Gene expression profiling of mouse Sertoli cell lines

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.Electronic Supplementary Material Supplementary material is available in the online version of this article at This work was supported by grants from the Deutsche Forschungsgemeinschaft (Confocal Research Group The Male Gamete: Production, Maturation, Function, grant FOR 197-3) and from the German Academic Exchange Service (DAAD) to P. Mathur     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Expression profiles of 109 apoptosis pathway-related genes in 82 mouse tissues and experimental conditions

Apoptosis is an important process in development and tissue homeostasis. To understand the similarities and differences in the apoptosis machinery in different normal, developmental, and diseased tissues, the expression profiles of 109 apoptosis-pathway-related genes in 82 mouse tissues and experimental conditions were examined using Incyte Mouse GEMI cDNA arrays. It has been found that the compositions of the apoptotic machinery vary among different tissues, developmental stages, and disease states, with subsets of apoptotic genes co-ordinately expressed in the 82 tissues and experimental conditions. Additional genes whose expression profiles resemble selected genes from the 109 apoptotic gene list were also identified. This study provides valuable information on possible molecular mechanisms of differential apoptotic responses to developmental signals, environmental stimuli, and therapeutic treatments in tissue-specific manner.     

Bioinformatic profiling of the transcriptional response of adult rat cardiomyocytes to distinct fatty acids

Diabetes mellitus, obesity, and dyslipidemia increase risk for cardiovascular disease, and expose the heart to high plasma fatty acid (FA) levels. Recent studies suggest that distinct FA species are cardiotoxic (e.g., palmitate), while others are cardioprotective (e.g., oleate), although the molecular mechanisms mediating these observations are unclear. The purpose of the present study was to investigate the differential effects of distinct FA species (varying carbon length and degree of saturation) on adult rat cardiomyocyte (ARC) gene expression. ARCs were initially challenged with 0.4 mM octanoate (8:0), palmitate (16:0), stearate (18:0), oleate (18:1), or linoleate (18:2) for 24 h. Microarray analysis revealed differential regulation of gene expression by the distinct FAs; the order regarding the number of genes whose expression was influenced by a specific FA was octanoate (1,188) > stearate (740) > palmitate (590) > oleate (83) > linoleate (65). In general, cardioprotective FAs (e.g., oleate) increased expression of genes promoting FA oxidation to a greater extent than cardiotoxic FAs (e.g., palmitate), whereas the latter induced markers of endoplasmic reticulum and oxidative stress. Subsequent RT-PCR analysis revealed distinct time- and concentration-dependent effects of these FA species, in a gene-specific manner. For example, stearate- and palmitate-mediated ucp3 induction tended to be transient (i.e., initial high induction, followed by subsequent repression), whereas oleate-mediated induction was sustained. These findings may provide insight into why diets high in unsaturated FAs (e.g., oleate) are cardioprotective, whereas diets rich in saturated FAs (e.g., palmitate) are not.     

Retinal ganglion cell differentiation in cultured mouse retinal explants

The availability of genetically engineered mice harboring specific mutations in genes affecting one or more retinal cell types affords new opportunities for investigating the genetic regulatory mechanisms of vertebrate retina formation. When identifying critical regulatory genes involved in retina development it is often advantageous to complement in vivo analysis with in vitro characterization. In particular, by combining classical techniques of retinal explant culturing with gene transfer procedures relying on herpes simple virus (HSV) amplicon vectors, gain-of-function analysis with genes of interest can be performed quickly and efficiently. Here, details are provided for isolating and culturing explants containing retinal progenitor cells and for infecting the explants with HSV expression vectors that perturb or rescue retinal ganglion cells, the first cell type to differentiate in the retina. In addition, the availability of sensitive techniques to monitor gene expression, including detection of reporter gene expression using antibodies and detection of endogenous marker gene expression using quantitative RT-PCR, provides an effective means for comparing wild-type and mutant retinas from genetically engineered mice.     

A novel type of self-beating cardiomyocytes in adult mouse ventricles

This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.     

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest.     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Gene expression profiling of mouse postnatal cerebellar development using oligonucleotide microarrays designed to detect differences in glycoconjugate expression

Differences in gene expression patterns between adult and postnatal day 7 (P7) mouse cerebellum, at the peak of granule neuron migration, were analyzed by hybridization to the GLYCOv2 glycogene array. This custom designed oligonucleotide array focuses on glycosyl transferases, carbohydrate-binding proteins, proteoglycans and related genes, and 173 genes were identified as being differentially expressed with statistical confidence. Expression levels for 11 of these genes were compared by RT-PCR, and their differential expression between P7 and adult cerebellum confirmed. Within the group of genes showing differential expression, the sialyltransferases (SiaTs) and GalNAc-Ts that were elevated at P7 prefer glycoprotein substrates, whilst the SiaTs and GalNAc-Ts that were elevated in the adult preferentially modify glycolipids, consistent with a role for gangliosides in maintaining neuronal function in the adult. Also within this group, three proteoglycans--versican, bamacan and glypican-2--were elevated at P7, along with growth factor midkine, which is known to bind to multiple types of proteoglycans, and fibroblast growth factor receptor 1, whose activity is known to be influenced by heparan sulfate proteoglycans. Two sulfotransferases that can modify the extent of proteoglycan sulfation were also differentially regulated, and may modify the interaction of a subset of proteoglycans with their binding partners during cerebellar development. Bamacan, glypican-2 and midkine were shown to be expressed in different cell types, and their roles in cerebellar development during granule neuron migration and maturation are discussed.     

Differential expression of connexin 43 in mouse mammary cells

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primary mouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group of genes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMEC cultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levels of expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early pregnancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting that connexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.     

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes

Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.