolonging lactation with continual litter cross-fostering. Over time, lactational capacity and mammary development declined in both WAP-DES and control mice. However, the rate of decline was 40% slower in WAP-DES mice. Mammary cell apoptosis increased by 3-fold in both groups during prolonged lactation but was not different between genotypes. Plasma concentrations of murine IGF1 were decreased in WAP-DES mice, while those of the transgenic human IGF1 were elevated during prolonged lactation. Phosphorylation of the mammary IGF1 receptor was increased in the WAP-DES mice, but only during prolonged lactation. Plasma prolactin decreased with prolonged lactation in nontransgenic mice but remained high in WAP-DES mice. The WAP-DES mice maintained a higher body mass and a greater lean body mass during prolonged lactation. These data support the conclusion that overexpressed des(1-3)IGF1 enhanced milk synthesis and mammary development during prolonged lactation through localized and direct activation of the mammary gland IGF1 receptor and through systemic effects on prolactin secretion and possibly nutrient balance.     

Variation in the lack of polyadenylation of the rat milk protein mRNAs during the lactation cycle

Translationally active milk protein mRNAs were found as nonpolyadenylated mRNAs in the rat mammary gland during pregnancy, lactation and involution. Analyses of whey protein mRNA and casein mRNA with the corresponding cDNAs showed that the lack of polyadenylation of these mRNAs at different time points of the lactation cycle is not consistent with the hypothesis that polyadenylation may be incomplete in the mammary gland when large amounts of mRNA are synthesized. The fraction of whey protein mRNA and casein mRNA that lacked polyadenylation was inversely proportional to the concentration of each sequence in the tissue during pregnancy, lactation and involution. A model is proposed to explain the finding that in each animal the ratio of casein mRNA to whey protein mRNA was similar in polyadenylated RNA and in nonpolyadenylated RNA at all stages of the lactation cycle.     

Metabolic proteomics of the liver and mammary gland during lactation

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by β-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and β-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than β-oxidation.     

Passive immunization of lactating mice with stanniocalcin-1 antiserum reduces mammary gland development, milk fat content, and postnatal pup growth

During pregnancy and lactation in rodents, stanniocalcin-1 (STC-1) production by the ovaries is upregulated markedly and released into the circulation. The mammary glands are one target of this systemically delivered hormone. The purpose of this study was to lower serum levels of STC-1 in lactating mice through passive immunization so as to monitor the effects on mammary gland function and postnatal pup growth. Passive immunization significantly reduced circulating hormone levels, and pup growth was significantly compromised (30%), even though control and experimental litters had ingested equal amounts of milk. When mammary glands were analyzed, the alveolar area was significantly reduced in antibody-treated mothers. An analysis of milk composition revealed no changes in lactose, protein, or electrolyte levels but an approximately 40% reduction in triglyceride levels. The latter was due to a significant reduction in mammary gland lipoprotein lipase activity and led to a significant buildup of triglycerides in the serum. Body fat content was also significantly reduced in pups from antibody-treated mothers, whereas pup fecal fat content was increased. In mothers, passive immunization also caused significant behavioral effects, in particular, increased locomotor and hindleg rearing activities. Collectively, the results suggest that systemically derived STC-1 has important effects on mammary gland development and the transfer of serum-based triglycerides into milk. Locomotor effects suggest that STC-1 also has a role in maternal behavior.     

Localization and expression of BCAT during pregnancy and lactation in the rat mammary gland

During lactation, branched-chain aminotransferase (BCAT) gene expression increases in the mammary gland. To determine the cell type and whether this induction is present only during lactation, female rats were randomly assigned to one of three experimental groups: pregnancy, lactation, or postweaning. Mammary gland BCAT activity during the first days of pregnancy was similar to that of virgin rats, increasing significantly from day 16 to the last day of pregnancy. Maximal BCAT activity occurred on day 12 of lactation. During postweaning, BCAT activity decreased rapidly to values close to those observed in virgin rats. Analyses by Western and Northern blot revealed that changes in enzyme activity were accompanied by parallel changes in the amount of enzyme and its mRNA. Immunohistochemical studies of the mammary gland showed a progressive increase in mitochondrial BCAT (mBCAT)-specific staining of the epithelial acinar cells during lactation, reaching high levels by day 12. Immunoreactivity decreased rapidly after weaning. There was a significant correlation between total BCAT activity and milk production. These results indicate that the pattern of mBCAT gene expression follows lactogenesis stages I and II and is restricted to the milk-producing epithelial acinar cells. Furthermore, BCAT activity is associated with milk production in the mammary gland during lactation.