Thoracic Mammary Glands Research Articles

The development of the terminal end bud in the prepubertal-pubertal mouse mammary gland

The mammary gland is one of the few organ systems in mammals that completes its morphologic development postnatally. Due to this unique developmental property, it serves as an excellent model for studying ductal morphogenesis beyond embryonic life. The primary objective of this study was to investigate the development of terminal end bud (TEB) structure in the prepubertal-pubertal mouse mammary gland. Seventy-five mice, weaned at 21 days of age, were fed a high fat, nonrestricted intake diet for optimum mammary gland growth. Whole-mount preparations and a computer-assisted image analyzer were used to record the size and number of TEB structures in the thoracic and inguinal mammary glands. Alight microscope was used to determine the number of cap cells present in select TEB structures. In the prepubertal age group, the size and number (No.) of TEBs in the No. 2 and No. 3 thoracic glands were significantly greater than those in the No. 4 inguinal gland. In the pubertal age group, the size and number of TEBs in the No. 2 gland significantly decreased, indicating that for this particular gland the largest size and greatest number of TEBs are present during the prepubertal growth period. In contrast, the size and number of TEBs in the No. 3 and No. 4 glands remained relatively constant throughout the prepubertal-pubertal growth period. The results of this study show: (1) like the rat mammary gland, there is asynchronous growth among the mouse mammary glands, (2) the maximum size and number of TEBs in the prepubertal-pubertal mouse mammary glands are a function of the gland of origin and the age of the mouse, and (3) the number of cap cells per TEB structure is directly correlated with the size of the TEB structure.

Uso da fluorsceína na identificação dos vasos linfáticos superficiais das glândulas mamárias em cadelas

Uma técnica empregando fluoresceína sódica 5% e luz ultravioleta foi desenvolvida para a identificação dos trajetos dos vasos linfáticos superficiais das glândulas mamárias em dez cadelas. A fluoresceína foi injetada por via intradérmica ao redor da base de cada mamilo e cada mama foi observada após 5, 15, 30 e 60 minutos. Em cada animal, primeiramente, foram avaliadas as mamas torácica cranial, abdominal cranial e inguinal das cadeias direita e esquerda e 48 horas após as mamas torácica caudal e abdominal caudal das cadeias direita e esquerda. De um total de 97 mamas injetadas, em 8 a fluoresceína não foi captada pelos linfáticos. O tempo de 30 minutos foi o mais adequado para a visualização total dos trajetos. Mostrou ser um método simples, rápido e inócuo de verificação in vivo de vasos linfáticos.

Topographie des Collecteurs Lymphatiques Mammaires de la Chienne

SummaryOn the topography of the lymph drainage from the mammary glands of the bitchThe topography of the lymph drainage was examined by in vivo injections of india ink into the mammary parenchyma of 73 female dogs. Only one gland was injected at a time on each side. Only the cranial abdominal mammary gland had direct drainage to the axillary as well as to the superficial inguinal nodes. The principal routes of drainage differ for each gland. The glands on each side are completely independent as regards their lymph drainage. The cranial thoracic mammary glands on each side had their own collecting duct which led to the sternal lymph nodes. The results of this study suggest to develop a new model for the surgical extirpation of mammary tumors in the bitch.

Differential characteristics of the thoracic and abdominal mammary glands from mice

The anatomical and physiological characteristics of thoracic and abdominal mammary glands were investigated in order to understand why the incidence of mammary tumors is higher in the former. Epithelium in explants from both sets of glands required DNA synthesis, insulin, cortisol, and prolactin for full differentiation as measured by α-lactalbumin accumulation. The temporal pattern and magnitude of response were the same with respect to both DNA synthesis and differentiation; however, the epithelium in explants from the thoracic glands required concentrations of hormones for α-lactalbumin accumulation only one-half to one-third those from abdominal glands. Tumor distribution did not appear to correlate with mammary gland histology, size, or epithelial content.

Influence of Melatonin on Mammary Gland Growth: In Vivo and In Vitro Studies

Our objective was to determine whether melatonin influenced mammary growth in response to mammogenic hormones. Prepubertal female BALB/c mice were injected for 9 days with 1 microgram of 17 beta-estradiol and 1 mg of progesterone or 17 beta-estradiol/progesterone plus 50, 100, or 200 micrograms of melatonin. Area of the parenchyma and total DNA content of the second thoracic gland were similar between controls and melatonin-injected mice. However, micrograms of DNA/100 mg of mammary tissue were lower in animals treated with 17 beta-estradiol/progesterone plus 200 micrograms of melatonin than in controls. Triglyceride content of mammary glands from animals treated with 100 or 200 micrograms of melatonin/day increased relative to controls. In an in vitro experiment, thoracic mammary glands of 21-day-old mice were cultured for 6 days in a mammogenic milieu of hormones (17 beta-estradiol/progesterone, aldosterone, bovine prolactin, growth hormone, and insulin) with 0 (control), 10(-6), 10(-9), or 10(-12) M melatonin. Relative to controls, 10(-12) M melatonin increased and 10(-6) M melatonin decreased mammary DNA and uptake of [methyl-3H]thymidine. We conclude that high doses of melatonin reduce mammary development in normal mice and that some of this effect may be mediated directly at the mammary tissue.

Estrogen and Progesterone Augment Growth Responsiveness of Mammary Tissue to Cholera Toxin

Second (thoracic) mammary glands of endocrine intact mice were removed intact and incubated in Dulbecco's Modified Eagle's Medium supplemented with insulin, aldosterone, and cholera toxin. Insulin and aldosterone resulted in relatively little mammary development. However, insulin, aldosterone, and cholera toxin substantially increased mammary development, as assessed by development scores and DNA after 6 d of culture. Ovariectomy abolished the ability of cholera toxin to augment mammary development in vitro. Estradiol and progesterone injections for 3 d partly restored responsiveness of mammary tissue to cholera toxin, whereas responsiveness was greater after 6 d of injection than in endocrine-intact mice. Additionally, cyclic AMP-dependent protein kinase (kinase A) activity of fourth (inguinal) mammae was increased after as little as 3 d of estradiol and progesterone treatment. Cholera toxin induced phosphorylation of at least one protein was also increased by estradiol and progesterone. Because cholera toxin is a potent activator of adenylate cyclase, these findings suggest that estradiol and progesterone interact with cyclic AMP active agents to promote mammary development. This interaction may be mediated, at least in part, by increased kinase A activity and increased kinase A substrate availability.

Actions of oestrogens and antioestrogens on rat mammary gland development: Relevance to breast cancer prevention

The proliferative actions of a series of antioestrogens on the development of the second thoracic mammary gland of ovariectomized immature Sprague-Dawley rats have been investigated. Evidence is presented that shows trans-tamoxifen, LY 117018 and LY 139481, like oestradiol-17β and cis-tamoxifen, promote full mammary gland ductal development and induce a high rate of cell proliferation in the undifferentiated epithelial cells of the terminal end buds, the main growth region for ductual growth. Conversely, ICI 164,384, a new antioestrogen, is without effect on ductal elongation. In vivo exposure of trans-tamoxifen and LY 117018 treated glands in medically castrated animals to the carcinogen DMBA, results in a high rate of mammary tumour development. Indeed, the actions of these so-called antioestrogens are equivalent to those observed in oestradiol-treated rats.

Release of catecholamines follows suckling or electrical stimulation of mammary nerve in lactating rats.

The hypothesis that catecholamines may be released by mammary gland stimulation during lactation was tested by measuring, with an HPLC electrochemical method, plasma epinephrine (E) and norepinephrine (NE) concentrations during suckling in conscious rats and during electrical stimulation (pulses: 1 msec duration, 10/sec at 5-30 V) of the central end of a cut abdominal mammary nerve in urethane-anesthetized rats. Plasma E and NE concentrations were significantly elevated in two different strains of rats (Wistar and Holtzman) within 5 min of suckling. The concentration of E and NE did not change in control unsuckled rats during the same time period. As a complementary indication of sympathetic activation, it was observed that piloerection occurred during suckling. Plasma E levels (but not NE levels) increased significantly within 30 sec of a 2-min period of nerve stimulation in lactating rats on either day 7 or day 21 of lactation, as well as in nonlactating rats. The effect was significantly greater in nonlactating rats. The levels of E and NE were not altered after sham stimulation, whereas adrenalectomy abolished the rise in plasma E after mammary nerve stimulation. Blockade of the rise in plasma E also occurred after rapid injection of 100 microliters milk intraductally into each of two thoracic mammary glands, 15 sec before the onset of mammary nerve stimulation. These results show that E and NE can be released in response to suckling, and that activation of ductal mechanoreceptors may inhibit such release. These mechanisms may operate to regulate the rate of milk removal during suckling in the rat.

Simultaneous occurrence of pregnancylike lobuloalveolar morphogenesis and casein-gene expression in a culture of the whole mammary gland.

Entire second thoracic mammary glands of estrogen- and progesterone-treated immature virgin BALB/c mice were stimulated to pregnancylike lobuloalveolar morphogenesis after 6 days of incubation with insulin (5 micrograms/ml), aldosterone (1 micrograms/ml), growth hormone (5 micrograms/ml), cortisol (5 micrograms/ml), and prolactin (80 ng/ml, present as a contaminant in 5 micrograms/ml growth hormone). The alveolar growth in the glands, as judged by morphological studies, was accompanied by an increase in cell number as a function of incubation time in the hormonal medium. Hybridization of the total RNA from these glands to the casein mRNA specific complementary DNA probe (cDNAcsn) revealed that the level of casein mRNA rises from 0.00012 to 0.005% between 1 and 6 days of incubation. Estimates showed that the concentration of casein mRNA per cell rises 17-fold from 70 molecules on Day 1 to 1200 molecules on Day 6, whereas the number of epithelial cells increases only twofold during the same incubation time. When the growth hormone preparation was totally replaced by 80 ng of prolactin during the 6-day incubation, casein-mRNA levels were found to be 0.0083%. These results demonstrate that a pregnancy-like morphogenesis and concurrent expression of the casein gene in vitro can be achieved in a controlled hormone environment containing high cortisol and low prolactin concentrations. This one-step mammogenesis-lactogenesis culture model should be useful for studying the mechanisms of hormonal regulation of casein-gene expression observed in prepartum mammary gland in vivo.

Absolute requirement of glucocorticoid for expression of the casein gene in the presence of prolactin.

Second thoracic mammary glands of immature BALB/c female mice were stimulated to pregnancy-like lobuloalveolar (LA) development aftr 6 days of incubation in a corticosteroid-free step I culture medium containing insulin, prolactin, estradiol, progesterone, and growth hormone. A low basal level (0.0009%) of casein mRNA (mRNAcsn) sequences was detectable in the LA glands by a specific cDNA probe. Subsequent incubation of the LA glands for 3 days in medium containing insulin and prolactin or insulin and cortisol failed to elicit mRNAcsn above the basal level, indicating that neither prolactin nor cortisol alone can support casein gene expression. However, an increase in mRNAcsn levels was observed when the 3-day incubation with insulin and cortisol or insulin and prolactin was followed by 3 days of culture in presence of insulin, prolactin, and cortisol. When a 3-day incubation with insulin and prolactin was followed by 3 days in insulin and cortisol medium, mRNAcsn levels in the gland remained similar to the basal level. However, a 20-fold increase in the mRNAcsn levels ensued when the LA glands were sequentially incubated for 3 days in insulin and cortisol and then for another 3 days in insulin and prolactin medium. After a preincubation in insulin and cortisol medium, the LA glands retained residual cortisol during subsequent incubation in insulin and prolactin medium, and the mRNAcsn levels in these glands were related to the level of residual cortisol present. When mRNAcsn and the residual cortisol level reached a minimum, addition of fresh cortisol to the medium caused a 20-fold increase in the mRNAcsn levels. This indicates that cortisol is a limiting factor in insulin and prolactin medium and its presence is absolutely required for casein gene expression.

Alterations in the Oxytocin-Induced Intramammary Pressure Response after Mechanical Stimulation of the Mammary Gland of the Anesthetized Lactating Rat*

Rhythmic, manual compression applied for 10–15 sec to two thoracic mammary glands of the urethane-anesthetized rat resulted in an immediate and significant reduction in amplitude of the intrammary pressure (IMP) response in abdominal glands induced by iv injected oxytocin. The period of reduced responsiveness to oxytocin lasted 13 min on the average; then the IMP responses gradually returned over the next few minutes to prestimulus heights. This invariably was followed by a phase of increased responsiveness to oxytocin lasting several minutes, during which the amplitude progressively increased to a maximum which was significantly above that seen before manual compression. A similar pattern of depression, recovery, and increased responsiveness of the IMP response to a given dose of oxytocin occurred if a single iv injection of 5 μg epinephrine was given instead of mammary compression. The IMP response was not reduced by manual compression of the thoracic glands if 1) the rat was pretreated with the Β-adren...

Hormone-inducible casein messenger RNA in a serum-free organ culture of whole mammary gland

The whole second thoracic mammary gland of estradiol-17beta + progesterone primed 3- to 4-week-old BALB/c female mice was induced to pregnancy-like lobulo-alveolar morphogenesis after 6-day cultivation in a serum-free culture medium containing a "growth promoting" hormone mixture, insulin + prolactin + growth hormone (somatotropin) + estradiol + progesterone. No radioimmunologically detectable casein was present in these glands. Subsequent cultivation for another 6 days in a "lactogenic" medium with the hormones insulin + prolactin + cortisol produced abundant milk-like secretory material in the alveolar lumen. RNA of the mammary gland after estradiol + progesterone priming or cultivation in the "growth-promoting" medium failed to show a measurable amount of casein mRNA activity when assayed in a cell-free protein synthesis system derived from Ehrlich ascites ribosomes, rabbit reticulocyte factors, and tRNA. However, the glands sequentially cultivated in the "growth-promoting" and the "lactogenic" media showed a high level of casein mRNA activity in the heterologous cell-free protein synthesis system. Sodium dodecyl sulfate/polyacrylamide gel electrophoretic characteristics of the immunoprecipitable (by antibody to mouse milk casein) polypeptides directed by the mammary RNA induced in organ culture medium containing the lactogenic hormones were similar to the characteristics of the polypeptides directed by mammary polysomes of lactating mice. These results demonstrate hormonal induction of a specific mRNA in a sequential two-step culture of an entire organ in a serum-free chemically defined medium.

Action of growth-promoting hormones on macromolecular biosynthesis during lobulo-alveolar development of the entire mammary gland in organ culture.

The entire 2nd thoracic mammary gland of the immature virgin BALB/c mouse was stimulated to full lobulo-alveolar (LA) growth after 120 h organ culture in hormone supplemented medium. The minimal hormonal combination required was insulin (I) + prolactin (Prl) + aldosterone (A). The corticosteroid was replaceable by oestradiol-17beta (E) + progesterone (P). The combination I alone or I + the steroid hormone(s) failed to induce the LA development and similar results were also evident in presence of Prl + the steroids. Incubation of the glands in medium with I + Prl + A activated a sequential rise of RNA, protein and DNA synthesis. A near maximal increase of RNA synthesis was present at 48 h in the medium with I + Prl, addition of the steroid hormones did not show further stimulatory effect. Supplementation of the medium with I + Prl and the adrenal or the ovarian steroids was needed for maximal activation of protein synthesis at 72 h and DNA replication at 96 h. The medium with I alone did not show a substantial rise of macromolecular biosynthesis in the mammary gland in organ culture. The highest level of DNA polymerase activity was observed at 72 h in glands cultivated in medium with I + Prl and A or E + P. Only a modest increase of DNA polymerase activity was present in glands cultivated with I alone or I + Prl. Prior treatment of the glands (cultivated with I + Prl + A) with actinomycin D or puromycin resulted into 44 and 40% reduction of DNA polymerase activity suggesting hormone-induced synthesis of the enzyme before the rise of DNA synthesis in the mammary cells at 96 h in organ culture. Significance of these results with respect to the action of the "growth-promoting" hormones in the mammary gland in organ culture and in the animal has been discussed.

Hormonal regulation of lobulo-alveolar growth, functional differentiation and regression of whole mouse mammary gland in organ culture.

The entire second thoracic mammary glands of 4-week-old BALB/c female mice primed with oestradiol plus progesterone were cultivate in organ culture medium containing the "growth-promoting" hormone combinations: insulin, prolactin, growth hormone, oestradiol, progesterone and aldosterone or insulin, prolactin and aldosterone. Full lobulo-alveolar development was induced after 5-6 days of incubation and could be maintained for 15-16 days in organ culture in medium containing either hormone combination. After the initial 5-6 days in the "growth-promoting" medium, subsequent cultivation of the glands in a medium with the "lactogenic hormones", insulin, prolactin plus cortisol, led to accumulation of "milk-like" secretory material in the ductal and alveolar lumina. Incubation of the lobulo-alveolar gland in medium with insulin alone for 7-9 days resulted in complete regression of the alveoli leaving only a ductal parenchyma. Incubation in insulin, prolactin, growth hormone or insulin plus the steriod hormones for 7-9 days led to considerable alveolar degeneration without a complete regression. The results indicate that both pituitary and steroid hormones are essential for development and maintenance of mammary alveoli; insulin can only sustain the basal ductal structure.

The estrogen-stimulation of mammary growth in ovariectomized immature mice and its inhibition by steroids.

The mammary growth following the estrogen injection into the ovariectomized immature mice of DS colony was studied using the ductal extension of the third thoracic mammary gland as a criterion. A rapid growth of the gland was achieved by the daily subcutaneous application of estradiol as little as 0.03μg a day for 9days after ovariectomy. A variety of steroids were subcutaneously injected daily along with the estrogen treatment for 9days. 5α-Androstan-17β-ol-3-one and its 2-substituents, dromostanolone propionate, oxymetholone and 17α-methyl dromostanolone, strongly inhibited the estrogen-stimulated mammary duct growth. The reduction of 3-one and/or oxidation of 17β-ol in 5α-androstan-17β-ol-3-one resulted in marked decrease of the inhibitory effect. The introduction of double bond at ring A slightly modified the activity of the parent androstane. On the other hand, potent estrogen antagonists on the uterus, such as 19-nortestosterone, norethisteron, progesterone and MER-25 were almost inactive. These results indicate that there is no direct correlation between the mammary growth inhibiting ability and the androgenic activity or anti-uterotropic activity of a steroid. The biological significance of these estrogen-steroid interactions on the mammary gland development was discussed.

Direct action of testosterone propionate on the rat mammary gland.

ABSTRACT Testosterone propionate in arachis oil was applied locally to the skin over the third left thoracic mammary gland (= experimental gland) of castrated male and female rats. Arachis oil was applied to the third right thoracic mammary gland (= control gland). After 21–23 days of local applications the development of the experimental and control glands was studied on whole mount preparations. It was found that small doses of testosterone (0.15 mg daily) stimulated slight but definite lobule-alveolar development in the experimental glands, while the control glands did not show any alveoli. Applications of higher doses of testosterone propionate (0.30 and 0.75 mg daily) stimulated extensive lobule-alveolar development in the experimental glands, while only few alveoli were produced in the control glands. It may therefore be concluded that the effect of testosterone on the rat mammary gland is not mediated through other endocrine glands, but is a direct, local effect on the mammary gland structures. The result is discussed in relation to our present knowledge of the hormonal control of different growth processes within the mammary glands.

Hybrid Vigor and Variation of Mammary Growth Response and Body Growth in F<SUB>1</SUB> Hybrid between Inbred Strains of Mice

Hybrid vigor and variability in F1 hybrid on the traits of the mammary growth response to estrogen and the body weight in mice were studied on 4 inbred strains and F1 hybrid between them. Strains bc and C (undergone full-sib mating for mcre than 25 generations) and KA and KB mice (undergone full-sib mation gor 12 generations), which have been selected for mammary growth response, were used in this study. All tested mice were recieved total dose of 12.5 I.U. of synthetic estrogen from 20 days of age. They were killed at 35 days and their third thoracic mammary glands were removed from the skin to prepare them by the whole mount method. The mammary growth response was expressed by the mammary duct area. Body weight were measured at birth, 12 days, 20 days and 35 days of age. In these characters, the potence ration ((F1-Pm)/|P-Pm|) of F1 hybrid, which indicated an effect of heterosis, were calculated. The presence of maternal influence was examined. Variations of body weight and mammary growth response were compared between inbred strains and F1 hybrid between them. The results obtained are briefly summarized in folllowing: (1)The potence rations of F1 hybrids were more than 1 in moset cases, and accordingly high heterosis in these characters seemde to be present in F1 hybrids. On the body weight, the values were the smallest at 12 days and thereafter increased gradually with advancing stages. (2)In mammary growth response the potence ratio of F1 hybrid between unselected strain × sekected was the smallest in both sexes. But the value of mammary growth response of selected × selected was the highest, and that of selected × unselected was next and that of unselected × unselected was the lowest. (3)Maternal influence on body weight was significant at 5% level at 35 days of age only. Proportion of the magnitude to total variance was considerably large at birth, and thereafter decrease with advancing stages. That of mammary growth response was significant at 5% level in female but not in male. (4)In body weight at any stage and mammary growth resoinse, F1 hybrid proved to be more unifrom than inbred strains. (5)Cariability of mammary growth response in F1 hybrid of selected × selected was the most unifrom, that of unselected × was next and that of unselected × unselected was the largest in both sexes. (6)Close relationship was seen between mammary growth response and body weight at 35 days of age within each inbred strain or F1 hybreid, but no relationship was seen inter inbred strain groups and inter F1 hybred proups in both sexes. A suggestion with regard to obtaining animals with the smaller phenotypic variablity for a given trait, was put forward.

Establishment of mice strains with special reference to mammary growth response. 1. Comparison of several measuring methods of mammary duct areas in immature mice treated with estrogen.

Present study was carried out to obtain simple and considerably accuratemethod for measuring mammary duct areas in mice treated with estrogen.Normal and gonadectomized Kasukabe mice in both sexes were injected onfive occasions subcutaneously with total dose of 12.5 I.U.of synthetic estrogenfrom 20 days old.On the 35th day mice were killed and the third thoracicmammary glands were prepared by whole mount method.Preparations wereenlarged on section paper and such four methods as A) Index of mammaryduct area method, B) Planimeter method, C) Calipers method-1, and D) Calipersmethod-2 were used to compare the properties of four measuring methods.Results obtained are as follows.1.Planimeter method is comparatively reliable among B), C) and D) method.2.There are close associations between mammary duct areas measured byA) and B) method.Correlation coefficients are 0.85 in female, 0.95 in castratedfemale, 0.94 in male and 0.77 in castrated male.3.The differences of mammary duct areas between normal and gonadectomizedgroup are not significant in each sex but the difference between sexes issignificant statistically.When normal and gonadectomized group's data in eachsex are combined, means with fiducial limits by B) method are 175.3±4.6 infemale and 179.8±8.2 in male when the mammary duct areas measured by A) method in each sex are placed as standards respectively.