Influence of neonatal diethylstilbestrol treatment on androgen and estrogen receptor levels in the mouse anterior prostate, ventral prostate and seminal vesicle

Perinatal exposure to the synthetic estrogen, diethylstilbestrol (DES), affects the structure of both male and female reproductive systems. Changes may also occur in the levels of steroid hormone receptors. Cytosolic and nuclear androgen and estrogen receptor levels (expressed per mg DNA) from the sex accessory glands of male BALB/c mice exposed neonatally to DES were analyzed by exchange assays. Neonatal DES exposure caused significant decreases in: (1) cytosolic androgen and cytosolic and nuclear estrogen receptor levels in the anterior prostate and (2) cytosolic estrogen receptor levels in the ventral prostate. A significant increase was seen in the cytosolic estrogen receptor levels in the seminal vesicle. Significant decreases in cytosolic protein levels occurred in all DES-exposed glands.     

Overexpression of aromatase in transgenic male mice results in the induction of gynecomastia and other biochemical changes in mammary glands

Our previous studies have shown that overexpression of aromatase in mammary glands results in the induction of hyperplastic and dysplastic changes in female transgenic mice. In this study we show that overexpression of aromatase in male transgenic mice results in increased mammary growth and histopathological changes similar to gynecomastia. Increased estrogenic activity also results in an increase in estrogen and progesterone receptor expression in the mammary glands of transgenic males as compared to the nontransgenic males, as well as an increase in the expression of various genes involved in cell cycle and cell proliferation. We have also observed an increase in certain growth factors, such as bFGF and TGFbeta, as a result of aromatase overexpression in the male transgenic mammary glands. In order to obtain a better understanding of the biological significance of gynecomastia, a reliable model is necessary to explain the mechanisms and correlations associated with human cancers. This model, can potentially serve as a predictable and useful tool for studying gynecomastia, hormonal carcinogenesis and action of other carcinogens on hormone induced cancers.     

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.     

Aromatase overexpression transgenic mice model: cell type specific expression and use of letrozole to abrogate mammary hyperplasia without affecting normal physiology

Our recent studies have shown that overexpression of aromatase results in increased tissue estrogenic activity and induction of hyperplastic and dysplastic lesions in female mammary glands and gynecomastia and testicular cancer in male aromatase transgenic mice. Both aromatase mRNA and protein are overexpressed in transgenic mammary glands and its expression is not limited to epithelial cells. However, it is more in epithelial than in stromal cells. Our results also indicate aromatase overexpression-induced changes in mammary glands can be abrogated with very low concentrations of the aromatase inhibitor, letrozole. Low concentration of letrozole had no effect on normal physiology as indicated by no significant change in the circulating levels of estradiol and follicle stimulating hormone as well as no change in estrogen responsive genes such as the progesterone receptor and lactoferrin in the uterine tissue. These observations indicate that the expression of aromatase in both epithelial and stromal cells can influence the complex interactions of biochemical pathways leading to mammary carcinogenesis and that the aromatase inhibitor, letrozole can be used as chemopreventive agents without affecting normal physiology.     

Region-specific regulation of cytochrome P450 aromatase messenger ribonucleic acid by androgen in brains of male rhesus monkeys

We demonstrated previously that testosterone regulates aromatase activity in the anterior/dorsolateral hypothalamus of male rhesus macaques. To determine the level of the androgen effect, we developed a ribonuclease protection assay to study the effects of testosterone or dihydrotestosterone (DHT) on aromatase (P450(AROM)) mRNA in selected brain areas. Adult male rhesus monkeys were treated with testosterone or DHT. Steroids in serum were quantified by RIA. Fourteen brain regions were analyzed for P450(AROM) mRNA. Significant elevations of its message over controls (P<0.05) were found in the medial preoptic area/anterior hypothalamus of both androgen treatment groups and the medial basal hypothalamus of the testosterone-treated males. Other brain areas were not affected by androgen treatment. We conclude that testosterone and DHT regulate P450(AROM) mRNA in brain regions that mediate reproductive behaviors and gonadotropin release. The P450(AROM) mRNA of other brain areas is not androgen dependent. Brain-derived estrogens may also be important for maintaining neural circuitry in brain areas not related to reproduction. The control of P450(AROM) mRNA in these areas may differ from what we report here, but it is equally important to understand the function of in situ estrogen formation in these areas.     

Roles of androgens in the development, growth, and carcinogenesis of the mammary gland

Androgens influence the development and growth of the mammary gland in women. Treatment of animals and cultured cells with androgens has either inhibitory or stimulatory effects on the proliferation of mammary epithelia and cancer cells; the mechanisms for these dual functions are still not very clear and are discussed in this review. Epidemiological data suggest that, similar to increased estrogens, elevated androgens in serum may be associated with the development of breast cancer. Experiments in rodents have also shown that simultaneous treatment of androgen and estrogen synergizes for mammary gland carcinogenesis. Similar synergistic effects of both hormones have been observed for carcinogenesis of the uterine myometrium of female animals and for carcinogenesis of the prostate and deferens of males. There are also clinical and experimental indications for a possible association of elevated levels of both androgens and estrogens with the development of ovarian and endometrial cancers. A hypothesis is thus proposed that concomitant elevation in both androgens and estrogens may confer a greater risk for tumorigenesis of the mammary gland, and probably other female reproductive tissues than an elevation of each hormone alone.     

Proteome analysis of combined effects of androgen and estrogen on the mouse mammary gland

We and other investigators have previously shown that estrogen and androgen have synergistic effects on the growth of mammary epithelial ducts and alveoli in the Noble mouse. However, the molecular mechanisms behind the synergy are unknown. In the present study, we treated female FVB mice with 17-estrodial (E2) and 5-dihydrotestosterone-bezonate (DHT-B) using slow-releasing hormone pellets for 7 months. Dissection showed that hormone treatment caused atypical hyperplasia of mammary ducts and alveoli. A functional proteomic approach was used to study the holistic protein changes in mammary glands. 2-DE was used to separate proteins. Twenty-five protein spots that were differentially expressed in hormone-treated tissues compared to the control were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF-MS, which include some proteins that are correlative with response to estrogen and androgen stimulation, cells differentiation and growth, signal transduction, metabolism, etc. Real-time RT-PCR was carried out to verify the different expression. These results offered some clues to understand the function of E2 and DHT-B.     

Low sex steroids, high steroid receptors: Increasing the sensitivity of the nonreproductive brain

Male aggressive behavior is generally regulated by testosterone (T). In most temperate breeding males, aggressive behavior is only expressed during the reproductive period. At this time circulating T concentrations, brain steroid receptors, and steroid metabolic enzymes are elevated in many species relative to the nonreproductive period. Many tropical birds, however, display aggressive behavior both during the breeding and the nonbreeding season, but plasma levels of T can remain low throughout the year and show little seasonal fluctuation. Studies on the year-round territorial spotted antbird (Hylophylax n. naevioides) suggest that T nevertheless regulates aggressive behavior in both the breeding and nonbreeding season. We hypothesize that to regulate aggressive behaviors during the nonbreeding season, when T is at its minimum, male spotted antbirds increase brain sensitivity to steroids. This can be achieved by locally up-regulating androgen receptors (ARs), estrogen receptors (ERs), or the enzyme aromatase (AROM) that converts T into estradiol. We therefore compared mRNA expression of AR, ERalpha, and AROM in free- living male spotted antbirds across reproductive and nonreproductive seasons in two brain regions known to regulate both reproductive and aggressive behaviors. mRNA expression of ERalpha in the preoptic area and AR in the nucleus taeniae were elevated in male spotted antbirds during the nonbreeding season when circulating T concentrations were low. This unusual seasonal receptor regulation may represent a means for the year-round regulation of vertebrate aggressive behavior via steroids by increasing the brain's sensitivity to sex steroids during the nonbreeding season.     

Estrogen receptor beta in health and disease

Estrogens, acting through its two receptors, ESR1 (hereafter designated ER alpha) and ESR2 (hereafter designated ER beta), have diverse physiological effects in the reproductive system, bone, cardiovascular system, hematopoiesis, and central and peripheral nervous systems. Mice with inactivated ER alpha, ER beta, or both show a number of interesting phenotypes, including incompletely differentiated epithelium in tissues under steroidal control (prostate, ovary, mammary, and salivary glands) and defective ovulation reminiscent of polycystic ovarian syndrome in humans (in ER beta-/- mice), and obesity, insulin resistance, and complete infertility (both in male and female ER alpha-/- mice). Estrogen agonists and antagonists are frequently prescribed drugs with indications that include postmenopausal syndrome (agonists) and breast cancer (antagonists). Because the two estrogen receptors (ERs) have different physiological functions and have ligand binding pockets that differ enough to be selective in their ligand binding, opportunities now exist for development of novel ER subtype-specific selective-ER modulators.     

The effects of aromatase overexpression on mammary growth and gene expression in the aromatase x transforming growth factor alpha double transgenic mice

The transforming growth factor alpha (TGF) and its receptor (EGFR) are expressed in many breast cancers. Typically, the progression of estrogen dependent primary breast cancers into a hormone-independent state, due to the loss of the estrogen receptor, is associated with increased levels of TGF and EGFR, leading to aggressive breast carcinomas. The relationship between breast tumorigenesis and TGF is evident in the transgenic mice overexpressing TGF in the mammary glands. In the aromatase transgenic mice, the mammary glands exhibit preneoplastic developments but do not form frank tumors. To test the interactions between growth factor overexpression with tissue estrogen, we have crossed the aromatase transgenic mice with the TGF transgenic mice to produce a double transgenic strain. The histological data for the mammary glands of aromatase x TGF double transgenic mice show that these mice develop hyperplastic changes similar to the aromatase parental strain but no tumors are formed. Consistently, the expression of cyclin D1 and PCNA is diminished in the double transgenic strain as compared to the parental strains. In addition, the expression of TGF, EGF and EGFR are also decreased in the double transgenic strain, suggesting that continuous estrogen presence in the tissue due to aromatase overexpression downregulates the expression of EGFR and its ligands.     

Seasonal changes of reproductive behavior and fecal steroid concentrations in Père David's deer.

From July 1997 to September 1997 and from March 1998 to July 1998, we studied reproductive behaviors of Père David's deer in Dafeng, China. During the field behavioral observations, we collected fresh voided fecal samples from the Père David's stags and hinds periodically and kept those samples under -20 degrees C until laboratory analysis. We analyzed the fecal testosterone, estradiol, and progesterone concentrations in those samples using radioimmunoassay. During this study, we also recorded 17 types of male reproductive behaviors and nine types of female reproductive behaviors. Reproductive behaviors and the fecal steroid concentrations showed overt seasonal fluctuations. There were statistically significant correlations between some male reproductive behaviors, such as anogenital sniffing, urine sniffing, urine spraying, wallowing, bellowing, antler adorning, Antler swags mud, chasing, herding hinds, chin resting, mounting and copulating, with the fecal testosterone concentrations. These results suggested that seasonal reproductive behaviors in stags are strongly associated with circulating testosterone. We also found that some female reproductive behaviors fluctuate corresponding with changes in fecal estrogen concentrations. Although there was no direct evidence to confirm the correlations between female reproductive behaviors and fecal estrogen in our experiment, we could not rule out that reproductive activities of hinds were largely related to ovarian estrogen secretion, and estrogen is necessary for inducing female reproductive behaviors.     

Cyclic nucleotides and protein phosphorylation in mouse mammary glands: effects of estrogen and progesterone administered in vivo

Ovariectomized mice were injected daily for 20 days with saline, 17 beta-estradiol (1 microgram/day), progesterone (1 mg/day), or estrogen + progesterone. Mammary glands were removed, homogenized, and analyzed for DNA, cAMP, cGMP, cAMP-dependent protein kinase (kinase A), cGMP-dependent protein kinase (kinase G), tyrosyl kinase (kinase T), and epidermal growth factor-stimulated tyrosyl kinase (EGF-T). Estrogen and progesterone, administered singly, increased DNA, cAMP, kinase A, kinase T, and EGF-T. In addition, progesterone, administered alone or with estrogen, decreased kinase G activity. cGMP concentrations were not altered by estrogen or progesterone. No evidence of a synergism between estrogen and progesterone on the levels of the cyclic nucleotides and the activities of kinase enzyme was observed, although an additive effect of these steroids was seen. These data indicate that ovarian steroid-induced growth of mouse mammary glands is accompanied by significant changes in protein phosphorylation, i.e., increased cAMP-dependent protein phosphorylation and tyrosyl phosphorylation and decreased cGMP-dependent protein phosphorylation.     

Development of mammary hyperplasia and neoplasia in MMTV-TGF alpha transgenic mice

To study the role of transforming growth factor alpha (TGF alpha) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGF alpha cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGF alpha in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenic mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGF alpha overexpression in salivary glands and reproductive organs. TGF alpha can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma.     

Protracted reproductive seasonality in the male giant panda (Ailuropoda melanoleuca) reflected by patterns in androgen profiles, ejaculate characteristics, and selected behaviors

The female giant panda (Ailuropoda melanoleuca) experiences a brief (24-72 h) seasonal estrus, occurring once annually in spring (February-May). Our aim was to determine the existence and temporal profile of reproductive seasonality in the male of this species. The study was facilitated by 3 yr of access to eight giant panda males living in a large breeding center in China. Seasonal periods for the male were defined on the basis of female reproductive activity as prebreeding, breeding (early, peak, late), and nonbreeding seasons. Testes size, fecal androgen excretion, ejaculated sperm density, and frequency of reproductive behaviors (i.e., locomotion, scent marking, vocalizations) increased (P < 0.05) from the prebreeding period (October 1-January 31) to the early breeding season (February 1-March 21). Testes volume and sperm concentration were maximal from March 22 through April 15, a period coinciding with maximal female breeding activity. The occurrence of male reproductive behaviors and fecal androgen concentrations began declining during peak breeding and continued from April 16 through May 31 (late breeding period), returning to nadir throughout the nonbreeding interval (June 1-September 30). Reproductive quiescence throughout the latter period was associated with basal testes size/volume and aspermic ejaculates. Our results reveal that testes morphometry, fecal androgen excretion, seminal quality, and certain behaviors integrated together clearly demonstrate reproductive seasonality in the male giant panda. The coordinated increases in testes size, androgen production, sperm density, and sexual behaviors occur over a protracted interval, likely to prepare for and then accommodate a brief, unpredictable female estrus.     

Sex-associated differences in the regulation of immune responses controlled by the MHC of the mouse

The immunologic potential of T lymphocytes and antigen-presenting cells (APC) from male and female mice were compared. Lymphocytes from female mice or from male mice that cannot produce and respond to testosterone (Tfm/y) were more reactive than male lymphocytes to alloantigens in MLR. Spleen cells from Tfm/y mice equipped with estrogen implants showed a higher responsiveness than control Tfm/y to alloantigens. The removal of suppressive adherent cells or the addition of T cell growth factor (TCGF) enhanced the proliferative activity of the cells in the MLR. The responsiveness of female cells to alloantigens, however, remained superior to that observed in male cells. Similarly, in the presence of TCGF, thymocytes from female mice react more effectively than male cells in MLR. In addition, Con A-stimulated spleen cells from female mice produce more interleukin 2 (IL 2) than do spleen cells from males or female mice treated with testosterone. Lymphocytes from immunized mice were tested for their ability to respond to soluble antigens (KLH and OVA) in vitro. Again, female immunocompetent cells respond more vigorously than male cells or cells originating in female mice with testosterone implants. APC from female spleen were more efficient than male APC in initiating a secondary response in primed lymphocytes from either males or female mice. Moreover, castration of male mice enhanced, and treatment of female mice with androgen reduced, the efficiency of antigen presentation. In conclusion, these data suggest that female cells are superior to male cells in immunologic functions that are known to be associated with reactions to and recognition of histocompatibility antigens, i.e., antigen presentation and MLR. Furthermore, our present data indicate that the differential reactivity of immunocytes between male and female mice depends on the hormonal balance of the animal.     

Effects of treatment of male and female rats in infancy with mifepristone on reproductive function in adulthood

Treatment of neonatal male and female rats with mifepristone (1 mg s.c. every 2 days from Day 1 to 15 or Day 4 to 18 of life) interfered with the normal development of their reproductive capacities and of the adrenal glands. The effect on the adrenal glands seemed only temporary. Effects on reproductive functions seemed permanent. Female rats developed abnormalities in structure of the oviduct and ovarian capsule reminiscent of effects reported after perinatal treatment with androgen or oestrogen. During adulthood, anovulatory polyfollicular ovaries developed, reminiscent of rats treated with a small dose of androgen in infancy. Males showed retardation of testicular growth and delay of puberty. During adulthood testes did not grow beyond 65% that of normal rats. Sexual behaviour was deficient in that ejaculations occurred only rarely; when ejaculations did occur, fertility was unimpaired. Males treated with mifepristone in infancy exhibited female sexual behaviour as adults after castration and injections of testosterone and oestradiol. All effects pointed to an insufficient action of testicular hormones in infancy to bring about normal 'masculinization'. Mifepristone therefore appears to show 'teratogenic' actions in rats which affect female reproductive tract development and, in males and females, development of the systems underlying normal reproductive activity and functions in adulthood.     

Estrogen and adiposity--utilizing models of aromatase deficiency to explore the relationship

Estrogen has an important role to play in energy homeostasis in both men and mice. Lack of estrogen results in the development of a metabolic syndrome in humans and rodents, including excess adiposity, hepatic steatosis (in male but not female aromatase knockout (ArKO) mice) and insulin resistance. Estrogen replacement results in a prompt reversal of the energy imbalance symptoms associated with estrogen deficiency. A corollary to the perturbed energy balance observed in the ArKO mouse is the death by apoptosis of dopaminergic neurons in the hypothalamic arcuate nucleus of male ArKO mice, an area of the brain pivotal to the regulation of energy uptake, storage, and mobilisation. An extension of our work exploring the relationship between estrogen and adiposity has been to examine the role played by androgens in energy balance. We have demonstrated that an increased androgen to estrogen ratio can promote visceral fat accumulation in the rodent by inhibiting AMPK activation and stimulating lipogenesis. Therefore, understanding the regulation of energy homeostasis is becoming an increasingly fascinating challenge, as the number of contributors, their communications, and the complexity of their interactions, involved in the preservation of this equilibrium continues to increase. Models of aromatase deficiency, both naturally occurring and engineered, will continue to provide valuable insights into energy homeostasis.     

Nafoxidine administered to newborn female GR mice arrests the development of their mammary glands

Mice of the GR strain develop many hormone-dependent mammary tumors in response to estrogen and progesterone stimulation. Since this strain is so sensitive to steroid hormones, we administered a single dose of the antiestrogen Nafoxidine to female GR mice within 24 hours after their birth. This treatment arrested the development of their mammary glands and when the mice were adults, 10 weeks old, they did not cycle normally but were in a state of persistent estrus. Whole mounts of mammary glands from Nafoxidine-treated mice revealed cystic areas within some ducts and bulbous swellings at the ends of others. No hyperplastic alveolar nodules (HAN) were identified in the glands. In contrast, a single dose of 17 beta estradiol administered within 24 h after birth, resulted in a highly branched gland displaying typical end buds, a few alveoli and more HAN than were observed in glands of control adult mice of the same strain. Thus Nafoxidine treatment not only arrested the development of the mammary glands in female GR mice (causing them to appear "masculinized") but it also produced abnormalities within the glands.