Studies of mitochondrial development prior to lactogenesis in the mouse mammary gland

Studies were performed to define mitochondrial development in relation to epithelial cell proliferation and differentiation prior to lactogenesis in the mammary gland of the mouse. Gland weight, total DNA, and total protein, selected as criteria of gland growth and proliferation, and various parameters of mitochondrial development were followed throughout the period. Gland weight and total DNA started increasing about Day 5 of pregnancy and reached maximal values by parturition. Total gland protein began to increase at the same time but did not reach maximal values until Day 4 of lactation. Total mitochondrial protein and the mitochondrial marker enzyme activities, succinate oxidase, succinate-linked ATP formation, and cytochrome oxidase increased gradually during pregnancy with rapid 2- to 3-fold increases occurring during the early days of lactation. Similarly, succinate oxidase activity per unit DNA of isolated mammary parenchymal cells increased gradually from mid-pregnancy to parturition with a precipitous, 2-fold increase occurring during early lactation.     

Characterization of WWOX inactivation in murine mammary gland development

The WW domain‐containing oxidoreductase (WWOX) is commonly inactivated in multiple human cancers, including breast cancer. Wwox null mice die prematurely precluding adult tumor analysis. Nevertheless, aging Wwox‐heterozygous mice at C3H genetic background develop higher incidence of mammary tumors. We recently generated a Wwox conditional knockout mouse in which loxp sites flank exon 1 in the Wwox allele and showed that total ablation of WWOX in these mice resembles that of conventional targeting of Wwox. Here, we report the characterization of WWOX ablation in mouse mammary gland using MMTV‐Cre transgenic line. We demonstrated that WWOX ablation leads to impaired mammary ductal growth. Moreover, targeted deletion of WWOX is associated with increased levels of fibronectin, a component of the extracellular matrix. In addition, we showed that shRNA knockdown of WWOX in MCF10A breast epithelial cells dramatically increased fibronectin and is associated with enhanced cell survival and impaired growth in three‐dimensional culture Matrigel assay. Taken together our results are consistent with a critical role for WWOX in normal breast development and tumorigenesis. J. Cell. Physiol. 228: 1391–1396, 2013. © 2012 Wiley Periodicals, Inc.     

Diet-induced obesity severely impairs myelinated aortic baroreceptor reflex responses

Diet-induced obesity (DIO) attenuates the arterial cardiac baroreceptor reflex, but the mechanisms and sites of action are unknown. This study tested the hypothesis that DIO impairs central aortic baroreceptor pathways. Normal chow control (CON) and high-fat-chow obesity-resistant (OR) and obesity-prone (OP) rats were anesthetized (inactin, 120 mg/kg) and underwent sinoaortic denervation. The central end of the aortic depressor nerve (ADN) was electrically stimulated to generate frequency-dependent baroreflex curves (5-100 Hz) during selective activation of myelinated (A-fiber) or combined (A- and C-fiber) ADN baroreceptors. A mild stimulus (1 V) that activates only A-fiber ADN baroreceptors induced robust, frequency-dependent depressor and bradycardic responses in CON and OR rats, but these responses were completely abolished in OP rats. Maximal activation of A fibers (3 V) elicited frequency-dependent reflexes in all groups, but a dramatic deficit was still present in OP rats. Activation of all ADN baroreceptors (20 V) evoked even larger reflex responses. Depressor responses were nearly identical among groups, but OP rats still exhibited attenuated bradycardia. In separate groups of rats, the reduced heart rate (HR) response to maximal activation of ADN A fibers (3 V) persisted in OP rats following pharmacological blockade of β(1)-adrenergic or muscarinic receptors, suggesting deficits in both parasympathetic nervous system (PNS) and sympathetic nervous system (SNS) reflex pathways. However, the bradycardic responses to direct efferent vagal stimulation were similar among groups. Taken together, our data suggest that DIO severely impairs the central processing of myelinated aortic baroreceptor control of HR, including both PNS and SNS components.     

Diet-induced obesity impairs endothelium-derived hyperpolarization via altered potassium channel signaling mechanisms

Background

The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study.

Methodology/Principal Findings

Membrane potential, vessel diameter and luminal pressure were recorded in 4^th order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16–20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK_Ca/IK_Ca) inhibition; with such activity being impaired in obesity. SK_Ca-IK_Ca activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK_Ca-mediated EDH contribution was increased in obesity, and associated with altered IK_Ca distribution and elevated expression. In contrast, the SK_Ca-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K_ir) and Na⁺/K⁺-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K_ir expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density.

Conclusion/Significance

In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK_Ca and Na⁺/K⁺-ATPase, and decreased K_ir underlie changes in the EDH mechanism.     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

Genomic analysis of early murine mammary gland development using novel probe-level algorithms

We describe a novel algorithm (ChipStat) for detecting gene-expression changes utilizing probe-level comparisons of replicate Affymetrix oligonucleotide microarray data. A combined detection approach is shown to yield greater sensitivity than a number of widely used methodologies including SAM, dChip and logit-T. Using this approach, we identify alterations in functional pathways during murine neonatal-pubertal mammary development that include the coordinate upregulation of major urinary proteins and the downregulation of loci exhibiting reciprocal imprinting.     

Manganese superoxide dismutase is dispensable for post-natal development and lactation in the murine mammary gland

Abstract

Mammary gland development is a multistage process requiring tightly regulated spatial and temporal signalling pathways. Many of these pathways have been shown to be sensitive to oxidative stress. Understanding that the loss of manganese superoxide dismutase (Sod2) leads to increased cellular oxidative stress, and that the loss or silencing of this enzyme has been implicated in numerous pathologies including those of the mammary gland, we sought to examine the role of Sod2 in mammary gland development and function in situ in the mouse mammary gland. Using Cre-recombination driven by the mouse mammary tumor virus (MMTV) promoter, we created a mammary-specific post-natal conditional Sod2 knock-out mouse model. Surprisingly, while substantial decreases in Sod2 were noted throughout both virgin and lactating adult mammary glands, no significant changes in developmental structures either pre- or post-pregnancy were observed histologically. Moreover, mothers lacking mammary gland expression of Sod2 were able to sustain equal numbers of litters, equal pups per litter, and equal pup weights as were control animals. Overall, our results demonstrate that loss of Sod2 expression is not universally toxic to all cell types and that excess mitochondrial superoxide can apparently be tolerated during the development and function of post-natal mammary glands.     

Exosome signaling in mammary gland development and cancer

Exosomes are 40-100 nm intraluminal vesicles that are released by cells upon fusion of multivesicular endosomes (MVEs) with the plasma membrane. The Rab family of small GTPases, including Rab27A and Rab27B, control different steps of exosome release, including transport of MVEs and docking at the plasma membrane. Exosomes are long range message particles that mediate communication between cells in physiological conditions such as mammary gland development and lactation, but also in pathology such as breast cancer. Metastasis is the culmination of cancer progression and involves a complex interaction with the local and distant environment. Exosome messaging contributes to tumor environment interactions such as immune escape, thrombosis and myofibroblast differentiation, thereby modulating metastatic niche preparation.     

Signaling pathways in mammary gland development.

Unlike most other organs, development of the mammary gland occurs predominantly after birth, under the control of steroid and peptide hormones. Once the gland is established, cycles of proliferation, functional differentiation, and death of alveolar epithelium occur repeatedly with each pregnancy. Although it is unique in this respect, the signaling pathways utilized by the gland are shared with other cell types, and have been tailored to meet the needs of this secretory tissue. Here we discuss the signaling pathways that have been adopted by the mammary gland for its own purposes, and the functions they perform.     

Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation.

Endogenous mannary gland peroxidase in acinar cells of prelactating and lactating rats is revealed in tannic acid-formaldehyde-glutaraldehyde-fixed tissue by means of the standard diaminobenzidine procedure. Diaminobenzidine cytochemical reaction product is present in perinuclear cisternae, in the granular endoplasmic reticulum and in Golgi apparatus of functionally differentiated secretory cells. The mammary gland peroxidase is thought to represent lactoperoxidase. Peroxidase staining is diminished or absent in acinar cells of hypophysectomized and ovariectomized rats, in normal rats during early pregnancy and in nonpregnant mature females. Endogenous peroxidase or a heme protein with peroxidatic activity may be considered an ultracytochemical marker enzyme for acinar cells actively engaged in lactogenesis.