Teresa A. Rose-Hellekant

Prolactin induces ERα-positive and ERα-negative mammary cancer in transgenic mice

The role of prolactin in human breast cancer has been controversial. However, it is now apparent that human mammary epithelial cells can synthesize prolactin endogenously, permitting autocrine/paracrine actions within the mammary gland that are independent of pituitary prolactin. To model this local mammary production of prolactin (PRL), we have generated mice that overexpress prolactin within mammary epithelial cells under the control of a hormonally nonresponsive promoter, neu-related lipocalin (NRL). In each of the two examined NRL-PRL transgenic mouse lineages, female virgin mice display mammary developmental abnormalities, mammary intraepithelial neoplasias, and invasive neoplasms. Prolactin increases proliferation in morphologically normal alveoli and ducts, as well as in lesions. The tumors are of varied histotype, but papillary adenocarcinomas and adenosquamous neoplasms predominate. Neoplasms can be separated into two populations: one is estrogen receptor alpha (ERα) positive (greater than 15% of the cells stain for ERα), and the other is ERα− (<3%). ERα expression does not correlate with tumor histotype, or proliferative or apoptotic indices. These studies provide a mouse model of hormonally dependent breast cancer, and, perhaps most strikingly, a model in which some neoplasms retain ERα, as occurs in the human disease.

Development of in vitro matured/in vitro fertilized bovine embryos into morulae and blastocysts in defined culture media

Abstract The techniques of in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes, with development of the resulting embryos to advanced preimplantation stages in vitro, have gained widespread popularity in recent years because of the potential for obtaining information about regulatory mechanisms, and for inexpensively mass-producing embryos for research or for transfer. However, the efficiency of the techniques (blastocyst yield) is unsatisfactory, due to inadequate information about the requirements of bovine embryos for development in culture, and of oocytes for achieving normal maturation. An experiment was designed to determine effects of using different media for bovine oocyte maturation on subsequent embryo development. Five of seven media tested on oocyte maturation resulted in higher levels of fertilization and/or first cleavage division. In addition, the data indicated that culture conditions for oocyte maturation also affect development to the morula/blastocyst stages. A second experiment evaluated culture conditions for embryo development, using two media (HECM-1 and TCM-199) with or without supplementation by serum and/or oviduct cell conditioning, in a 2×2×2 factorial design. Neither serum supplementation nor conditioning nor the two together increased morula/blastocyst formation (35–46% in all treatments). Serum had a biphasic effect, suppressing the first cleavage division but enhancing morula compaction. No block was observed at the 8- to 16-cell stage with TCM-199 + serum, contrary to many other studies. The data support the hypothesis that the reported stimulation of bovine IVM/IVF embryo development by somatic cell conditioning is due to removal of inhibitory influences from the culture environment. The ability to support development of IVM/IVF embryos to the morula and blastocyst stages in defined media (i.e., without serum or somatic cell conditioning) will help to elucidate the metabolic and nutritional requirements of bovine oocytes and embryos, and to formulated improved culture media. These advances will increase the utility of bovine IVM/IVF for both basic and applied resarch, and for commercial embryo production.

Energy substrates and amino acids provided during in vitro maturation of bovine oocytes alter acquisition of developmental competence.

Energy substrates and amino acids were evaluated for supporting acquisition of developmental competence by bovine cumulus-oocyte complexes during in vitro maturation. The basic culture medium (Basic Medium-3) used for in vitro maturation of oocytes was modified to produce six media containing glucose or glutamine with lactate or pyruvate, or glucose + glutamine, or glucose + 11 amino acids; a seventh (control) medium was TCM199. All media contained polyvinyl alcohol, gonadotropins, epidermal growth factor and oestradiol. Following maturation, oocytes were incubated in medium TALP for fertilisation, then cumulus cells were removed and presumptive embryos cultured for 48 h in a chemically defined medium (HECM-6) followed by 120 h in medium TCM199 + bovine calf serum. Six substrate treatments yielded similar first cleavage responses (66-78%) at 72 h post-insemination; however, blastocyst development at 192 h varied significantly. Oocytes matured in medium with glucose + 11 amino acids gave the best blastocyst development: 21% of inseminated oocytes or 25% of 2-cell embryos. Cumulus expansion in HECM-6 required glucose with either glutamine, 11 amino acids or lactate, or glutamine + lactate. We conclude that (1) the type of energy substrate or nutrient supplied during in vitro maturation of oocytes profoundly affects subsequent developmental competence; (2) oocyte maturation in simple medium containing glucose with lactate or 11 amino acids or glutamine, or lactate + glutamine, can support development equally as well as the complex medium, TCM199; and (3) media supporting at least moderate cumulus expansion during oocyte maturation also support subsequent blastocyst development.

Roles of protein kinase A and C in spontaneous maturation and in forskolin or 3-isobutyl-1-methylxanthine maintained meiotic arrest of bovine oocytes.

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H‐89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP‐stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDDβ) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDDβ followed by cotreatment with forskolin, or (b) with H‐89 or H‐7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H‐89 was added to spontaneously maturing COCs at intervals 0–18 hr to test if H‐89 interfered with the transition between meiosis I and II. The results were as follows: H‐89 interfered with forskolin or IBMX arrested oocytes in a dose‐response manner (IBMX ED50 = 41 μM for COCs; forskolin ED50 = 9 μM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDDβ followed by PDDβ + forskolin: 41–47% germinal vesicle (GV) oocytes; forskolin alone: 90–95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109230x, 85% GV; forskolin, 67–80% GV). Additionally, pretreatment of COCs with H‐89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H‐89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro.

Transforming growth factor alpha- and c-myc-induced mammary carcinogenesis in transgenic mice

The growth factor transforming growth factor alpha (TGFα) and the nuclear transcription factor c-myc often are overexpressed by human breast cancer cells. To produce models of breast disease with these etiologies, mice were generated that carried TGF-α- or c-myc-encoding transgenes. Transgene targeting employed the whey acidic protein (WAP) gene promoter, which is expressed in pregnant and lactating mammary epithelial cells. Non-virgin WAP-TGFα transgenic mice displayed accelerated mammary development during pregnancy, delayed post-parturient mammary involution, a progressive increase in the number of hyperplastic alveolar nodules (HANs), and development of mammary carcinoma with a mean latency of 9 months. Non-virgin WAP-c-myc transgenic mice displayed accelerated mammary gland development during pregnancy and development of mammary carcinomas with a latency of 8 months. Bitransgenic mice carrying both WAP-TGFα and WAP-c-myc displayed a dramatic acceleration of tumor development. These models identify the overexpression of TGFα or c-myc as etiological factors in the development of mammary neoplasia and demonstrate the increased severity of disease when both molecular alterations are present in the same cell.

Strain Background Alters Mammary Gland Lesion Phenotype in Transforming Growth Factor-α Transgenic Mice

Whey acidic protein (WAP)-transforming growth factor (TGF)-alpha transgenic mice acquire both cancerous and noncancerous mammary lesions. For this study, we evaluated the effect of mouse strain background on the incidence, latency, and histotype of two noncancerous lesions, hyperplastic alveolar nodules (analogous to typical hyperplasias in women), and macrocysts. These lesions display characteristics of fibrocystic changes observed in breasts of women, and in both mice and humans are associated with an uncertain risk of progression to neoplasia. Virgin transgenic mice of the (C57BL/6J;SJL)F2 background developed very few hyperplastic alveolar nodules and no macrocysts. In contrast, when the WAP-TGF-alpha transgene was carried on the FVB/N strain, congenic virgin transgenic mice acquired both lesion types with approximately 100% penetrance. In the (FVB;C57BL/6J)F1 background, hyperplastic alveolar nodule incidence was reduced to approximately the nontransgenic mouse level, and macrocyst latency was increased dramatically. Crossing into C57BL/6 resulted in elimination of the macrocyst phenotype. Finally, FVB strain transgenic mammary epithelium transplanted into nontransgenic recipients of the FVB/N or (FVB;C57BL/6J)F1 backgrounds displayed macrocyst latency characteristic of the recipient, and not donor, mouse strain. Quantitative real-time polymerase chain reaction analysis demonstrated that, despite the difference in macrocyst incidence between (FVB;C57BL/6J)F1 and C57BL/6 virgin transgenic mice (81% versus 0%), the level of TGF-alpha expression was not different. FVB strain transgenic mice expressed only twofold more TGF-alpha than the other backgrounds. These findings indicate that C57BL/6J modifier alleles inhibit mammary lesion incidence and macrocyst latency in a semidominant manner, and that suppression of lesion development can involve host factors that are independent of mammary epithelial genotype.

Bioactive polybrominated diphenyl ethers from the marine sponge Dysidea sp.

A new polybrominated diphenyl ether ( 9), together with eight known compounds, were isolated from the crude organic extract of the marine sponge Dysidea sp. collected from the Federated States of Micronesia. Their structures were elucidated on the basis of various NMR spectroscopic data. These compounds exhibited inhibitory activities against Streptomyces 85E in the hyphae formation inhibition (HFI) assay and displayed antiproliferative activities against the human breast adenocarcinoma cancer cell line MCF-7. Compound 6 was selected for further evaluation in a cell cycle progression study.

Estrogen receptor-positive mammary tumorigenesis in TGFα transgenic mice progresses with progesterone receptor loss

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFα) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFα mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/−) p53 mice did not acquire mammary lesions, p53+/− mice carrying the NRL-TGFα transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFα expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR−, and when combined with a heterozygous p53 genotype, ER−/PR−.

Dysregulation of mammary Stats 1, 3 and 5 and PRL receptors by overexpression of TGFα

Mammary TGFalpha overexpression results in delayed involution and eventually mammary cancer in transgenic mice. We hypothesized that STATs and PRL receptors (PRLR), critical regulators of mammary function, are altered in these animals and may contribute to this phenotype. We examined these factors late in the first pregnancy (d.18) and during normal involution (d.4 post-lactation) in WAP-TGFalpha transgenic mice and non-transgenic controls. Long form PRLR mRNA in WAP-TGFalpha glands at both pregnant d.18 and d.4 post-lactation was significantly reduced compared to controls, and PRLR-S3 failed to rise during involution. Total and pTyr STAT 1,3,5a and 5b also were altered. STAT 3 was higher at both times in WAP-TGFalpha glands. STAT 5a and 5b were lower at late pregnancy, but higher post-lactation; however, pTyr(694) STAT 5 was abnormally low at both times. Thus overexpression of TGFalpha has direct or indirect effects on both STATs and PRL responsiveness in vivo, which may reflect mechanisms of TGFalpha-induced mammary epithelial abnormalities.

CALHM1 Deletion in Mice Affects Glossopharyngeal Taste Responses, Food Intake, Body Weight, and Life Span

Stimulation of Type II taste receptor cells (TRCs) with T1R taste receptors causes sweet or umami taste, whereas T2Rs elicit bitter taste. Type II TRCs contain the calcium channel, calcium homeostasis modulator protein 1 (CALHM1), which releases adenosine triphosphate (ATP) transmitter to taste fibers. We have previously demonstrated with chorda tympani nerve recordings and two-bottle preference (TBP) tests that mice with genetically deleted Calhm1 (knockout [KO]) have severely impaired perception of sweet, bitter, and umami compounds, whereas their sour and salty tasting ability is unaltered. Here, we present data from KO mice of effects on glossopharyngeal (NG) nerve responses, TBP, food intake, body weight, and life span. KO mice have no NG response to sweet and a suppressed response to bitter compared with control (wild-type [WT]) mice. KO mice showed some NG response to umami, suggesting that umami taste involves both CALHM1- and non-CALHM1-modulated signals. NG responses to sour and salty were not significantly different between KO and WT mice. Behavioral data conformed in general with the NG data. Adult KO mice consumed less food, weighed significantly less, and lived almost a year longer than WT mice. Taken together, these data demonstrate that sweet taste majorly influences food intake, body weight, and life span.