Mammary Glands Of Virgin Mice Research Articles

Autocrine prolactin induced by the Pten–Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten-PI3K-Akt pathway. Conditional activation of the PI3K-Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K-Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)-Jak-Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl(-/-), Prlr(-/-), and Stat5(-/-) mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1(-/-);Akt2(+/-) mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K-Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K-Akt pathway.

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis.

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.

Retraction: Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis.

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.

Developmental Changes in Insulin‐like Growth Factor I Receptor Gene Expression in the Mouse Mammary Gland

The insulin‐like growth factor I receptor (IGF‐IR), which mediates the mitogenic action of IGF‐I, has been shown to play an essential role in normal growth and development. However, the precise role of IGF‐IR in the growth and differentiation of the mammary gland has not been elucidated. This study examines the profile of the IGF‐IR gene and protein expression during normal postnatal mammary gland development in order to gain further insight into the role of the IGF‐I/IGF‐IR during mammary gland morphogenesis. Gene and protein expression were examined in developing mouse mammary glands (virgin, pregnant, lactating, involuting) by real time PCR analysis and Western blotting. Both IGF‐IR gene and protein expression levels were high during early pregnancy. Interestingly, the level of gene expression was significantly down‐regulated during late pregnancy (5.4 fold) and lactation (9–13 fold) and significantly up‐regulated (3.9 fold) during late involution, to the level observed in the virgin mammary gland. By in situ hybridization, the IGF‐IR transcripts were localized to the proliferating ductal epithelium of the mammary glands of virgin mice and to the differentiating ductal and alveolar epithelium of the mammary glands during pregnancy and lactation. In the involuting gland, the transcripts were localized to the regressing ductal epithelium. These data are direct evidence that IGF‐IR expression is important for alveolar cell proliferation and suggest that the progression of involution may require the down‐regulation of IGF‐IR gene expression. Altogether, these results demonstrate that a developmental IGF‐IR gene expression pattern exists in the mouse mammary gland and that increases in gene expression at specific phases of development may reflect an important role for IGF‐I/IGF‐IR at those phases of development.

Expression of xanthine oxidoreductase in mouse mammary epithelium during pregnancy and lactation: regulation of gene expression by glucocorticoids and prolactin.

In the mammary gland of virgin mice, xanthine oxidoreductase (XOR) enzymic activity is barely measurable. a high increase in the levels of the enzyme is observed during the last days of pregnancy and during lactation, and this is parallelled by an elevation in the amounts of the respective protein and transcript. In situ hybridization experiments demonstrate that the XOR mRNA is specifically expressed in the alveolar epithelial cells of the mammary gland. In HC11 cells, a model culture system for normal breast epithelium, the levels of XOR enzymic activity are dose- and time-dependently induced by dexamethasone, and a further synergistic augmentation is observed in the presence of dexamethasone plus prolactin. Increased XOR gene expression is consequent on glucocorticoid receptor activation, as indicated by sensitivity to the specific receptor antagonist RU486. In addition, the phenomenon is likely to involve protein phosphorylation and dephosphorylation events, as suggested by modulation of XOR mRNA by tyrosine kinase and phosphatase inhibitors.

Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary gland explants.

A method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.

Morphological changes induced in mouse mammary gland by porcine and human relaxin.

The effects of pure porcine relaxin and of human decidual extracts with relaxin-like activity on the mammary gland of virgin mice primed with estrogen have been studied by the light microscope. Porcine relaxin enhanced the changes induced by estrogen alone; the effect was different in the various mammary tissues. In the stroma, relaxin only slightly increased the loosening of connective tissue, the extent of the adipose tissue and of the capillary bed, as well as the degranulation of the mast cells. The changes in the parenchyma, such as elongation and branching of ducts, are strikingly enhanced. Moreover, relaxin seems to promote differentiation of the cells forming the walls of distal ducts, and of the myoepithelial cells. Tissue extracts of human decidua with relaxin-like activity induce changes in the mammary gland similar to those due to porcine relaxin. Such data indicate that relaxin synergizes with estrogen to cause growth of ducts of the mammary gland and that tissue extracts of human decidua have a similar effect, thus providing further evidence that decidua may be a source of relaxin in humans.

Loss of differentiative potential of the mammary gland in ovariectomized mice: Altered estrogen responsiveness in parous mice

Previously, estrogen had been shown to be essential for the maintenance of differentiative potential in the mammary gland of virgin mice; the purpose of this study was to evaluate the role of these steroids in nonpregnant, nonlactating parous mice. To this end, parous mice were either ovariectomized or adrenalectomized and ovariectomized for 4–6 weeks before hormonally induced differentiation was attempted in culture. Although mammary glands from virgin mice lose their differentiative potential in vitro following ovariectomy, tissue from castrated parous mice exhibit only a partial loss of activity. Since combined adrenalectomy and ovariectomy in parous animals virtually abolished differentiation and since exogenous estradiol preserved it, it would appear that mammary gland differentiation in parous mice, as in virgins, requires estrogens, but that tissue from the former is more sensitive. This difference between virgins and parous mice is not related to differences in either the capacity or affinity of the cytosol estrogen receptor, as these parameters are identical in all experimental groups.

The nonresponsiveness of lactating mammary gland to estradiol.

The effect of estradiol on mammary glucose metabolism and DNA synthesis has been studied. Estradiol causes a significant increase in both the rate of glucose oxidation and DNA synthesis in the mammary glands of virgin mice, but is without any stimulatory effect on the glands of lactating mice. The reason for the inability of lactating mammary gland to respond to estradiol appears to reside in the lactating tissue itself and does not seem to be related to the hormonal milieu of lactation. Previously, we have shown that, in contrast to mammary gland of virgin mice, lactating mammary glands do not respond to estradiol with an increase in progesterone receptor levels. Therefore, all of these studies taken together reveal that during lactation, mammary glands may be nonresponsive with respect to most known parameters of estrogen action.

MAMMARY CELL PROLIFERATION IN MICE DURING PREGNANCY AND LACTATION IN RELATION TO MILK YIELD

Measurements have been made of body weight, mammary weight and total mammary DNA content (DNAt) during the second half of gestation and throughout lactation in mice. When corrected for conceptus weight and mammary weight, body weight was not significantly changed with advancing gestation but did increase during lactation. Mammary weight increased progressively until day 10 of lactation, then decreased until the end of lactation. On day 4 of involution mammary weight was not significantly different from that on day 12 of gestation. Mammary DNAt increased exponentially from day 12 of gestation to day 5 of lactation, with a mean doubling time of 6 d. No further increase was apparent at day 10, thereafter it fell in a similar manner to mammary weight. The incorporation of [3H]thymidine into mammary tissue was studied in vitro. [3H]Thymidine incorporation per unit weight of tissue was highest at mid‐gestation, fell during late gestation and then rose to a peak value 48 h post partum. For the remainder of lactation incorporation was very low. Incorporation calculated relative to DNAt showed similar, although less marked, changes. Milk yield of lactating mice has been estimated by daily weighing of the litter. Three phases were apparent: (i) a period from parturition to day 6 when milk yield was rising; (ii) a peak plateau from day 6 to day 10; (iii) a lower plateau from day 11 to day 15. Total daily litter weight gain was not significantly different between litters of nine and eighteen pups from day 7 to day 13 of lactation. Virgin and lactating mice were treated daily for seven days with 0·6 mg progesterone plus 0·2 µg oestradiol benzoate. This treatment significantly stimulated [3H]thymidine incorporation in the mammary glands of virgin mice and of mice in late lactation (days 9–15) but not in those of mice in early lactation (days 2–8). Milk yield was significantly reduced in the early‐lactation group, but not in the late‐lactation group.

Distribution of mouse mammary tumour virus antigens in RIII mice as detected by immunofluorescence on tissue sections and by immunoassays in sera and organ extracts.

Organs of RIII mice at various physiological stages were tested for mouse mammary tumour virus (MMTV) antigen expression. Indirect immunofluorescence was used with three monospecific antisera to localize one envelope glycoprotein, gp47, and two core proteins, p28 and p8. These virus-specific antigens gave characteristic fluorescent patterns in the mammary tissues and were also detected in thymus and salivary gland sections of some mice. The amounts of antigens gp47 and p28 were measured by immunoassay in sera and organ extracts of corresponding samples of mice. Sera of mice of both sexes contained virus antigens from the suckling age onwards. Although ingested virus could be traced in suckling mice, weanlings were characterized by the absence of virus expression except in lymph nodes. This location points to the possible role of lymphoid tissue in producing the antigens of the blood and in disseminating the infection. In adult animals, virus antigens were present in salivary glands, digestive tract, lymph nodes, male genital organs and female mammary glands. Antigen expression, even found in the mammary glands of virgin mice, was strikingly increased by pregnancy, lactation and (or) ageing, the highest values being found in mammary tumours. The results for milk-borne MMTV infection in RIII mice are compared with those obtained previously in Swiss albino mice.

Effect of oestradiol on progesterone receptors in normal mammary glands and its relationship with lactation.

Mammary glands of lactating mice, either intact or ovariectomized, do not contain detectable amounts of progesterone receptors and this lack of receptors persists also in tissues of animals treated with oestradiol. In contrast with lactators, mammary glands of virgin mice contain progesterone receptors whose amounts are augmented by oestradiol administration.

Regulation of messenger RNA and specific milk protein in mammary gland.

Growth and functional differentiation of the mammary gland are dependent on the modulating action of multiple hormones (steroid and protein) both in vivo and in organ culture of the whole gland. There is little or no RNA synthesis in the mammary gland of virgin mice; rapidly labeled RNA synthesis is evident at pregnancy and it rises at lactation. In the animal, adrenalectomy causes a reduction of rough endoplasmic reticulum (RER), and larger ribosomal aggregates (polysomes) in lac tating mammary gland. Adrenalectomy‐induced reduction of casein (determined by immunoprecipitation) synthesis activity of lactating mammary polysomes, evident in a cell‐free protein synthesis system, can be prevented by cortisol therapy of the postpartum mice. A direct assay of the lactating mammary cell messenger RNA (mRNA) in a heterologous cell‐free protein synthesis system, derived from ascites tumor cell ribosomes (S30), rabbit reticulocyte factors (0.5 M KCI, S30 wash), and transfer RNA, shows that the specific activity of...

Effect of procaine on development of ‘ridges’ and ‘domes’ in primary cell cultures from mammary glands of untreated virgin mice: The role of cell density in the occurrence of ‘domes’

Primary cell cultures from mammary glands of virgin mice that were not pretreated with hormones were subjected to: (1) procaine; (2) insulin+ prolactin +hydrocortisone; (3) a combination of (1) and (2). Procaine caused a ‘ridge’ effect similar to that of the hormones. The combination of procaine with the hormones caused a still stronger ‘ridge’ effect as well as the formation of ‘domes’. The formation of ‘domes’ is suggested to be dependent on cell density.

Rapidly-labelled RNA in the mouse mammary gland before and during lactation.

SUMMARY Linear sucrose density gradient analysis showed that the synthesis of rapidly-labelled high molecular weight RNA was virtually absent in the mammary glands of virgin mice. The rapidly-labelled RNA was first evident in the mammary gland in pregnancy and was also present during lactation. The bulk of this newly-made nuclear RNA sedimented as 45S and 32S fractions after a 15-min [3H]uridine pulse period in vivo. No labelled 18S RNA was detectable in the nuclear fraction after the 15-min pulse but it was present in the cytoplasm, suggesting that the 18S RNA migrates to the cytoplasm almost immediately after its formation. Thirty minutes after injection of [3H]uridine, the initial radioactivity of the 45S region migrated to the 32S fraction and a labelled 28S peak was also present in the cytoplasmic RNA at 60 min, suggesting that the processed 28S ribosomal RNA in the mammary gland began to migrate to the cytoplasm between 30 and 60 min after the nuclear synthesis of the precursor molecule.

DNA polymerase activity in mammary gland of virgin mice after ovarian hormone treatment.

SUMMARY The effect of ovarian hormones on DNA polymerase activity in the mouse mammary gland was determined. The rate of [3H]thymidine triphosphate incorporation into DNA during incubation in vitro was used as a measure of DNA polymerase activity in the post-microsomal supernatant fraction of the mammary tissue. A negligible amount of DNA polymerase activity was present in the mammary tissue of ovariectomized mice which had no hormone treatment. Daily subcutaneous injections of 1 μg oestradiol-17β plus 1 mg progesterone induced a sixfold increase of DNA polymerase activity in the mammary tissue after two injections. A single intraperitoneal injection of the same dose also induced a similar rise of DNA polymerase activity and the effect was evident within hours after the treatment. The results provide evidence that stimulation of cellular biosynthesis by the growth-promoting ovarian hormones is associated with the activation of a specific enzyme involved in DNA replication.