High-precision targeting workflow for volume electron microscopy.

Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)-SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

Cell influx and contractile actomyosin force drive mammary bud growth and invagination.

The mammary gland develops from the surface ectoderm during embryogenesis and proceeds through morphological phases defined as placode, hillock, bud, and bulb stages followed by branching morphogenesis. During this early morphogenesis, the mammary bud undergoes an invagination process where the thickened bud initially protrudes above the surface epithelium and then transforms to a bulb and sinks into the underlying mesenchyme. The signaling pathways regulating the early morphogenetic steps have been identified to some extent, but the underlying cellular mechanisms remain ill defined. Here, we use 3D and 4D confocal microscopy to show that the early growth of the mammary rudiment is accomplished by migration-driven cell influx, with minor contributions of cell hypertrophy and proliferation. We delineate a hitherto undescribed invagination mechanism driven by thin, elongated keratinocytes-ring cells-that form a contractile rim around the mammary bud and likely exert force via the actomyosin network. Furthermore, we show that conditional deletion of nonmuscle myosin IIA (NMIIA) impairs invagination, resulting in abnormal mammary bud shape.

Expression of Grainyhead-like 2 in the Process of Ductal Development of Mouse Mammary Gland.

Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial-mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone-dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts.

Exposure to oxidized soybean oil induces mammary mitochondrial injury in lactating rats and alters the intestinal barrier function of progeny.

Similar to other food contaminants, dietary oxidized soybean oil (OSO) is also a toxic xenobiotic for animal and human nutrition. This research evaluated the effects of maternal OSO exposure during lactation on mammary mitochondrial injury and intestinal barrier of sucking progeny. Twenty-four female adult SD rats were fed a fresh soybean oil (FSO) homozygous diet (7%) or an OSO homozygous diet (7%) during lactation. On day 21 of lactation, upregulated mRNA expression of Sirt3 and PRDX3 and downregulated mRNA expression of Mfn2 were observed in mammary tissues in the OSO group compared to the control group (P < 0.05). Maternal OSO consumption increased the FasL transcriptional level in the mammary glands of rat dams (P < 0.05), while the mRNA expression of Bax, Bcl-2, Caspase3, and Fas was not different from that in the control group (P > 0.05). OSO enhanced the Nrf2 transcriptional level and decreased the expression of Keap1 and PPARα in mammary tissues (P < 0.05). In addition, the contents of CAT, MDA, SOD were not affected by dietary OSO (P > 0.05), while the concentration of H2O2 was significantly decreased in the OSO-treated mammary glands of rat dams (P < 0.05). Maternal OSO exposure during lactation did not affect the organ coefficients of pups (P > 0.05). However, maternal OSO consumption influenced the intestinal tight junction protein expression of progeny (P < 0.05). In summary, the present study demonstrated that dietary OSO may aggravate mammary injury and mitochondria dysfunction, but the OSO-induced damage was self-alleviating via the promotion of Sirt3 and PRDX3 expression and further scavenging of oxidative products.

Gambogic acid alleviates inflammation and apoptosis and protects the blood-milk barrier in mastitis induced by LPS.

Mastitis is one of the most common diseases among dairy cows. There is still much debate worldwide as to whether antibiotic therapy should be given to dairy cows, or if natural products should be taken as a substitute for antibacterial therapy. As the antibiotic treatment leads to the bacterial resistance and drug residue in milk, introducing natural products for mastitis is becoming a trend. This study investigates the mechanisms of the protective effects of the natural product gambogic acid (GA) in lipopolysaccharide (LPS)-induced mastitis. For in vitro treatments, it was found that GA reduced IL-6, TNF-α, and IL-1ß levels by inhibiting the phosphorylation of proteins in the nuclear factor κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathway. GA also maintained a stable membrane mitochondrial potential and inhibited the overproduction of reactive oxygen species, which protected the cells from apoptosis. On the other hand, in vivo treatments with GA were found to reduce pathological symptoms markedly, and protected the blood-milk barrier from damage induced by LPS. The results demonstrate that GA plays a vital role in suppressing inflammation, alleviating the apoptosis effect, and protecting the blood-milk barrier in mastitis induced by LPS. Thus, these results suggest that the natural product GA plays a potential role in mastitis treatment.

Electron Tomography Revels that Milk Lipids Originate from Endoplasmic Reticulum Domains with Novel Structural Features.

Lipid droplets (LD) are dynamically-regulated organelles that originate from the endoplasmic reticulum (ER), and function in the storage, trafficking and metabolism of neutral lipids. In mammary epithelial cells (MEC) of lactating animals, intact LD are secreted intact into milk to form milk lipids by a novel apocrine mechanism. The secretion of intact LD and the relatively large amounts of lipid secreted by lactating MEC increase demands on the cellular processes responsible for lipid synthesis and LD formation. As yet these processes are poorly defined due to limited understanding of LD-ER interactions. To overcome these limitations, we used rapid-freezing and freeze-substitution methods in conjunction with 3D electron tomography and high resolution immunolocalization to define interactions between LD with ER in MEC of pregnant and lactating rats. Using these approaches, we identified distinct ER domains that contribute to lipid droplet formation and stabilization and which possess unique features previously unrecognized or not fully appreciated. Our results show nascent lipid droplets within the ER lumen and the association of both forming and mature droplets with structurally unique regions of ER cisternae, characterized by the presence of perilipin-2, a protein implicated in lipid droplet formation, and enzymes involved in lipid synthesis. These data demonstrate that milk lipids originate from LD-ER domains with novel structural features and suggest a mechanism for initial droplet formation in the ER lumen and subsequent maturation of the droplets in association with ER cisternae.

In utero exposure to commercial artificial sweeteners affects mice development and mammary gland structure.

Commercial artificial sweeteners present in the market are usually made of combination of nutritive and artificial sweeteners such as sorbitol and aspartame. The aim of this research was to study the effect of in utero exposure to commercial artificial sweeteners on the mouse development and on mammary gland in different stages (18-day embryos and 4-week-old mice). Pregnant mice of treated groups were given 50 mg/kg body weight of commercial artificial sweetener. The dose was given on day 1 of pregnancy until 3-week nursing, while the controls were given distilled water. Congenital malformations were seen in treated 18-day fetus and 4-week-old mice, such as a significant decrease in the diameter of the placenta and the weight of the fetuses, while in 4-week-old mice, a significant decrease in the length of the body, limbs, and tail was seen compared to the controls. The result of this study showed that in 18-day fetuses, clusters of mammary gland in the treated mice seemed to be more differentiated than the controls. In 4-week-old mice, the number of mammary gland ducts in the treated group was significantly more than the control group, and the lumen of the ducts in the treated sections seemed to be narrower than the controls, also many regressing terminal end buds (TEBs) were seen in the treated group. A significant increase in the mammary gland area of treated group was seen compared to the controls.

Mammary-specific ablation of Cyp24a1 inhibits development, reduces proliferation and increases sensitivity to vitamin D.

Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24 A1), and provides local control of 1,25(OH)2D bioactivity. Several reports of the association between elevated CYP24 A1 activity and breast cancer incidence, suggest that CYP24 A1 may be a target for therapeutic intervention. Whether CYP24 A1 activity within the mammary epithelium regulates 1,25(OH)2D levels and mammary gland development is yet to shown. We have used a conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis at early pregnancy, by inhibiting proliferation but not apoptosis in both basal and luminal MECs. In vitro study showed increased sensitivity of luminal MECs to lower levels of 1,25(OH)2D with the ablation of Cyp24a1 activity. In summary, Cyp24a1 within MECs plays an important role in modulating postnatal and pregnancy-associated mammary gland development which provides support for inhibiting CYP24 A1 as a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.

Expression of iron-related proteins in feline and canine mammary gland reveals unexpected accumulation of iron.

Dysregulation of cellular iron homeostasis in human breast cancer is reflected by the altered expression of regulatory proteins. The expressions of iron-related proteins in the mammary glands of cats and dogs have not been assessed. We evaluated the expressions of ferritin, ferroportin, hepcidin and transferrin receptor 1 in benign and malignant mammary gland lesions in cats and dogs. Iron deposition was detected using Perls' Prussian blue staining. We found no major differences in the expression of iron-related proteins between benign and malignant mammary gland lesions in either cats or dogs; however, these species exhibited accumulation of iron in benign lesions. Our findings provide an explanation for the absence of higher iron requirements by tumor cells in these animals. Further investigation of local iron homeostasis in cats and dogs and differences in their physiology compared to human breast cancer is required.

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

Processing and characterization of canine mixed mammary tumor using transmission electron microscopy.

Canine mammary gland tumors represent the second most frequent type of neoplasm in dogs, being an important problem within veterinary medical field. Canine mixed mammary tumors are the most common; the use of a transmission electron microscope (TEM) can contribute as a tool in its diagnosis by determining the characteristics of cellular components from numerous neoplasms. The aim of this study was to characterize cytologically canine mammary mixed tumor by the use of the TEM. A biopsy collected from an 11 years old bitch Shih-Tzu and analyzed by histopathology was used for ultrastructural analysis. Specimens obtained were double stained using uranyl acetate and lead citrate prior to observation in the TEM. The protocol established to transmission electron microscopy observation allowed the identification of main cellular characteristics of canine mixed mammary tumors; however, it was not possible a detailed visualization of the organelles due to the preservation of the biopsy in formaldehyde.

Understanding the Inguinal Sinus in Sheep (Ovis aries)-Morphology, Secretion, and Expression of Progesterone, Estrogens, and Prolactin Receptors.

Post-parturient behavior of mammalian females is essential for early parent-offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

Mammary alveolar epithelial cells convert to brown adipocytes in post-lactating mice.

During pregnancy and lactation, subcutaneous white adipocytes in the mouse mammary gland transdifferentiate reversibly to milk-secreting epithelial cells. In this study, we demonstrate by transmission electron microscopy that in the post-lactating mammary gland interscapular multilocular adipocytes found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X-Gal-stained and uncoupling protein 1-positive interscapular multilocular adipocytes. These data suggest that during mammary gland involution some milk-secreting epithelial cells in the anterior subcutaneous depot may transdifferentiate to brown adipocytes, highlighting a hitherto unappreciated feature of mouse adipose organ plasticity.

Hyaluronan modulates growth factor induced mammary gland branching in a size dependent manner.

Mammary gland morphogenesis begins during fetal development but expansion of the mammary tree occurs postnatally in response to hormones, growth factors and extracellular matrix. Hyaluronan (HA) is an extracellular matrix polysaccharide that has been shown to modulate growth factor-induced branching in culture. Neither the physiological relevance of HA to mammary gland morphogenesis nor the role that HA receptors play in these responses are currently well understood. We show that HA synthase (HAS2) is expressed in both ductal epithelia and stromal cells but HA primarily accumulates in the stroma. HA accumulation and expression of the HA receptors CD44 and RHAMM are highest during gestation when gland remodeling, lateral branch infilling and lobulo-alveoli formation is active. Molecular weight analyses show that approximately 98% of HA at all stages of morphogenesis is >300kDa. Low levels of 7-114kDa HA fragments are also detected and in particular the accumulation of 7-21kDa HA fragments are significantly higher during gestation than other morphogenetic stages (p<0.05). Using these in vivo results as a guide, in culture analyses of mammary epithelial cell lines (EpH4 and NMuMG) were performed to determine the roles of high molecular weight, 7-21kDa (10kDa MWavg) and HA receptors in EGF-induced branching morphogenesis. Results of these assays show that while HA synthesis is required for branching and 10kDa HA fragments strongly stimulate branching, the activity of HA decreases with increasing molecular weight and 500kDa HA strongly inhibits this morphogenetic process. The response to 10kDa HA requires RHAMM function and genetic deletion of RHAMM transiently blunts lateral branching in vivo. Collectively, these results reveal distinct roles for HA polymer size in modulating growth factor induced mammary gland branching and implicates these polymers in both the expansion and sculpting of the mammary tree during gestation.

[NUCLEAR STRUCTURE IN THE SECRETORY CELLS OF MAMMARY GLANDS IN LACTATING AND NON-LACTATING RATS].

The features of structural and functional organization of the main nuclear compartments and distribution of their key molecular components (chromatin-remodeling protein ATRX, RNA polymerase I and II, and the splicing factor SC35) has been studied in the nuclei of mammary gland cells at different functional states. No significant differences between the nuclei of the cells in the lactating and non-lactating mammary glands have been revealed at the ultrastructural level. At the same time, photometric analysis has revealed higher intensity of nucleoplasmic immunofluorescent staining of mammary glands in the lactating animals when antibodies against the proteins ATRX and SC35 were used. Apparently, this observation reflects the changes of the structural and functional status of chromatin as well as the redistribution of splicing factors between the sites of their deposition and transcription.

Isolation of Endoplasmic Reticulum Fractions from Mammary Epithelial Tissue.

In the mammary glands of lactating animals, the mammary epithelial cells that surround the lumen of the acini produce and secrete copious amounts of milk. Functional differentiation of these mammary epithelial cells depends on the development of high-efficiency secretory pathways, notably for protein and lipid secretion. Protein secretion is a fundamental process common to all animal cells that involves a subset of cellular organelles, including the endoplasmic reticulum and the Golgi apparatus. In contrast, en masse secretion of triglycerides and cholesterol esters in the form of milk fat globules is a unique feature of the mammary epithelial cell. Cytoplasmic lipid droplets, the intracellular precursors of milk fat globules, originate from the endoplasmic reticulum, as do most milk-specific proteins. This organelle is therefore pivotal in the biogenesis of milk components. Fractionation of the cell into its subcellular parts is an approach that has proven very powerful for understanding organelle function and for studying the specific role of an organelle in a given cell activity. Here we describe a method for the purification of both smooth and rough microsomes, the membrane-bound endoplasmic reticulum fragments that form from endoplasmic reticulum domains when cells are broken up, from mammary gland tissue at lactation.

Essential role for a novel population of binucleated mammary epithelial cells in lactation.

The mammary gland represents a unique tissue to study organogenesis as it predominantly develops in the post-natal animal and undergoes dramatic morphogenetic changes during puberty and the reproductive cycle. The physiological function of the mammary gland is to produce milk to sustain the newborn. Here we view the lactating gland through three-dimensional confocal imaging of intact tissue. We observed that the majority of secretory alveolar cells are binucleated. These cells first arise in very late pregnancy due to failure of cytokinesis and are larger than mononucleated cells. Augmented expression of Aurora kinase-A and Polo-like kinase-1 at the lactogenic switch likely mediates the formation of binucleated cells. Our findings demonstrate an important physiological role for polyploid mammary epithelial cells in lactation, and based on their presence in five different species, suggest that binucleated cells evolved to maximize milk production and promote the survival of offspring across all mammalian species.

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development.

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.

XBP1 Regulates the Biosynthetic Capacity of the Mammary Gland During Lactation by Controlling Epithelial Expansion and Endoplasmic Reticulum Formation.

Cells composing the mammary secretory compartment have evolved a high capacity to secrete not only proteins but also triglycerides and carbohydrates. This feature is illustrated by the mouse, which can secrete nearly twice its own weight in milk proteins, triglycerides and lactose over a short 20-day lactation. The coordination of synthesis and export of products in other secretory cells is orchestrated in part by the transcription factor X-box binding protein 1 (XBP1). To assess the role of XBP1 in mammary epithelial cells (MEC), we studied floxed XBP1 female mice lacking (wild type; WT) or expressing the Cre recombinase under the control of the ovine ß-lactoglobulin promoter (ΔXBP1(MEC)). Pregnant ΔXBP1(MEC) females had morphologically normal mammary development and gave birth to the same number of pups as WT mice. Their litters, however, suffered a weight gain deficit by lactation day 3 (L3)3 that grew to 80% by L14. ΔXBP1(MEC) dams had only modest changes in milk composition (-21% protein, +24% triglyceride) and in the expression of associated genes in isolated MEC. By L5, WT glands were fully occupied by dilated alveoli, whereas ΔXBP1(MEC) glands contained fewer, mostly unfilled alveoli and retained a prominent adipocyte population. The smaller epithelial compartment in ΔXBP1(MEC) glands was explained by lower MEC proliferation and increased apoptosis. Finally, endoplasmic reticulum ribbons were less abundant in ΔXBP1(MEC) at pregnancy day 18 and failed to increase in abundance by L5. Collectively, these results show that XBP1 is required for MEC population expansion during lactation and its ability to develop an elaborate endoplasmic reticulum compartment.

Poros Nucleares y Función Celular en Epitelio Mamario / Nuclear Pores and Cellular Function

El presente artículo tiene como objetivo central evidenciar la interesante relación que se establece entre la función celular y el número de poros nucleares, relación que modula el activo intercambio nucleo-citoplasmatico en distintas etapas del ciclo celular de la estirpe HC11.

The main objective of this article is related to the study of different existing relationships between cellular function and the number of nuclear pores in order to explain the amount of nuclear-cytoplasmatic exchange through HC11 cell cycle stages.