Stage-Specific Effects of Androgens and Estradiol-17beta on the Development of Late Primary and Early Secondary Ovarian Follicles of Coho Salmon (Oncorhynchus kisutch) In Vitro

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.     

Comparative 2D-DIGE Proteomic Analysis of Bovine Mammary Epithelial Cells during Lactation Reveals Protein Signatures for Lactation Persistency and Milk Yield

Mammary gland is made up of a branching network of ducts that end with alveoli which surrounds the lumen. These alveolar mammary epithelial cells (MEC) reflect the milk producing ability of farm animals. In this study, we have used 2D-DIGE and mass spectrometry to identify the protein changes in MEC during immediate early, peak and late stages of lactation and also compared differentially expressed proteins in MEC isolated from milk of high and low milk producing cows. We have identified 41 differentially expressed proteins during lactation stages and 22 proteins in high and low milk yielding cows. Bioinformatics analysis showed that a majority of the differentially expressed proteins are associated in metabolic process, catalytic and binding activity. The differentially expressed proteins were mapped to the available biological pathways and networks involved in lactation. The proteins up-regulated during late stage of lactation are associated with NF-κB stress induced signaling pathways and whereas Akt, PI3K and p38/MAPK signaling pathways are associated with high milk production mediated through insulin hormone signaling.     

Effects of dietary fat on mammary development relative to age and hormones in BALB/c mice

Earlier studies reported that mammary ducts grew faster if the 10% fat in the diet was composed of oils containing polyunsaturated fatty acids (corn oil: CO) compared to hydrogenated cottonseed oil (HCTO), which is devoid of such fatty acids. These experiments were primarily carried out in immature mice and left unanswered questions regarding the effects of dietary fats on more differentiated stages of mammary development. The use of transplanted ducts permitted the study of mammary growth rates in adult mice. If the diet was started when the animals were adults, there was no difference in the growth rate of those fed HCTO diet compared to those fed CO diet. However, when the diets were fed to immature mice, the mammary gland grew slower in mice fed the HCTO diet, confirming our earlier observations. The HCTO and CO diets caused no difference in the growth rate or morphology of fine ducts and alveoli that developed during pregnancy. Furthermore, no differences were seen in female mice following 6 weeks of progesterone administration begun at 3 weeks of age. Experiments that used male mice to examine the initial stages of mammary duct growth also showed that the effect of dietary fat was not observed when estrogen (E) or E and progesterone (P) were injected. In addition, there was no effect of dietary fat in ovariectomized 3-week-old females when any dose of E was administered from 0.01 to 1 microgram/day. Examination of the ovaries from mice fed either HCTO or CO diets from 3 to 9 weeks or 3 to 13 weeks of age showed that mice fed HCTO diet did not develop corpora lutea, while those fed CO diet had normal appearing ovaries. The HCTO diet inhibits normal maturation of the follicle and corpus luteum formation. We conclude that the effect of the dietary fat on the developing mouse is on the maturation of the ovary and subsequently on mammary growth.     

Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression.

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17beta estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by approximately sixfold (P:<0.0001) and increased mammary epithelial estrogen receptor (ERalpha) mRNA expression by approximately 50% (P:<0.0001; ERbeta mRNA was not detected in the primate mammary gland). Progesterone did not alter E2's proliferative effects, but testosterone reduced E2-induced proliferation by approximately 40% (P:<0.002) and entirely abolished E2-induced augmentation of ERalpha expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a approximately threefold increase in mammary epithelial proliferation (P:<0.01), but tamoxifen also reduced ERalpha expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid-responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen-induced down-regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifen's androgen-like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifen's protective action on mammary gland may involve androgenic effects.     

The estrogen-regulated 52K-cathepsin-D in breast cancer: From biology to clinical applications

We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis. In estrogen receptor positive human breast cancer celllines (MCF7, ZR75-1) estrogens specifically increase the production into the culture medium of a 52,000 daltons (52K) glycoprotein. Several high affinity monoclonal antibodies to the partially purified secretory 52K protein have allowed to purify to homogeneity this protein and its cellular processed products. The 52K protein has been identified as the secreted precursor of a cathepsin-D like protease bearing mannose-6-phosphate signals and routed to lysosomes via mannose-6-phosphate receptor. The protease is mitogenic in vitro on estrogen deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its activation. The cellular related proteins, as detected by immunohistochemistry and immunoassay are more concentrated in proliferative mammary ducts than in resting ducts and their concentration in breast cancer cytosol appears to be more correlated with lymph nodes invasion and disease free survival (with S. Thorpe, Copenhagen) than with the estrogen receptor (RE) level. The protein is also produced constitutively by RE-negative cell lines, while in some antiestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in λgt11 has allowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the normal human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness, In addition to other estrogen regulated growth factors, the precursor of lysosomal proteases has the potential to stimulate the proliferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

Integrin-interacting protein Kindlin-2 induces mammary tumors in transgenic mice

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.

     

Androgen action on hepatic vitellogenin synthesis in the eel, Anguilla japonica is suppressed by an androgen receptor antagonist

Involvement of additional hormones other than estrogen in the control of vitellogenin (Vg) synthesis has been suggested in fish. However, no satisfactory explanation on the mechanism of the action of these hormones has been reported. In this study, we have exploited the possibility of androgen receptor mediation during the androgen action on the pathway of Vg synthesis. Hepatocytes were prepared from sexually immature Japanese eel Anguilla japonica and treated with estradiol-17beta (E2), 17alpha-methyltestosterone (MT), growth hormone, tamoxifen or flutamide, or in combination of these. Spent culture media were analysed by SDS-PAGE for Vg detection. Results from the chemical treatments demonstrated the necessity of E2 as the primary factor for Vg synthesis and requirement of additional hormones for the full expression of Vg. The effects of E2 and MT were effectively blocked by tamoxifen, an estrogen receptor antagonist and flutamide, an androgen receptor antagonist, respectively, indicating ER-mediated estrogen action and AR-mediated androgen action on Vg synthesis in this species.     

Establishment and initial characterization of the ovine mammary epithelial cell line nish

Summary Analysis of the molecular mechanisms involved in the differentiation and formation of the characteristic three-dimensional structures of the developing mammary gland of the major milk-producing livestock (ducts, end buds, and alveoli) requires in vitro model cell cultures. The few cell lines that have been established from dairy animals do not fully reproduce the entire program of mammary differentiation. Here we present the initial characterization of a unique mammary epithelial cell line derived spontaneously from midpregnant sheep (NISH). These cells form in vitro functional structures resembling ducts, lateral buds, and alveoli that secrete β-lactoglobulin (BLG) in an ECM (extracellular matrix)-dependent manner. Interestingly, the presence of growth hormone dramatically increased BLG secretion from NISH cells cultured on ECM. It appears that GH is required not only to establish the structural organization but also is continuously needed to maintain BLG expression. Stable transfection of NISH cells with BLG/Human Serum Albumin (HSA) hybrid gene constructs revealed that the relative level of expression was comparable to the in vivo secretion of HSA in transgenic mice carrying these gene sequences. No expression could be detected in cells transfected with hybrid genes carrying either HSA cDNA or the entire HSA gene, and HSA expression was dependent on the presence of intronic sequences. These results demonstrate that NISH cells may prove a useful tool for studying the differentiation and organogenesis of mammary epithelial cells under defined culture conditions. Furthermore, transfected NISH cells may be an alternative for the transgenic mouse model in evaluating the potential of gene constructs to be efficiently expressed in the mammary gland of transgenic farm animals.     

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.     

Steroid hormone responsive cells in serum-free culture--analyses of hormone-mediated gene expression and cell growth

Steroid hormone-responsive cell lines were clones from mouse mammary cancer (Shionogi Carcinoma 115) and Leydig cell tumor. SC-3 and SC-4 cells from Shionogi Carcinoma were androgen-responsive and -unresponsive in a serum-free medium, respectively. SC-3 cells secreted FGF-like growth factor as well as 24 K glycoprotein in response to androgen stimuli. B-1 and B-1F cells from mouse Leydig cell tumor were growth-stimulated in a serum-free medium by estrogen, androgen or retinoic acid. Transfection of ERE-TK-CAT gene into B-1F cells revealed that both estrogen and retinoic acid activated the CAT activity. In addition, the presence of corresponding receptors for steroid hormones or retinoic acid was demonstrated by hormone binding assays and/or Northern blot analysis. Thus, these serum-free culture systems seem to be very useful for analysing hormone action mechanisms in vitro.     

Gender dimorphism of tumor growth: role of gonadal hormones in differential regulation of apoptosis of a murine T cell lymphoma

The present investigation was undertaken to study if a gender-dependent differential induction of tumor cell apoptosis is responsible for the manifestation of gender dimorphism observed in the growth of a transplantable murine T cell lymphoma, designated as Dalton’s lymphoma (DL). Tumor cell samples obtained from male tumor-bearing mice showed a higher number of cells with apoptotic morphology compared to that observed in female tumor-bearing mice. In this report we demonstrate that male hormone androgen and female hormone estrogen can differentially modulate tumor cell proliferation and apoptosis through alteration in the expression pattern of cell death regulating genes: p53 and CAD. DL cells were shown to express mRNA for androgen and estrogen receptors. Further these gonadal hormones also induced tumor cells to produce tumor growth regulating proteins: VEGF, TGF-β, IL-2, IL-2R, SOCS, Hsp-70 and IFN-γ which in turn either through autocrine action on tumor cells or via TAM-derived NO were observed to regulate tumor cell apoptosis leading to gender dimorphism of tumor growth. This study also discusses the possible mechanism involved. The study has clinical significance as these results will helps in understanding the mechanism of gender dimorphism with respect to the progression of T-cells tumors.     

Postnatal mammary ductal growth: three-dimensional imaging of cell proliferation,effects of estrogen treatment,and expression of steroid receptors in prepubertal calves

Cows may provide insights into mammary development that are not easily obtained using mouse models. Mammary growth in control and estrogen-treated calves was investigated to evaluate general patterns of proliferation and relationship to estrogen receptor (ER) expression. After in vivo labeling with bromodeoxyuridine (BrdU), serial histological sections of mammary tissue were used to generate three-dimensional reconstructions. BrdU-labeled cells were present throughout the highly branched terminal ducts. ER and progesterone receptors (PR) were colocalized in nuclei of ductal epithelial cells. However, basal cells and epithelial cells that were located in the central region of epithelial cords and those that lined the lumen of patent ducts were ER- and PR-negative, as were stromal cells. Cells along the basal portion of the epithelium were not myoepithelial. ER in mammary epithelial cells but not stromal cells is analogous to patterns in human breast but contrasts with localization in murine mammary gland. After estrogen stimulation, 99% of BrdU-labeled (and Ki67-labeled) epithelial cells were ER-negative. Data suggest that proliferation in response to estrogen treatment was initiated within ER-positive epithelial cells of the developing mammary gland and the signal was propagated in paracrine fashion to stromal elements and ER-negative epithelial cells.     

Immunocytochemical Localization of Sex Steroid Hormone Receptors in Normal Human Mammary Gland

The sex steroids, estrogens, progesterone, and androgens, all play a role in mammary development and function. To precisely identify the sites of action of these steroids, we studied the localization of the estrogen receptor α (ERα) and ERβ, the progesterone receptor A (PRA) and PRB, and androgen receptors (AR) in the normal human mammary gland. Immunocytochemical localization of ERα, ERβ, PRA, PRB, and AR was performed with reduction mammoplasty specimens from premenopausal women. ERα, PRA, PRB, and AR were localized mostly to the inner layer of epithelial cells lining acini and intralobular ducts, as well as to myoepithelial cells scattered in the external layer of interlobular ducts. AR was also found in some stromal cells. ERβ staining was more widespread, resulting in epithelial and myoepithelial cells being labeled in acini and ducts as well as stromal cells. These results suggest that all sex steroids can directly act on epithelial cells to modulate development and function of the human mammary gland. Estrogens and androgens can also indirectly influence epithelial cell activity by an action on stromal cells. (J Histochem Cytochem 58:509–515, 2010)     

Effects of intravaginal dehydroepiandrosterone on vaginal histomorphology, sex steroid receptor expression and cell proliferation in the rat

OBJECTIVE: Recent clinical studies have shown that postmenopausal hormone therapy with estrogen plus progestogen increases breast cancer risk. Moreover, intravaginal estrogen-containing pills, creams and rings lead to significant systemic exposure to estrogen, thus indicating the need for a completely novel approach to alleviate vaginal atrophy in postmenopausal women. DESIGN: We have studied the effect of intravaginal application of dehydroepiandrosterone at daily doses of 0.33 mg, 0.66 mg or 1mg in ovariectomized animals for 2 weeks, with the objective of inducing local beneficial effects in the vagina without significant systemic action. RESULTS: After 2 weeks, serum dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol and dehydroepiandrosterone-sulfate were increased over a 4h time period, but serum testosterone, estradiol, estrone and dihydrotestosterone remained below detectable levels. The suppository vehicle alone produced minimal epithelial thickening limited to the vaginal distal half. The morphological effects of dehydroepiandrosterone on vaginal mucosa were observed at the lowest dose and consisted mainly of a typical androgenic effect of epithelial mucification. No change in morphological features related to cell proliferation was observed at any dehydroepiandrosterone dose on uterus, mammary gland and skin. At the highest dose, body weight showed a significant decrease, thus indicating a systemic effect on lipid accumulation. Immunohistochemistry for androgen, estrogen alpha and progesterone receptors did not reveal any significant systemic effects in the uterus, mammary gland and skin except some suggestion of increased androgen receptor labeling in mammary gland and skin at the highest dehydroepiandrosterone dose. CONCLUSION: The present data show that intravaginal dehydroepiandrosterone can exert beneficial effects limited to the vagina.     

Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.