Immunocytochemical technique for detection of prolactin (PRL) and growth hormone (GH) in hyperplastic and neoplastic lesions of dog prostate and mammary gland.

An immunocytochemical technique was described to test for immunoreactive prolactin (PRL) and growth hormone (GH) in spontaneous and experimentally induced hyperplastic and neoplastic lesions of the prostate and mammary gland. The dog was used as an animal model. The specificity and validity of the immunocytochemical staining procedure and of the antisera to canine PRL and canine GH can be regarded as established for the demonstration of PRL- and GH-dependent staining respectively. In mammary and prostatic tissues, both endogenous PRL and GH as well as intracellular free binding sites (for exogenous PRL and GH) were detected immunocytochemically. The technique presented seems to be an important tool to localize putative target sites for pituitary hormones in hormone-dependent hyperplasia and neoplasia.     

Aging: hypothalamic catecholamines, neuroendocrine-immune interactions, and dietary restriction

The decline in hypothalamic catecholamine (CA) activity with age in rats leads to a reduction in hormone secretion by the neuroendocrine system, and results in decreased reproductive function, a reduction in protein synthesis, development of numerous mammary and pituitary tumors, and probably contributes to the decline in immune function. Some of these same effects can be produced in young rats by administration of drugs that lower hypothalamic CA activity. Administration of drugs to old rats that elevate hypothalamic CA activity can inhibit or reverse the reproductive decline, increase protein synthesis, induce regression of mammary and pituitary tumors, decrease disease incidence, probably elevate immune function, and significantly extend the life span. Therefore, hypothalamic CA have a critical role in the development of aging processes. When young or mature rats or mice are fed a caloric restricted diet, aging processes are inhibited and life span is significantly lengthened. These effects are believed to be mediated primarily via the neuroendocrine system, since calorie restriction results in decreased secretion of hypothalamic, pituitary, and target gland hormones. The decline in hormone secretion leads to a reduction in most body functions, lowers whole body metabolism, and reduces gene expression, and thereby results in a decreased rate of aging of body tissues and longer life. These effects of caloric restriction can be counteracted by administration of hormones, providing evidence that the favorable effects on aging are mediated by reducing hormone secretion.     

Mammary carcinogenesis in different rat strains after irradiation and hormone administration

Radiation carcinogenesis of the rat mammary gland was investigated with the objective of investigating the combined effect of oestrogen administration and irradiation. Three rat strains, Sprague-Dawley, Wistar WAG/Rij and Brown Norway, with different susceptibilities to the induction of mammary cancer, have been irradiated with X-rays and mono-energetic neutrons. Increased hormone levels were obtained by subcutaneous implantation of pellets with oestradiol-17 beta (E2). The tumour incidence results were corrected for competing risks and were analysed with a continuous failure time distribution. The latency period for the hormone-treated animals is considerably shorter than for animals with normal endocrinological levels. Administration of the hormone results in an appreciable increase in the proportion of rats with malignant tumours. At the level of hormone administration applied in this study, radiation and hormones appear to produce an additive effect. The effect of hormone administration and irradiation for mammary tumourigenesis is equal for hormone administration one week prior to, or 12 weeks after irradiation. The RBE values for induction of mammary carcinomas after irradiation with 0.5 MeV neutrons have a maximum value of 20 and are not strongly dependent on the hormone levels.     

Effect of monthly administration of GHRH (fragment 1-29) with osmotic pump on the rat anterior pituitary

Growth Hormone-Releasing Hormone (GHRH) is the main factor, which regulates GH secretion and somatotrope proliferation. However, its chronic effect on anterior pituitary gland is still unknown. It is known that excessive GHRH secretion in patients with gastroenteropancreatic tumors secreting GHRH results in acromegaly and somatotrope hyperplasia. In mice transgenic for GHRH somatotrope tumors develop. Thus, the aim of this paper was to examine the effect of GHRH chronic administration on somatotrope secretion, their percentage and cell proliferation in anterior pituitary gland in rats. The experiment was performed on male Fischer 344 rats weighing 200+/-20 g. The animals were divided into two groups: group I-controls (13 rats) received solvent for GHRH (5% ethanol in demineralized water); group II (10 rats) received GHRH (Growth Hormone Releasing Factor, fragment 1-29 amide) at a dose of 5 microg/day. The substances were given for 1 month via osmotic pump (ALZET), which were implanted subcutaneously in the dorsal region under ketamin anesthesia. After 4 weeks all rats were decapitated and the blood was collected. In the microscopic preparations of anterior pituitary gland the morphology of pituitary (Herlant staining) and the percentage of somatotrope cells and proliferation index based on PCNA staining were assessed. It was found that the chronic treatment with GHRH caused a statistically significant increase in serum rGH concentration and in percentage of somatotropes, but did not change proliferation index and did not induce pathological changes in the morphology of the anterior pituitary gland when compared to the control group. Summing up, monthly GHRH administration did not induce somatotrope adenomas but it caused serum GH level elevation, what seems to depend partially on the increase of somatotrope number.     

Carcinogenicity and hormone studies with the tissue‐selective estrogen receptor modulator bazadoxifene

Bazedoxifene Acetate (BZA) is a selective estrogen receptor modulator (SERM) that is approved for the prevention and/or treatment of osteoporosis in postmenopausal women. To assess for carcinogenic potential, BZA was administered ad libitum in the diet to rats for 2 years. BZA caused an increase in benign ovarian tumors in female rats and decreased incidences of mammary tumors (females) and pituitary tumors (males and females). In addition, BZA provided a significant survival benefit at all dosages tested, which correlated with a significant reduction in pituitary and mammary gland tumors and decreased body weight gain (both genders). Additional studies were subsequently conducted in rats and monkeys to further explore the mechanisms likely responsible for the observed effects. Results from studies in hypophysectomized and chemically castrated female rats indicated that BZA did not directly stimulate formation of ovarian cysts, but an intact pituitary was required for cyst formation. Further, BZA increased estradiol concentrations in rats and monkeys. In monkeys, BZA increased concentrations of luteinizing hormone (LH) after onset of treatment and prohibited the preovulatory surge of LH until after cessation of treatment. These hormonal changes suggest that BZA inhibited both the positive and negative feedback effects of estrogen on gonadotropins and the resulting increase in LH caused formation and persistence of ovarian cysts, which eventually transformed into benign ovarian granulosa cell tumors in the rat carcinogenicity study. These results also suggest that the reductions in pituitary and mammary gland tumors were attributed to BZA‐related antagonism of endogenous estrogens at the estrogen receptors. J. Cell. Physiol. 228: 724–733, 2013. © 2012 Wiley Periodicals, Inc.     

Hormonal activation of mammary gland peroxidase.

Ultrastructural cytochemistry was used to detect an endogenous peroxidase in the rat mammary gland. The enzyme was identified only during the latter half of pregnancy and during lactation, indicating its possible dependence upon hormones. To test this hypothesis, specific hormones associated with the development and differentiation of the mammary gland were used both in vivo and in vitro in an effort to induce, or unmask, the activity of the enzyme. Estrogen injected into nonpregnant rats induced some peroxidase activity in the mammary glands of a few animals. Two hormone combinations tested in organ cultures of mouse mammary gland were able to activate the enzyme: (1) dexamethasone + insulin and (2) dexamethasone + insulin + prolactin.     

Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis

Activating Ras mutations can induce either proliferation or senescence depending on the cellular context. To determine whether Ras activation has context-dependent effects in the mammary gland, we generated doxycycline-inducible transgenic mice that permit Ras activation to be titrated. Low levels of Ras activation - similar to those found in non-transformed mouse tissues expressing endogenous oncogenic Kras2 - stimulate cellular proliferation and mammary epithelial hyperplasias. In contrast, high levels of Ras activation - similar to those found in tumours bearing endogenous Kras2 mutations - induce cellular senescence that is Ink4a-Arf- dependent and irreversible following Ras downregulation. Chronic low-level Ras induction results in tumour formation, but only after the spontaneous upregulation of activated Ras and evasion of senescence checkpoints. Thus, high-level, but not low-level, Ras activation activates tumour suppressor pathways and triggers an irreversible senescent growth arrest in vivo. We suggest a three-stage model for Ras-induced tumorigenesis consisting of an initial activating Ras mutation, overexpression of the activated Ras allele and, finally, evasion of p53-Ink4a-Arf-dependent senescence checkpoints.     

Enhancement of spontaneous mammary tumorigenesis at advanced age in mice by temporary stimulation of mammary growth during youth

As a possible step to clarify the relationship between mammary gland growth during youth and mammary tumorigenesis at advanced age, the long-term effects of the temporary stimulation of mammary gland growth at youth on spontaneous mammary tumorigenesis was studied in a high mammary tumor strain of SHN virgin mice. They received a single pituitary graft under the kidney capsule or a subcutaneous implantation of progesterone for 60 days between 30 and 90 days of age. Both treatments enhanced mammary gland growth and the effect of pituitary grafting was higher. Spontaneous mammary tumorigenesis paralleled mammary gland conditions at youth. The results indicate that the stimulation of mammary gland growth during youth enhances spontaneous mammary tumorigenesis at advanced age in mice, which is quite contrary in rats, and they explain the cause of the higher mammary tumor potential in breeders than in virgin mice.     

Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.     

Reduction of 1-methyl-1-nitrosourea-induced mammary gland carcinoma by in vivo application of immunostimulatory CpG motifs in Sprague-Dawley rats

In the present work the role of 13-cis retinoic acid and CpG oligodeoxynucleotides (CpG-ODN) in a 1-methyl-1-nitrosourea (MNU)-induced mammary gland carcinoma animal model was investigated. Treatment with both components, applied either alone or in combination, induced a significant decrease of the tumour burden and the volume of tumours only in rats that received CpG-ODN (p = 0.046, compared to the MNU control group). The data indicate that the Th-1 biased immunostimulatory capacities of CpG motifs may play a significant role in induction of protective immune responses against mammary gland tumours in Sprague-Dawley rats.     

Histological changes of various organs in aged SD-JCL rats (author's transl)

Age-related histological changes were studied in various organs from SD-JCL rats reared throughout their lifespan. In aged-male rats examined at 5 to 36 months of age were frequently observed nephropathy, periarteritis, skeletal muscle degeneration, pigmentation of the follicular epithelium in the thyroid, fibrosis of the pancreatic islets, hyperplasia of the parathyroid epithelium, and changes of the acini in the extraorbital lacrymal gland. In aged-female rats sinusoid dilatation of the adrenal, and atrophy of the ovary were also noted. Perilobular fat deposition of the liver, dilatation of the gastric gland and severe hemosiderosis of the spleen were observed similar frequency in the both sexes. The nephropathy and cardiovasculopathy were major factors to cause death for males, while the main cause of death for females was tumors, especially of the mammary and pituitary glands. Enlargement of the parathyroid gland, bone resorption and metastatic calcification in the solf tissues were found in rats with severe nephropathy.     

The effect of pituitary homografts on the accessory sex organs and serum hormonal levels with or without androgen in mature male rats

The effect of pituitary homografts on the accessory sex organs and hormonal levels were studied in Wistar mature male rats. Grafted rats were further divided into four experiments: rats were bled once daily via a jugular vein cannula for seven days to investigate when serum prolactin began to rise after transplantation. rats were decapitated on the seventh day after transplantation to test whether 7 days were long enough to show the effect of pituitary grafts on the weight of prostate and seminal vesicles. rats were orchiectomized or orchiectomized and adrenalectomized on the seventh day after transplantation and then decapitated 4 weeks later to test a long term action of pituitary grafts and hormonal levels on the accessory sex organs without androgen. Rats grafted with several pieces of muscle were used as controls in each experiment. The initial rise in serum prolactin level was observed on the fourth day after pituitary transplantation, and then a higher serum prolactin level was maintained thereafter. Despite the higher prolactin level in the pituitary-grafted rat than in the control, no significant differences from the control in the weight of prostates and seminal vesicles and adrenal gland and the concentrations of serum luteinizing hormone (LH) and follicular stimulating hormone (FSH) were measured. This result showed that the weight of accessory sex organs was not affected by a higher serum prolactin within seven days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous pituitary abnormalities and mammary hyperplasia in FVB/NCr mice: implications for mouse modeling

The FVB/N mouse strain is widely used in the generation of transgenic mouse models. We have observed that mammary glands of wild-type virgin female FVB/NCr mice frequently have the morphologic and histologic appearance of a gland during pregnancy. By 13 months of age, the mammary glands of more than 40% of the mice examined had lobuloalveolar hyperplasia that was characterized by the presence of secretory alveoli and distended ducts apparently containing secretory material. The prevalence of this phenotype further increased with age. The mammary phenotype was highly correlated with the presence of proliferative, prolactin-secreting lesions in the pituitary gland. In mice aged 18 to 23 months, hyperplasia of the pars distalis was seen in 11 of 21 mice (52%), and a further 4 of 21 mice (19%) had pituitary adenomas. Pituitary hyperplasia was already evident in some mice as young as nine months. The pituitary phenotype was also associated with high prevalence (4/6 mice) of spontaneous mammary tumors in aged multiparous, but not virgin FVB/NCr mice. This high prevalence of pituitary abnormalities and their effects on the mammary gland have important consequences for the interpretation of new phenotypes generated in transgenic models using this mouse substrain.     

Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription

We investigated the consequences of augmented c-myc gene expression in the mammary gland of transgenic mice. For this purpose we directed the expression of a mouse c-myc transgene to the differentiating mammary epithelial cells by subjecting the protein coding region to the 5' regulatory sequences of the murine whey acidic protein gene (Wap). Analogous to the expression pattern of the endogenous Wap gene, the Wap-myc transgene is abundantly expressed in the mammary gland during lactation. The tissue-specific and hormone-dependent expression of the Wap-myc transgene results in an 80% incidence of mammary adenocarcinomas. As early as two months after the onset of Wap-myc expression, tumours occur in the mammary glands of the transgenic animals. The tumours express not only the Wap-myc transgene, but also the endogenous Wap and beta casein genes. The expression of the milk protein genes becomes independent of the lactogenic hormonal stimuli and persists even in transplanted nude mouse tumours.     

A sparing procedure to clear the mouse mammary fat pad of epithelial components for transplantation analysis

Transplantation of epithelial cells into cleared fat pads is a widely used technique in the study of mammary gland biology. It was first described in 1959 and has remained a valuable technique, most recently in conjunction with the analysis of mammary anlagen from knockout mice with an embryonic lethal phenotype or reproductive defect, and for mammary epithelial stem-cell assays or analysis of precancerous cells. Mammary glands, unlike most other organs, mainly develop postnatally. When the small amount of endogenous epithelium present in the fat pad of a prepubertal mouse is removed, this clearance leaves a natural microenvironment that can be repopulated with exogenously supplied epithelial cells. Cells with the appropriate developmental potential (stem cells or progenitor cells) can regenerate the epithelial portion of the mammary gland after puberty and pregnancy. The conventional clearance of the fat pad is an involved surgical procedure. We have improved the technique and minimized surgery and recovery time, while maintaining an efficient removal of endogenous epithelium from the mammary fat pad.     

Precocious development of UDP-glucuronyltransferase activity in chick-embryo liver after administration of adrenocorticotropic hormone and of certain 11beta-hydroxy corticosteroids.

1. Precocious development of UDP-glucuronyltransferase (EC 2.4.1.17) and of glucuronidation by endogenous compounds of known chemical composition is reported for the first time. 2. This development occurs precociously in chick-embryo liver after administration to the egg of mammalian adrenocorticotropic hormone, of Synacthen (a synthetic compound possessing adrenocorticotropic activity), or of certain corticosteroids possessing a hydroxy or an oxo group at C-11. 3. Corticosterone-dependent transferase development parallels the rise of infused corticosterone in plasma, but does not require the presence of embryo pituitary in ovo, and is demonstrable in embryo liver explants in vitro. 4. Competence of embryo liver transferase to respond to corticosterone (or dexamethasone) begins over days 13-14, the time of competence to respond to grafted pituitary gland. 5. The transferase appearing after treatment with corticosterone or adrenocorticotropic hormone, like that appearing after pituitary grafting or on natural development and unlike that from the untreated embryo, is markedly activated by membrane-perturbation procedures, suggesting it appears through induction, not activation. 6. Thyroxine and tri-iodothyronine accelerate transferase development after treatment with adrenocorticotropic hormone or corticosteroid to the rate seen after pituitary grafting. 7. A wide range of other hormones and steroids did not obviously influence transferase development in this system. 8. We suggest that grafted pituitary gland evokes precocious transferase development in embryo liver through production of adrenocorticotropic hormone and hence of the active corticosteroids; thyrotropin and thyroxine hasten the process. The role of this mechanism in the natural development of UDP-glucuronyltransferase is discussed.     

The beagle dog and contraceptive steroids.

The mandatory use of beagle dogs for chronic toxicological testing of contraceptive steroids has eliminated some useful products from the market, and stopped development of new compounds. Mammary tumour formation in beagle bitches is induced by some progestogens, including progesterone, but not in other laboratory species, such as rodents or monkeys. Most beagle mammary tumours induced by progestogens are of the benign mixed type, though a small minority are malignant. As all the evidence to date suggests that oral contraceptives have a protective effect on the occurrence of breast neoplasms in women, the beagle findings are irrelevant to the human situation. Possible reasons for the unique response of the beagle to progestogens are discussed.     

Effect of insulin and adrenaline on the 59Fe transferrin uptake of lactating mouse mammary gland cells.

The results of insulin action (0.4 IU per mouse) are demonstrated in intact animals only. This action leads to a higher uptake of 59Fe. rabbit transferrin of isolated cells from lactating mouse mammary gland. It is suggested that most inactive transferrin receptors in the cell membrane are incorporated by the hormone action or some new receptors are synthesized. On the contrary, adrenaline in a dose 0.5 micrograms per animal demonstrated an opposite effect--a lower uptake of 59Fe. human transferrin from lactating mouse mammary gland. This is probably due to a redistribution of some part (about 28%) of the iron. Instead of flowing to the mammary gland it flows towards other organs for overcoming the stress situation. An alternative explanation could be the inhibition of endogenous insulin secretion by adrenaline. From our data it follows that insulin and adrenaline have an antagonistic effect on regulation of Fe transport in lactating mouse mammary gland.