A quantitative histotopologic analysis of the variation in lobulo-alveolar mitotic activity in the Lewis/Mai rat mammary gland during the estrous cycle

Quantitative microscopic techniques were employed to determine relative mitotic activity of lobulo-alveolar tissue in thick sections obtained from the inguinal mammary glands of colchicine-treated post-pubertal Lewis/Mai rats sacrificed at different phases of the estrous cycle. It was found that the magnitude of lobulo-alveolar cell mitotic activity and the proportion of the lobulo-alveolar tissue with mitotic activity undergo significant fluctuations during the estrous cycle. The frequency distribution of mitotic activity in lobulo-alveolar tissue was different and heterogenous at each phase of the estrous cycle. The frequency distribution of nuclei in lobulo-alveolar tissue during the estrous cycle showed only minor variations and could not explain the variation in mitotic activity. Similarly, no morphological differences were observed in the mammary gland which could account for the variation observed in mitotic activity. It was concluded that the variation in mitotic activity of lobulo-alveolar tissue is probably related to alterations in the generation time of lobulo-alveolar cells imposed by cyclical variation in ovarian hormones during the estrous cycle.     

Effect of neonatal androgenization on hypothalamic biogenic monoamines and pituitary function in rats

Injection of testosterone propionate into female Wistar rats on the 2nd–4th day after birth did not change the serotonin concentration but sharply reduced the noradrenalin and dopamine conconcentrations in the hypothalamus of the animals at the age of 3.5 months. This was accompanied by an increase in the prolactin content in the adenohypophysis despite preservation of normal somatotropic activity. The results of this investigation point to a role of catecholamines in the pathogenesis of anovulatory sterility and give greater precision to modern views on the regulation of pituitary gonadotropic function.Laboratory of Neurohormonal Regulation of Reproduction and Laboratory of Biosynthesis of Protein Hormones, Kiev Research Institute of Endocrinology and Metabolism. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Yudaev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1112–1114, September, 1976.     

Induction and prevention of carcinogen-induced precancerous lesions in mouse mammary gland organ culture

Mouse mammary glands respond to growth promoting hormones in organ culture. In the presence of insulin, prolactin, aldostrone, and hydrocortisone, the glands exhibit extensive proliferation within 10 days of culture mimicking the mammary alveolar structures observed during pregnancy. However withdrawal of prolactin and steroids from the medium for an additional 14 days results in the disintegration of the alveolar structures resembling the mammary morphology observed during the involution stage. During the growth promoting phase if the glands are exposed to 7, 12, dimethylbenz(a)anthracene (DMBA) for 24 hours and cultured through the entire 24 days of culture period, they develop precancerous lesions. This model is highly reproducible and extensively utilized to evaluate efficacy of potential chemopreventive agents against carcinogen-induced mammary lesions.     

Social play soliciting by male and female juvenile rats: effects of neonatal androgenization and sex of cagemates

Male and female juvenile rats were individually exposed to nonplayful juvenile social stimuli in a novel test of play-soliciting behavior to examine hormonal and experiential determinants of sex differences. In Experiment 1, neonatally androgenized females engaged in play soliciting at a level equal to that of male controls and greater than that of nonandrogenized female controls. In Experiment 2, males and females were reared in unisexual and bisexual groups in order to compare long-term sex-related social experience effects on juvenile play soliciting. Males exposed only to other young males engaged in greater play soliciting than males exposed to both sexes; females, in contrast, were unaffected by sex of cagemates. Within rearing conditions, however, males engaged in greater play soliciting than females. The combined results suggest that perinatal gonadal androgen exposure effects on social play are prepotent and contribute essentially to sex differences in the initiation of social play behavior.     

Endocrinologic milieu and susceptibility of the rat mammary gland to carcinogenesis.

The incidence of DMBA-induced mammary carcinomas in Sprague-Dawley rats depends upon their previous reproductive histories. Young virgin rats (YV) are highly susceptible to the carcinogen, while old virgin rats (OV) are less susceptible, and parous rats (P) are resistant. The authors performed endocrinologic studies in these three groups of rats in order to determine whether the different susceptibility to carcinogenesis, according to the reproductive history, is or is not related to the hormonal milieu. The pituitary, the ovaries, the adrenals, and the mammary glands were processed for light microscopy. Pituitary prolactin (PRL), follicle-stimulating hormone, and luteinizing hormone cells were immunostained by peroxidase-antiperoxidase and quantitated with an image analyzer. Radioimmunoassays of serum and pituitary PRL and serum estradiol were also done. The results showed no differences in the hormonal milieu of YV, OV, and P rats at the time of carcinogen treatment. Several changes were observed after DMBA administration, the most conspicuous being 1) hyperplasia of pituitary PRL cells, 2) high serum PRL levels, 3) nodular hyperplasia of the adrenal cortex, 4) high serum estradiol levels, and 5) lack of adrenal necrosis in P rats and some OV rats. These modifications did not correlate with the degree of susceptibility of YV, OV, and P rats to carcinogenesis, supporting the concept of the importance of the mammary gland differentiation at the moment of carcinogen administration. (Am J Pathol 1982, 109:47-56).     

The effects of neonatal androgenization of female hamsters on adult preference for female hamster vaginal discharge

The effects of neonatal exposure of hamsters to androgen on the development of an adult preference for female hamster vaginal discharge (FHVD) were investigated. Three groups treated on Days 2-3 postpartum (females administered testosterone propionate (TP) in a peanut oil base, females administered oil alone, and males administered oil alone) were combined factorially with four sequential adult conditions (intact, post-gonadectomy, post-gonadectomy plus TP therapy, and post-gonadectomy) to produce twelve experimental conditions. Statistical analysis indicated that neonatal oil females did not show a significant FHVD preference in any adult condition. Neonatal TP females displayed a significant FHVD preference only in the post-gonadectomy plus TP condition. Males retained a significant FHVD preference in all the adult conditions, although the preference shown in the two adult hormonal conditions (intact and post-gonadectomy plus TP) was significantly higher than the preference shown in the two nonhormonal conditions.     

Identification and function of neonatal Fc receptor in mammary gland of lactating mice.

In addition to its proposed function in regulating serum IgG levels, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a role in IgG transfer across rodent yolk sac and neonatal intestine. In contrast to humans, for which transplacental transfer of IgG appears to be the only mechanism of maternal IgG delivery, the transmission of IgG in mice occurs both antenatally (yolk sac) and neonatally (transport from mother's milk across intestinal epithelial cells). In the current study, a possible role for FcRn in regulating IgG transfer into milk has been investigated. FcRn has been shown to be present in functional form in the mammary gland of lactating mice, and is localized to the epithelial cells of the acini. Analysis of the transfer of Fc fragments and IgG which have different affinities for FcRn indicate that, unexpectedly, these proteins are transferred in inverse correlation with their binding affinity for FcRn. Thus, in the lactating mammary gland FcRn appears to play a role in recycling IgG in a mode that may have relevance to FcRn trafficking during the maintenance of constant serum IgG levels.     

Overexpression of human Cripto-1 in transgenic mice delays mammary gland development and differentiation and induces mammary tumorigenesis

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.     

Morphometric evaluation of effects of two sex steroids on mammary gland of female rats

OBJECTIVE: The purpose of this study was to evaluate the effects of two sex hormones on normal mammary gland of female rats. METHODS: Forty 250-day-old female rats, 20 of them with offspring and 20 not, were ovariectomized and, divided into 4 subgroups in order to receive one of the following subcutaneous treatment: estradiol benzoate (EB), medroxyprogesterone (MPA), EB+MPA or placebo, for 10 weeks. After treatment, mammary glands were studied with optical microscope. Whole gland, lobule, ductule and lumen compartments were evaluated by morphometric methods. Also a qualitative evaluation were performed seeking for secretion, microcalcification and trophic status. RESULTS: It was found that (a) MPA-only and placebo were similar for all parameters; (b) the same between EB and EB+MPA; (c) EB and EB+MPA increased lobule, ductule and lumen compartments significantly compared to MPA-only or placebo; (d) EB increased epithelium but without significance and EB+MPA increased it significantly compared to placebo or MPA; (e) EB and EB+MPA incremented secretion. CONCLUSIONS: In normal mammary gland of female rats: progestin action depends on estrogen presence. MPA does not revert estrogen-dependent proliferation, but it magnifies estradiol effect. Both EB and EB+MPA stimulate differentiation. Rats without offspring presented a greater epithelial proliferation under treatment with these sex hormones.     

Histamine synthesis by mouse mammary gland epithelial cells in primary culture. The effects of mammary gland differentiation stage (pregnancy and lactation)

Inflammation Research -     

Cocaine-induced effects on isolation stress in neonatal rats.

The effects of cocaine administration on isolation-induced vocalizations and activity levels in 10-day-old rat pups were examined. Day 10 pups given cocaine (1.25, 2.5, 5, 10, & 20 mg/kg ip) vocalized significantly less than their caffeine- (10 mg/kg) and saline-administered siblings during a 5-min isolation period. Cocaine- and caffeine-administered pups also demonstrated a significant increase in overall activity compared with controls. In addition, intraperitoneal administration of the dopamine antagonist haloperidol (0.5 & 1.0 mg/kg) before 1.25 and 2.5 mg/kg cocaine produced a significant elevation in vocalizations compared with saline pretreatment, which indicates a blocking of cocaine's effect on calling behavior. These results suggest that the endogenous dopamine system involved with reinforcement and reward may quell the stress of isolation in the infant rat.     

Maternal behavior in F344/N and LEW/N rats. Effects on carrageenan-induced inflammatory reactivity and body weight

Inbred Fischer (F344/N) and Lewis (LEW/N) rats differ on a myriad of behavioral and physiological endpoints, such as inflammatory, startle and drug responsivity. These differences point to underlying genetic differences between the strains. However, genetic models of hypertension have shown the importance of the maternal environment in the development of high blood pressure, suggesting that maternal influences might also play a role in adult phenotypes of the LEW/N and F344/N strains. This was tested in the present series of experiments in which the effects of crossfostering on carrageenan-induced inflammation and on body weight were examined in the two strains. Following the demonstration that the two strains differed in maternal behavior (Experiment 1), which was independent of the pup being reared (Experiment 2), crossfostered and in-fostered pups from the LEW/N and F344/N strains were injected with carrageenan (at 60 days of age) and subsequently assessed for the accumulation of exudate in response to the injection. Body weights were also monitored from birth through 60 days of age. Although crossfostering affected body weight of the two strains, specifically, reducing weights in LEW/N pups reared by F344/N dams and increasing weights of F344/N pups reared by LEW/N dams, crossfostering did not affect inflammatory reactivity to carrageenan. Specifically, LEW/N pups had a greater level of exudate than F344/N pups, independent of the conditions under which they were reared, suggesting that differences in the inflammatory response between these two strains are under a high degree of genetic control. These results were discussed in terms of genetic factors mediating the early form of immune reactivity induced by carrageenan.     

Effects of dietary equol administration on the mammary gland in ovariectomized Sprague-Dawley rats

OBJECTIVE: To evaluate the effects of dietary equol, a metabolite of soy-derived daidzein or formononetin present in red clover, on the mammary gland of ovariectomized Sprague-Dawley rats. DESIGN: Sixty ovariectomized rats were divided into five groups (n = 12) and fed soy-free chow with the addition of equol (50 mg/kg chow and 400 mg/kg chow) or estradiol-3 benzoate (E2B) (4.3 mg/kg chow and 17.3 mg/kg chow). The control group received soy-free chow only. After 3 months animals were killed, blood was collected, and the mammary glands were removed for histological and immunohistochemical evaluation. RESULTS: Equol and E2B treatment significantly increased serum equol and 17beta-estradiol concentrations, respectively. Serum prolactin in animals treated with high-dose equol was also significantly higher than in the controls. Animals treated with high-dose equol had a significantly higher number of terminal ducts and type II lobules compared with controls. This was also apparent in animals treated with low- and high-dose E2B, but a higher number of type I lobules also was seen. Compared with controls, animals treated with high-dose equol had a significantly higher percentage of proliferating cell nuclear antigen-positive cells in terminal ducts and type II lobules. The percentage of progesterone receptor-positive cells in animals treated with high-dose equol was significantly higher only in type II lobules. In animals treated with low- and high-dose E2B, the percentage of proliferating cell nuclear antigen- and progesterone receptor-positive cells was significantly higher in all the mammary structures. Low-dose equol did not have any effects on the parameters listed above. CONCLUSIONS: High-dose dietary equol administration to ovariectomized rats exerts clear mammotropic effects.     

Excess neonatal thyroxine: effects on learning in infant and adolescent rats.

Previous reports have suggested that neonatal thyroxine administration may enhance learning ability in rats as infants but the rats become deficient in learning as they grow older. This experiment compared neonatally thyroxine-injected rats with injected and nonhanded controls on passive and active avoidance and a geotactic cold-escape discrimination. Thyroxine-injected infant rats were superior to controls on active avoidance, inferior on passive avoidance, and equal on discriminated escape. Adolescent rats which had received postnatal thyroxine were inferior to controls in Lashley III maze acquisition whether or not they had received supplementary thryoxine at the time of training. These data suggest that differences in locomotor activity, rather than in learning ability, account for early behavioral effects and that changes other than endocrine disturbances account for the adult deficits. The results are compatible with previous contentions that excess neonatal thyroxine disrupts the timing of brain development.     

Secretory effects of 5-hydroxytryptamine following neonatal sympathectomy in rat parotid gland.

Unilateral sympathetic denervation of rat parotid glands was performed within 4 h after birth. Nine weeks later the glands were used for in-vitro studies of amylase secretion, and 86Rb+ was used as a marker for potassium efflux. The non-denervated contralateral glands served as controls. The tissue concentrations of 5-hydroxytryptamine and its metabolite 5-hydroxyindole acetic acid were also measured. 5-Hydroxytryptamine caused a significant dose-dependent increase in amylase secretion, which was inhibited by methysergide. There was no difference between controls and denervated glands. 5-Hydroxytryptamine was without effect on potassium efflux from either denervated or control glands. The sympathectomy caused increased levels of 5-hydroxytryptamine and 5-hydroxyindole acetic acid as compared with contralateral controls. The results suggest that 5-hydroxytryptamine influences the two main secretory processes in rat parotid gland differently. A significant amylase discharge was seen following 5-hydroxytryptamine stimulation, whereas no effect was seen on 86Rb+ efflux. Although it is also proposed that there are no 5-hydroxytryptamine-associated nerves in the superior cervical ganglion innervating parotid tissue, it seems that there is a complex connection between the sympathetic pathway and the serotoninergic system.     

Gene expression in fibroadenomas of the rat mammary gland in contrast to spontaneous adenocarcinomas and normal mammary gland.

Fibroadenomas are considered a benign lesion in rodent carcinogenicity studies. However, the entity adenocarcinoma arising in fibroadenoma does exist and in humans there is evidence of certain forms of fibroadenomas to confer greater risk of subsequent breast cancer. In this study, we aim to elucidate the molecular features of both spontaneous fibroadenomas and adenocarcinomas. The gene expression of the two tumour types is examined and compared to mammary gland in the same developmental state and examined for similarities which might indicate common molecular pathways. In the present study no similarities were discovered. We conclude that in the tumours examined here, no progression to adenocarcinoma is likely. Further studies are needed, examining a greater number of tumours and including cases of adenocarcinoma arising in fibroadenoma.