Mammary gland development is delayed in mice deficient for aminopeptidase N

Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.     

Mammary gland involution is associated with rapid down regulation of major mammary Ca-ATPases

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca²⁺-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca²⁺-ATPases and mammary calcium transport is unknown. We found that 24 h after stopping milk production, PMCA2 and secretory pathway Ca²⁺-ATPases 1 and 2 (SPCA1 and 2) expression decreased 80-95%. PMCA4 and Sarco/Endoplasmic Reticulum Ca²⁺-ATPase 2 (SERCA2) expression increased with the loss of PMCA2, SPCA1, and SPCA2 but did not increase until 72-96 h of involution. The rapid loss of these Ca²⁺-ATPases occurs at a time of high mammary tissue calcium. These results suggest that the abrupt loss of Ca²⁺-ATPases, required by the mammary gland to regulate the large amount of calcium associated with milk production, could lead to accumulation of cell calcium, mitochondria Ca²⁺ overload, calcium mediated cell death and thus play a part in early signaling of mammary involution.     

The SV40 T-antigen induces premature apoptotic mammary gland involution during late pregnancy in transgenic mice

Transgenic animals of the line 8 contain the WAP-SV-T transgene. Females of this line synthesise the SV40 T-antigen in mammary gland epithelial cells during pregnancy and the lactation period. All females are ‘milk-less’ and the offspring have to be nursed by foster mothers. The reason for this phenomenon is a premature apoptosis during late pregnancy. Nonetheless a significant number of mammary epithelial cells escape apoptosis and all transgenic females develop breast cancer after the first lactation period.     

Accelerated mammary gland development during pregnancy and delayed postlactational involution in vitamin D3 receptor null mice

The vitamin D receptor (VDR) is present in mammary gland, and VDR ablation is associated with accelerated glandular development during puberty. VDR is a nuclear receptor whose ligand, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] is generated after metabolic activation of vitamin D by specific vitamin D hydroxylases. In these studies, we demonstrate that both the VDR and the vitamin D 1-alpha hydroxylase (CYP27B1), which produces 1,25-(OH)(2)D are present in mammary gland and dynamically regulated during pregnancy, lactation, and involution. Furthermore, we show that mice lacking VDR exhibit accelerated lobuloalveolar development and premature casein expression during pregnancy and delayed postlactational involution compared with mice with functional VDR. The delay in mammary gland regression after weaning of VDR knockout mice is associated with impaired apoptosis as demonstrated by reductions in terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling staining, caspase-3 activation and Bax induction. Under the conditions used in this study, VDR ablation was not associated with hypocalcemia, suggesting that altered mammary gland development in the absence of the VDR is not related to disturbances in calcium homeostasis. Furthermore, in the setting of normocalcemia, VDR ablation does not affect milk protein or calcium content. These studies suggest that the VDR contributes to mammary cell turnover during the reproductive cycle, and its effects may be mediated via both endocrine and autocrine signaling pathways. Unlike many mammary regulatory factors that exert transient, stage-specific effects, VDR signaling impacts on mammary gland biology during all phases of the reproductive cycle.     

Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart

The serine-threonine kinase, Akt, inhibits cardiomyocyte apoptosis acutely both in vitro and in vivo. However, the effects of chronic Akt activation in the heart are unknown. To address this issue, we generated transgenic mice (TG+) with cardiac-specific expression of a constitutively active mutant of Akt (myr-Akt) driven by the myosin heavy chain-alpha promoter. Three TG+ founders (9-19 weeks) died suddenly with massive cardiac dilatation. Two viable TG+ lines (TG564 and TG20) derived from independent founders demonstrated cardiac-specific transgene expression as well as activation of Akt and p70S6 kinase. TG564 (n = 19) showed cardiac hypertrophy with a heart/body weight ratio 2.3-fold greater than littermates (n = 17, p < 0.005). TG20 (n = 18) had less marked cardiac hypertrophy with a heart/body weight ratio 1.6-fold greater than littermates (n = 17, p < 0.005). Isolated TG564 myocytes were also hypertrophic with surface areas 1.7-fold greater than littermates (p < 0.000001). Echocardiograms in both lines demonstrated concentric hypertrophy and preserved systolic function. After ischemia-reperfusion, TG+ had a 50% reduction in infarct size versus TG- (17 +/- 3% versus 34 +/- 4%, p < 0.001). Thus, chronic Akt activation is sufficient to cause a spectrum of phenotypes from moderate cardiac hypertrophy with preserved systolic function and cardioprotection to massive cardiac dilatation and sudden death.     

Mammary tumor heterogeneity in wt-ErbB-2 transgenic mice

Phenotypic and biological heterogeneity was studied in a single transgenic mouse model to determine the level of biological variance. We analyzed 1,258 tumors from 417 MMTV-wt-ErbB-2 transgenic mice, subdivided by casein or soy-based dietary randomization and hormonal treatment. Variance in tumor histologic features, growth pattern, invasion, metastases, and multi-focality were detected in untreated and treated mice. Ninety-three percent (1,174/1,258) of tumors had the solid growth pattern widely reported in this model. However, among the solid tumors, a spectrum of growth patterns, from well-circumscribed tumors with a pseudocapsule to locally invasive or highly aggressive, metastatic subtype, was observed. Of the non-solid tumors, glandular features were prominent in 84 (7%). Adenocarcinomas included papillary, acinar/glandular, and adenosquamous subtypes. Adenosquamous tumors were exclusively observed in the group of mice treated on a short-term basis with estrogen. In contrast to the reported literature for this transgenic mouse model, mammary tumors were multifocal in the majority of cases (303 of 417 mice, or 73%). Results of this extensive study of a single transgenic model of mammary tumorigenesis indicate phenotypic and biological heterogeneity not previously associated with this transgenic mouse. These data support a complex, multistep process of carcinogenesis and clonal evolution, with biological and phenotypic variance similar to that observed in human mammary cancer development.     

Mammary gland neoplasia: insights from transgenic mouse models

Current theories of breast cancer progression have been greatly influenced by the development and refinement of mouse transgenic and gene targeting technologies. Early transgenic mouse models confirmed the involvement of oncogenes, previously implicated in human breast cancer, by establishing a causal relationship between overexpression or activation of these genes and mammary tumorigenesis. More recently, the importance of genes located at sites of loss of heterozygosity in human breast cancer have been examined in mice by their targeted disruption via homologous recombination. The union of these two approaches allows the generation of complex animal models that more accurately reflect the multistep nature of human breast cancer. This review will examine how the study of transgenic mice has increased our understanding of the molecular events responsible for oncogenic transformation of the mammary gland. BioEssays 22:554-563, 2000.     

High-level salivary gland expression in transgenic mice

A 7.1 kb mini-gene construct containing cloned DNA from the murine parotid secretory protein (PSP) gene with 6.2 kb of the promoter, has previously been shown to direct specific mRNA expression to the salivary glands in transgenic mice. However, the level of transgene expression in the parotid gland was only a few percent of the endogenous level. This indicated that elements necessary for high-level expression are still to be found. In this study, we have searched for such regulatory elements in additional flanking regions by using a 25 kb clonedPsp ^b fragment containing the complete structural gene, 11.4 kb of 5-flanking sequence, and 2.5 kb 3-flanking sequence as a transgene. To distinguish the expression of the transgene from that of the endogenous gene, we took advantage of an allelic difference, using an oligonucleotide that recognized the mRNA fromPsp ^b and the transgene but not that from the other allele,Psp ^a . The expression of the transgene was examined in animals homozygous forPsp ^a . Three independent integrations all exhibited a level of parotid-gland-specific expression that corresponded to that of the endogenous gene. Thus, sequences responsible for this high-level PSP mRNA expression are situated within the genomic DNA of the transgene.     

Mammary blood flow and cardiac output during initiated involution of the mammary gland in the rabbit

1. Cardiac output and its distribution to the mammary gland, kidneys, heart, liver and gastrointestinal tract were measured in conscious rabbits at day 1, day 3 and day 6 after removal of the young at day 0. 2. There was no change in cardiac output, proportion of cardiac output delivered to the mammary gland or mammary blood flow 24 hr after the last suckling period. After a further 48 hr there was a significant reduction in the cardiac output, proportion of the cardiac output and blood flow to the mammary gland compared to the values measured in lactating animals. 3. There was no significant difference in blood flow to the other organs although there were changes in the proportion of the cardiac output delivered to the heart, kidney and liver during this period. 4. The change in uptake of glucose, acetate, non-esterified fatty acids and triacylglycerols by the mammary gland are discussed in relation to the reabsorption of milk components.     

Multiple cystic sweat gland tumors in transgenic mice

Here we describe gross and microscopic sweat gland tumors found in a transgenic mouse model of breast cancer, which had transforming growth factor α under the control of mouse mammary tumor virus promoter (MMTV-TGFα). Initially, 20% of the mice in the colony were affected. Cystic lesions formed on the phalanges, palmar surfaces of the metacarpals, and plantar surfaces of the metatarsals. The lesions were multifocal and nonulcerated with straw-colored fluid, ranging in size from 1 to 30 mm at the largest dimension. The colony was monitored for 6 mo; during that time, the prevalence of lesions increased to 52% of the mice. Histologically, in most cases the cyst walls were lined by 1 or 2 layers of normal-appearing epithelial cells that resembled basal cells, indicating adenoma. However, 2 cysts from 2 different mice had papillary proliferative projections and extensive disorganized glandular structures that protruded into the cyst cavities, indicating adenocarcinoma. In these 2 cases, the neoplastic cells revealed architectural and cytologic atypia with rare mitoses. Similar findings have previously been observed in sweat gland tumors; however, multiple sweat-gland tumors have not been reported in mice.     

Mfge8 is critical for mammary gland remodeling during involution

Apoptosis is a critical process in normal mammary gland development and the rapid clearance of apoptotic cells prevents tissue injury associated with the release of intracellular antigens from dying cells. Milk fat globule-EGF-factor 8 (Mfge8) is a milk glycoprotein that is abundantly expressed in the mammary gland epithelium and has been shown to facilitate the clearance of apoptotic lymphocytes by splenic macrophages. We report that mice with disruption of Mfge8 had normal mammary gland development until involution. However, abnormal mammary gland remodeling was observed postlactation in Mfge8 mutant mice. During early involution, Mfge8 mutant mice had increased numbers of apoptotic cells within the mammary gland associated with a delay in alveolar collapse and fat cell repopulation. As involution progressed, Mfge8 mutants developed inflammation as assessed by CD45 and CD11b staining of mammary gland tissue sections. With additional pregnancies, Mfge8 mutant mice developed progressive dilatation of the mammary gland ductal network. These data demonstrate that Mfge8 regulates the clearance of apoptotic epithelial cells during mammary gland involution and that the absence of Mfge8 leads to inflammation and abnormal mammary gland remodeling.     

Akt is activated in response to an apoptotic signal

Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.     

Mammary gland development and alpha-lactalbumin production in hypophysectomized, pregnant mice

Swiss Webster mice were hypophysectomized on Day 10 of pregnancy and the effect of the ablation on mammary gland development was estimated by measuring the total weight and the DNA, RNA, and alpha-lactalbumin contents and concentrations of the mammary gland on Days 14 and 18 of gestation. Although a significant increase in mammary gland weight occurred in the hypophysectomized animals between Days 10 and 18 of pregnancy, the mammary gland weight of the hypophysectomized mice was significantly reduced when compared with intact and sham-operated mice on both Days 14 and 18 of pregnancy. The total RNA content of the mammary gland was also reduced in the hypophysectomized mice, although it increased significantly from Day 10 to Day 18. The alpha-lactalbumin content of the mammary gland increased only slightly between Days 10 and 14 of gestation in the intact and sham-operated mice, but a large increase was found on Day 18 in both groups. There was, on the other hand, no increment in the alpha-lactalbumin content of the mammary gland in the hypophysectomized mice either on Day 14 or 18 of gestation. The DNA content of the mammary gland was not affected by hypophysectomy when estimated on Days 14 and 18 of pregnancy. The effects of hypophysectomy on the concentrations of mouse placental lactogen II (mPL-II), progesterone, corticosterone, and thyroxine in the maternal serum were also assessed. The concentration of mPL-II was significantly elevated in the hypophysectomized mice, whereas the circulating concentrations of both corticosterone and thyroxine were greatly reduced. The serum progesterone concentration was not significantly altered by hypophysectomy. (ABSTRACT TRUNCATED AT 250 WORDS)     

Akt/protein kinase B promotes organ growth in transgenic mice

One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.     

Activation of guanylate cyclase by arachidonic acid: In Mammary gland homogenates from mice

Arachidonic acid stimulated guanylate cyclase activity about two fold in homogenates of mammary glands obtained from midpregnant mice; effects of arachidonic acid were observed during incubation periods between 5 and 20 minuted. Stimulatory effects of arachidonic acid on guanylate cyclase activity were observed when 10 to 100 μg arachidonic acid was added to the reaction mixtures (150 μl). When 250 μg or more arachidonic acid was added to the reaction mixtures, the activity of guanylate cyclase was inhibited. Other fatty acids including linoleic acid, linolenic acid and oleic acid also stimulated guanylate cyclase activity but neither arachidic acid nor stearic acid had an effect. The arachidonic acid stimulation of guanylate cyclase activity was abolished by incubation with indomethacin and aspirin, thus suggesting the arachidonic acid effect may be carried out via the prostaglandins. A variety of prostaglandins, however, at several concentrations did not stimulate guanylate cyclase activity when added to the reaction mixtures. The failure of the prostaglandins to have an effect may be due to several reasons which are discussed.     

Mammary gland growth and response to hormones in mastomys compared with mice

Mammary gland growth with or without hormone manipulation was examined in virgin mastomys (Praomys (mastomys) natalensis) and compared with C3H/He mice having a low mammary tumor incidence. Mammary glands of mastomys consisted mostly of duct systems even at 720 days of age, whereas conspicuous formation of normal end-buds and preneoplastic hyperplastic alveolar nodules were seen in the glands of mice after 180 days of age. Mammary glands of mastomys showed a higher response to estrogen or progesterone rather than prolactin, which is much different from other rodents.     

转基因小鼠乳腺表达G-CSF的研究

用PCR法从正常中国人脐带血提取总DNA作为模板,扩增出1.5 kb的人G-CSF基因组基因。序列分析证实其正确性。将其插入小鼠乳清酸蛋白(WAP)基因的起始密码子ATG前的KpnⅠ位点,使其受控于2.6kb的WAP调控序列,构建成乳腺表达载体pWGG。回收经EcoRⅠ酶切后的8.7kb片段用于显微注射。共注射1200枚受精卵,移植34受体母鼠,产仔鼠85只。经PCR检测和DNA印迹分析,证实获得两只整合有人G-CSF基因的雄性鼠,整合率为2.37％。建立的转基因鼠系表明,采用ELASA方法对F1代雌鼠乳汁检测,成功地表达出人G-CSF。表达量为120～250ng/ml。这一结果表明转基因的表达具有乳腺特异性。这为在大动物中实施转基因提供了依据。     

Demonstration of pineal gland and pituitary involution in mice poisoned with carbon tetrachloride]

Ingestion of carbone tetrachloride by male C3H Orleans mice determines an involution of the pineal gland and of the hypophysis, as well as extensive lesions of the hepatic parenchyma. There may exist in these animals a higher blood-level of glucocorticoids, as this has been shown to be the case in men suffering from cirrhosis or hepatitis. Involution of the pineal gland in cases of stress, as has been shown by various authors, would result from an entirely different mechanism, that is by increased secretion of corticoadrenal hormones.