Transplantation of a mammary stromal cell line into a mammary fat pad: development of the site-specific in vivo analysis system for mammary stromal cells

The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.     

Transplantation of a Mammary Stromal Cell Line into a Mammary Fat Pad: Development of the Site-Specific in Vivo Analysis System for Mammary Stromal Cells

The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.     

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.¹ and the sparing procedure developed by Brill B et al.², followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn''t occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal³, in the identification of mammary stem cells by transplanting cells in limited dilution^4,5, determining if hyperplastic nodules proceed to mammary tumors⁶, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium^7,8.Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.Download video file.^{(99M, mp4)}     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Biology of mammary fat pad in fetal mouse: capacity to support development of various fetal epithelia in vivo

Epithelia from the lobular part of submandibular salivary gland, glandular stomach, intestine and colon of 14-day C3H/HeN fetuses, and from pituitary gland and pancreas of 12-day fetuses were recombined with 14-day mammary fat pad precursor tissue and syngrafted under the kidney capsule. The normal organogenetic development typical of the epithelium occurred. The same epithelia taken from earlier stage fetuses did not develop normally. Thus, 14-day fetal mouse mammary fat pad precursor tissue has the capacity to support normal organogenesis of various fetal epithelia of developmentally advanced stages. This supportive capacity is decreased in the fat pad precursor tissue of 17- to 18-day fetal mice and is entirely lost postnatally.     

The effect of irradiation on the growth of human laryngeal tumors transplanted under the kidney capsule of immunocompetent mice

A study was made of the influence of gamma-radiation on the growth of human larynx squamous cell carcinoma transplanted under the capsule of the kidney of immunocompetent mice. The transplants were shown to increase in size 6 days after transplantation. Irradiation of animals 24 h after transplantation inhibited considerably the tumor growth. However, the preirradiation (24 h before operation) inhibited the growth of nonirradiated transplants to the same extent as the exposure of mice with the transplanted tumor fragments did: the radiation dose that induced 50% inhibition of the growth was 4.5 Gy and 5.3 Gy, respectively. Preliminary data indicate that tumor fragment of patients with the unfavourable prognosis increase in size and respond to radiation to a lesser extent.     

The proliferation of mouse mammary epithelial cells in response to specific mitogens is modulated by the mammary fat pad in vitro

Summary The ability of the murine mammary fat pad to directly stimulate the growth of mammary epithelial cells and to modulate the effects of various mammogenic agents has been investigated in a newly described, hormone- and serum-free coculture system. COMMA-1D mouse mammary epithelial cells were cultured for 5 or 7 d with various supplements in the absence or presence of epithelium-free mammary fat pad explants from virgin female BALB/c mice. Cocultured fat pad stimulated increases in the DNA content of COMMA-1D cultures by two- to threefold or six-to eightfold after 5 or 7 d, respectively. The mitogenic effect was additive to that of 10% fetal calf serum and could not be attributed to the release of prostaglandin E₂ or synthesis of prostaglandins by epithelial cells. In addition, bovine serum albumin attenuated (P<0.05) the mitogenic effect of cocultured mammary fat pad. Added alone, insulinlike growth factor-I, epidermal growth factor, and insulin increased (P<0.05) total DNA of COMMA-1D cultures by 2.5-, 3.7-, and 2.3-fold, respectively. Cocultured mammary fat pad markedly interacted (P<0.01) with these mitogens to yield final DNA values that were 21.2-, 13.3-, and 22.1-fold greater than in basal medium only. Associated with this proliferation was the formation of numerous domes above the COMMA-1D monolayer. There was no proliferative response to growth hormone or prolactin in the absence or presence of cocultured fat pad (P>0.05). Whereas hydrocortisone did not alter cell number, it attenuated (P<0.05) the mitogenic effect of cocultured mammary fat pad. These results indicate that the murine mammary fat pad is not only a direct source of mitogenic activity, but also modulates the response of mammary epithelial cells to certain mammogens.     

Organ culture passage enhances the oncogenicity of carcinogen-induced hyperplastic mammary nodules

Summary The purpose of this study was to determine whether culturing hyperplastic mammary nodules in hormone-free medium would enhance their oncogenicity following subsequent transplantation into mammary fat pads. The underlying hypothesis is that the proliferation of transformed cells within the nodules is inhibited by hormone-dependent normal cells also present in the nodules. Accordingly, both primary hyperplastic nodules and tissues from a hyperplastic outgrowth of a primary nodule were maintained as organ cultures for varying periods in hormone-free Medium 199. The results show that whereas noncultured nodules developed mammary tumors at an incidence of only 15%, those passaged in organ culture gave rise to mammary tumors at an incidence of 40 to 43%. This threefold enhancement in the oncogenicity of mammary nodules is interpreted to be due, at least in part, to a reduction in the normal mammary cell content of nodules. Consistent with this interpretation is the observation that cultured nodules gave rise to mammary outgrowths that were predominantly hyperplastic, whereas noncultured nodules generated outgrowths with varying proportions of hyperplastic and normal ductal mammary tissue. This investigation was supported by National Cancer Institute Grant CA-17862.     

P190A RhoGAP is required for mammary gland development

P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5 day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Sensitivity and specificity of bioassay of estrogenicity on mammary gland and uterus of female mice

Young intact (18 days of age) and adult ovariectomized (OV-X, ovariectomized between 21 to 24 days of age) C3H/Di mice were used to measure the estrogenicity on the basis of the growth response of mammary epithelial structures and weight of the uterus. The percentage area of the mammary fat pad occupied by mammary epithelial structures was progressively increased by 17beta estradiol from dose 0.001 microg.d(-1). The maximum effective dose of estradiol was 0.01 microg.d(-1) and the dose 10 microg.d(-1) of estradiol decreased mammary size to control levels (inverted-U-shaped dose-response curve). Progesterone alone progressively stimulated mammary growth in young intact females from dose 125 microg.d(-1), in adult OV-X animals from dose 1000 microg.d(-1). Both in young intact and adult OV-X animals, uterine weight progressively increased during estradiol treatment. Progesterone alone had no effect on uterine weight in young intact animals; in adult OV-X animals, uterine weight was increased starting from dose 250 microg.d(-1). Progesterone acted synergistically with estradiol to produce higher mammary growth than that in females treated with estradiol alone. The effects of a combination of estradiol plus progesterone in the mammary gland were mimicked by norethindrone acetate and inhibited by cortisol in both young intact and adult OV-X animals. Testosterone inhibited estradiol plus progesterone stimulated growth of mammary gland only in OV-X animals, but stimulated uterine weights in both young intact and adult OV-X animals. Spleen weight and size of mammary lymph nodes were not affected by estradiol, progesterone, norethindrone acetate or testosterone, but were decreased by cortisol. Cortisol also decreased the percent area of the mammary fat pad occupied by mammary epithelial structures, but had no effect on weight of the uterus. These results show that bioassay of estrogenicity in females is not specific. Mammary and uterine growth is stimulated not only by estrogens but also by progesterone and testosterone, respectively.     

IGF-IR Mediated Mammary Tumorigenesis Is Enhanced during Pubertal Development

Although breast cancer typically develops in women over the age of 40, it remains unclear when breast cancer initiating events occur or whether the mammary gland is particularly susceptible to oncogenic transformation at a particular developmental stage. Using MTB-IGFIR transgenic mice that overexpress type I insulin-like growth factor receptor (IGF-IR) in a doxycycline-inducible manner, mammary tumorigenesis was initiated at different developmental stages. Tumor multiplicity was significantly increased while tumor latency was significantly decreased when the IGF-IR transgene was expressed during pubertal development compared to post-pubertal transgene expression. Moreover, metastatic spread of mammary tumors to the lungs was approximately twice as likely when IGF-IR was overexpressed in pubertal mice compared to post-pubertal mice. In addition, engraftment of pubertal MTB-IGFIR mammary tissue into cleared mammary fat pads of pubertal hosts produced tumors more frequently and faster than engraftment into adult hosts. These experiments show that the mammary microenvironment created during puberty renders mammary epithelial cells particularly susceptible to transformation.     

The mouse mammary gland requires the actin-binding protein gelsolin for proper ductal morphogenesis

Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication.     

An MMP13-selective inhibitor delays primary tumor growth and the onset of tumor-associated osteolytic lesions in experimental models of breast cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.     

An InCytes from MBC Selection: Regulation of Mammary Gland Branching Morphogenesis by EphA2 Receptor Tyrosine Kinase

Eph receptor tyrosine kinases, including EphA2, are expressed in the mammary gland. However, their role in mammary gland development remains poorly understood. Using EphA2-deficient animals, we demonstrate for the first time that EphA2 receptor function is required for mammary epithelial growth and branching morphogenesis. Loss of EphA2 decreased penetration of mammary epithelium into fat pad, reduced epithelial proliferation, and inhibited epithelial branching. These defects appear to be intrinsic to loss of EphA2 in epithelium, as transplantation of EphA2-deficient mammary tissue into wild-type recipient stroma recapitulated these defects. In addition, HGF-induced mammary epithelial branching morphogenesis was significantly reduced in EphA2-deficient cells relative to wild-type cells, which correlated with elevated basal RhoA activity. Moreover, inhibition of ROCK kinase activity in EphA2-deficient mammary epithelium rescued branching defects in primary three-dimensional cultures. These results suggest that EphA2 receptor acts as a positive regulator in mammary gland development, functioning downstream of HGF to regulate branching through inhibition of RhoA. Together, these data demonstrate a positive role for EphA2 during normal mammary epithelial proliferation and branching morphogenesis.     

Influence of dietary macronutrient composition on adiposity and cellularity of different fat depots in Wistar rats

The aim of this study was to investigate the role of dietary macronutrient content on adiposity parameters and adipocyte hypertrophy/hyperplasia in subcutaneous and visceral fat depots from Wistar rats using combined histological and computational approaches. For this purpose, male Wistar rats were distributed into 4 groups and were assigned to different nutritional interventions: Control group (chow diet); high-fat group, HF (60% E from fat); high-fat-sucrose group, HFS (45% E from fat and 17% from sucrose); and high-sucrose group, HS (42% E from sucrose). At day 35, rats were sacrificed, blood was collected, tissues were weighed and fragments of different fat depots were kept for histological analyses with the new softwareAdiposoft. Rats fed with HF, HFS and HS diets increased significantly body weight and total body fat against Control rats, being metabolic impairments more pronounced on HS rats than in the other groups. Cellularity analyses usingAdiposoft revealed that retroperitoneal adipose tissue is histologically different than mesenteric and subcutaneous ones, in relation to bigger adipocytes. The subcutaneous fat pad was the most sensitive to the diet, presenting adipocyte hypertrophy induced by HF diet and adipocyte hyperplasia induced by HS diet. The mesenteric fat pad had a similar but attenuated response in comparison to the subcutaneous adipose tissue, while retroperitoneal fat pad only presented adipocyte hyperplasia induced by the HS diet intake after 35 days of intervention. These findings provide new insights into the role of macronutrients in the development of hyperplastic obesity, which is characterized by the severity of the clinical features. Finally, a new tool for analyzing histological adipose samples is presented.     

Analysis In Vitro of Capacity of Fetal Fat Pad to Support Mammary Gland Embryogenesis

Fourteen-day fetal mammary fat pad precursor tissue (FP) has the capacity to support various fetal epithelia allowing them to accomplish their characteristic development in vivo , without their own mesenchyme (1). This capacity decreases with age of fetal fat pad and is lost postnatally. To analyse the molecular mechanism of such interaction, a method for in vitro duplication of organogenesis is necessary. In the present paper, a co-culture system of fetal epithelium with prospective mammary fat pad is described. The explanted mammary epithelium started budding, then grew out forming branched mammary ducts with end buds. Ultrastructurally, the developing ductal structures exhibited the typical mammary gland morphogenesis.
³H-Thymidine incorportion assessed by autoradiography showed that the mammary gland morphogenesis in vitro was due to the proliferation of epithelial cells, not merely to a change of the shape of the epithelium. This supportive capacity of 14-day FP also decreased with aging; explanted mammary epithelium did not grow into 17-day FP. When insoluble, non-living biomatrix was used in place of living FP the epithelium grew into the matrix but the resulting structures lacked characteristic morphology of epithelium on living fetal FP. The difference of capacity between 14-day and 17-day tissues was also lost.     

Pathway of mesocercariae of Alaria marcianae (Trematoda) through the mammary glands of lactating mice

Lactating mice were infected with mesocercariae of Alaria marcianae to demonstrate more precisely how these parasites migrate within the mammary glands. The infected dam that first transmitted larvae to all of her young was necropsied and her mammary glands removed and sectioned serially. Mesocercariae penetrated the dense connective tissue surrounding the lobules. Within the stroma the larvae migrated along tracks of fat cells and were consistently found in pools of milk created from the destruction of alveoli. These pools of milk led directly into the large lactiferous ducts. It was notable that no mesocercaria was found in the lactiferous or galactiferous ducts indicating that clearance from these vessels was rapid. No larva was found in any blood vessel nor was any significant hemorrhage demonstrable. Lack of an inflammatory response surrounding the worm was characteristic, although large numbers of neutrophils were scattered diffusely throughout the lobules, and multiple, proliferative lymph nodules were present.     

The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.