Alveolar Lobules Research Articles

New insights into lineage restriction of mammary gland epithelium using parity-identified mammary epithelial cells

IntroductionParity-identified mammary epithelial cells (PI-MECs) are an interesting cellular subset because they survive involution and are a presumptive target for transformation by human epidermal growth factor receptor 2 (HER2)/neu in mammary tumors. Depending on the type of assay, PI-MECs have been designated lobule-restricted progenitors or multipotent stem/progenitor cells. PI-MECs were reported to be part of the basal population of mammary epithelium based on flow cytometry. We investigated the cellular identity and lineage potential of PI-MECs in intact mammary glands.MethodsWe performed a quantitative and qualitative analysis of the contribution of PI-MECs to mammary epithelial cell lineages in pregnant and involuted mammary glands by immunohistochemistry, fluorescence-activated cells sorting (FACS), and quantitative polymerase chain reaction. PI-MECs were labeled by the activation of Whey Acidic Protein (WAP)-Cre during pregnancy that results in permanent expression of yellow fluorescent protein.ResultsAfter involution, PI-MECs are present exclusively in the luminal layer of mammary ducts. During pregnancy, PI-MECs contribute to the luminal layer but not the basal layer of alveolar lobules. Strikingly, whereas all luminal estrogen receptor (ER)-negative cells in an alveolus can be derived from PI-MECs, the alveolar ER-positive cells are unlabeled and reminiscent of Notch2-traced L cells. Notably, we observed a significant population of unlabeled alveolar progenitors that resemble PI-MECs based on transcriptional and histological analysis.ConclusionsOur demonstration that PI-MECs are luminal cells underscores that not only basal cells display multi-lineage potential in transplantation assays. However, the lineage potential of PI-MECs in unperturbed mammary glands is remarkably restricted to luminal ER-negative cells of the secretory alveolar lineage. The identification of an unlabeled but functionally similar population of luminal alveolar progenitor cells raises the question of whether PI-MECs are a unique population or the result of stochastic labeling. Interestingly, even when all luminal ER-negative cells of an alveolus are PI-MEC-derived, the basal cells and hormone-sensing cells are derived from a different source, indicating that cooperative outgrowth of cells from different lineages is common in alveologenesis.

Identification of stable and oestrus cycle-independent housekeeping genes in the rat mammary gland and other tissues

The function and development of the rat mammary gland is dependent on the oestrus cycle. Normalization of gene expression in mammary gland samples assessed by quantitative RT-PCR therefore requires housekeeping genes (HKGs) which are stably expressed during the oestrus cycle. mRNA expression of 10 HKGs was measured in the rat mammary gland at different phases of the oestrus cycle. In addition, mRNA expression of the HKGs was measured in a panel of other rat tissues comprising laser microdissected mammary gland alveolar lobules and interlobular connective tissue and macrodissected mammary gland, liver, skeletal muscle, colon and ovary samples. Expression and ranking of HKGs varied between tissues and oestrus cycle phases and several HKGs were necessary for normalization between samples. In the mammary gland samples, three HKGs (Sdha, Tbp, and Atp5b) were identified as the optimal combination of stably expressed genes across oestrus cycle phases. For normalization between samples from the entire panel of rat tissues, eight HKGs (Rps18, Eef1a1, B2m, Actb, Tbp, Hprt, Pgk1, and Sdha) were identified as the optimal combination. These HKGs are of general relevance for studies comparing gene expression between different rat tissues.

A Role for KAI1 in Promotion of Cell Proliferation and Mammary Gland Hyperplasia by the gp78 Ubiquitin Ligase

Expression of gp78, an E3 ubiquitin ligase in endoplasmic reticulum-associated degradation, is associated with tumor malignancy. To study gp78 overexpression in mammary gland development and tumorigenicity, we generated murine mammary tumor virus (MMTV) long terminal repeat-driven gp78 transgenic mice. Embryos carrying the gp78 transgene cassette were implanted in FVB surrogate mothers, and two founders with high copy integration showed elevated gp78 expression at both transcript and protein levels at the virgin stage and at 12 days gestation. Transgenic mammary glands showed increased ductal branching, dense alveolar lobule formation, and secondary terminal end bud development. Bromodeoxyuridine staining showed increased proliferation in hyperplastic ductal regions at the virgin stage and at 12 days gestation compared with wild type mice. Reduced expression of the metastasis suppressor KAI1, a gp78 endoplasmic reticulum-associated degradation substrate, demonstrates that gp78 ubiquitin ligase activity is increased in MMTV-gp78 mammary gland. Similarly, metastatic MDA-435 cells exhibit increased gp78 expression, decreased KAI1 expression, and elevated proliferation compared with nonmetastatic MCF7 cells whose proliferation was enhanced upon knockdown of KAI1. Importantly, stable gp78 knockdown HEK293 cells showed increased KAI1 expression and reduced proliferation that was rescued upon KAI1 knockdown, demonstrating that gp78 regulation of cell proliferation is mediated by KAI1. Mammary tumorigenesis was not observed in repeatedly pregnant MMTV-long terminal repeat-gp78 transgenic mice over a period of 18 months post-birth. Elevated gp78 ubiquitin ligase activity is therefore not sufficient for mammary tumorigenesis. However, the hyperplastic phenotype observed in mammary glands of MMTV-gp78 transgenic mice identifies a novel role for gp78 expression in enhancing mammary epithelial cell proliferation and nontumorigenic ductal outgrowth.

ESTROGENS AND CORTICOIDS IN RELATION TO MAMMARY GLAND GROWTH IN MALE RATS.

SummaryThe reaction of the mammary glands to testosterone propionate (Tp) was studied in gonadectomized (gdx), adrenalectomized (adx) rats given estrone (E) and cortisone acetate (Ca) singly or together. Compared with controls with intact adrenals the mammary responses to Tp were reduced alter adrenalectomy. With few exceptions alveolar lobules failed to develop in gdx adx rats injected with 0.1 mg Tp every other day. When daily injections of 0.125 mg Ca were added to the treatment with 0.1 mg Tp the number of glands presenting alveoli increased slightly. E, 0.05 μg daily, failed to modify the mammary response to 0.1 mg Tp. Combined treatment with Ca, 0.125 mg, and E, 0.05 μg, daily and Tp, 0.1 or 0.2 mg, every other day resulted in development of alveolar lobules in a great majority of mammary glands of gdx adx rats. The findings support the view that the mammary response to androgens is dependent on concomitant actions of estrogens and adrenal cortex steroids.

Interactions of oestrogens and androgens on the mammary gland and growth of other tissues in hypohysectomized rats treated with insulin, cortisone and thyroxine.

ABSTRACT In a previous study it was found that oestrogens are necessary for androgens to elicit a development of alveolar lobules in the mammary glands of rats. Since androgens and oestrogens exert synergistic as well as antagonistic actions on mammary glands of e. g. rabbits, the significance of oestrogens in the response of the mammary gland to androgens was further investigated in the rat. The experiments were designed according to the same principles as previously, that is, the response of the gland itself to oestrogens was modified. This was achieved by treatment of hypophysectomized rats with a) thyroxine (negligible response) and b) thyroxine, cortisone and insulin (marked response). The effect of endogenous androgens was studied in males injected with PMS with or without oestrogens. Gonadectomized rats were injected with testosterone and oestrogens. No oestrogens given: The mammary glands of hypophysectomized males injected with PMS showed an abnormal response, irrespective of simultaneous treatment with thyroxine, cortisone and insulin in various combinations. The result confirms previous work with testosterone. Negligible response of the mammary glands to oestrogens: In hypophysectomized rats treated with thyroxine, oestrogens and PMS or testosterone, the response of the glands was uniform and abnormal. The absence of end buds indicated that the response to oestrogens, if present at all, was suppressed by the androgens. Marked response of the mammary glands to oestrogens: In hypophysectomized rats treated with thyroxine, cortisone and insulin another response of the glands to androgens and oestrogens was found. Besides abnormal structures, alveolar lobules were present. The changes produced with different doses of testosterone and oestrone indicated a complicated interplay of the two hormones. Confirming previous observations, records of the length and weight of the body and of the weight of the liver and heart revealed marked growth in the hypophysectomized rats treated with thyroxine, cortisone and insulin.

The growth responses of mammary glands and other tissues in hypophysectomized female rats treated with thyroxine, insulin, cortisone, and pregnant mare serum gonadotrophin.

ABSTRACT The reaction of the mammary gland of the hypophysectomized rat to endogenous ovarian hormones produced under the influence of serum gonadotrophin (PMS) was studied and the effect of treatment with insulin, thyroxine, and cortisone in various combinations on the reaction examined. Changes in body weight, body length, weight of liver, and weight of heart ventricles were also followed. In hypophysectomized rats treated with PMS alone (group 1) the ovarian weight increased approximately tenfold and the hormones secreted caused vaginal cornification. Mammary growth was absent. With PMS, thyroxine and cortisone (group 2) the ovaries reacted as with PMS alone and vaginal cornification was again observed. Growth of end buds occurred in the mammary glands. The weight of the heart ventricles increased. With PMS, insulin and cortisone (group 3) ovarian weight and function was similar to that in the previous groups but the effect on the mammary gland was greater in that the size was increased, there were numerous side buds and end buds, and a few alveolar lobules. Body and liver weight was also increased. With PMS, thyroxine, insulin and cortisone (group 4) the effect on the ovaries and mammary glands was qualitatively similar to that seen in group 3 but ovarian weight was lower, and mammary growth more extensive. The rats grew in length, increased in weight, and the weight of the liver and heart ventricles was close to that of control animals. The above results support the view that the growth of the mammary gland is dependent upon the metabolic state of the individual.

Testosterone and the browth of mammary glands and other tissues of hypophysectomized rats treated with thyroxine, insulin and cortisone.

ABSTRACT The reaction of the mammary glands to testosterone propionate was studied in gonadectomized, hypophysectomized rats treated with thyroxine, insulin and cortisone in various combinations. The weight and length of the body and the weight of the liver, heart ventricles, adrenal glands, seminal vesicles and ventral prostate was also recorded. Growth of alveolar lobules, such as produced by injections of testosterone into rats with intact pituitary gland did not occur in any of the 3 groups of hypophysectomized rats studied, that is, neither after treatment with testosterone and 1) thyroxine and cortisone, 2) insulin and cortisone, nor 3) thyroxine, insulin and cortisone. An abnormal elongation of side buds and a hyperplasia and proliferation of ductal epithelium did occur and was most prominent in the group receiving insulin and cortisone. A marked increase in body length, as well as in body weight, was observed in the group treated with testosterone, thyroxine, insulin and cortisone. The results are discussed with regard to the enhanced growth response to ovarian hormones observed in this laboratory under similar conditions.