In-vitro proliferation of germ cells and supporting cells in the neonatal mouse testis

Summary Testicular cells were prepared from neonatal (48 h after birth) mice by enzymatic dissociation and were cultured in serum-supplemented medium to investigate cell proliferation in vitro. The cultured cells were composed mostly of germ cells, identified by immunocytochemistry using a germ cell-specific antiserum, and supporting (immature Sertoli) cells. After 36 h in culture, the cells were pulse-labeled with ³H-thymidine and fixed at 2-h intervals for 36 h after labeling. Numbers of labeled and unlabeled metaphases of germ cells and supporting cells were counted, and percent labeled metaphases for both cell types were determined for cell-cycle analysis. The results indicate that germ cells, as well as supporting cells, incorporate ³H-thymidine and progress through the cell cycle in vitro. From the curve of the percent labeled metaphases for the supporting cells, the total cell cycle and intervals of DNA synthesis were estimated to be 27.2 h and 13.2 h, respectively.     

Localization of DJ-1 protein in the murine brain

Mutations in the DJ-1 gene have been identified to cause Parkinson's disease. In humans, nonmutated DJ-1 is expressed in specific brain areas but seems to be expressed by astrocytes rather than by neurons. In contrast, DJ-1 mRNA is mainly found in neurons in the mouse brain. We have investigated the distribution of DJ-1 protein in the mouse brain and found that DJ-1 protein is predominantly expressed by neurons but can also be detected in astrocytes. Consistent with a global role of DJ-1 in the brain, we found immunoreactivity, for example, in cortical areas, hippocampus, basolateral amygdala, the reticular nucleus of the thalamus, zona incerta, and locus coeruleus. Within the substantia nigra, however, DJ-1 is localized in both neuronal and nonneuronal cells, suggesting a distinct role in this area.     

Physiological response of bovine subcommissural organ to endothelin 1 and bradykinin

Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system x_c ⁻ (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na⁺-dependent and –independent uptake of [³H] glutamate was assessed as a measure of GLAST and system x_c ⁻ function, respectively. Hyperglycemia did not alter the uptake of [³H] glutamate by GLAST or system x_c ⁻; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by ~10% but increased system x_c ⁻ activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in V_max, but not K_m. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system x_c ⁻. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system x_c ⁻ activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione. This work was supported by NIH R01 EY014560.     

Comparison of different culture modes for long-term expansion of neural stem cells

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed more βIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer culture mode for large-scale expansion of NSCs.     

Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precursor cells

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce proliferation of neural precursor cells from several central nervous system regions in vitro. We have previously described two neural precursor cell populations from 13.5 days postcoitium (dpc) mesencephalon, one forming colonies in response to EGF, present in the ventral mesencephalon, and other forming colonies in response to EGF + bFGF, mainly present in the dorsal mesencephalon. In the present work, we show that 13.5 dpc dorsal mesencephalic cells required bFGF only for 1 h to form colonies in response to EGF alone, indicating that these two growth factors act in sequence rather than simultaneously. Absence of bFGF at the beginning of the culture gave rise to very few colonies, even after the addition of EGF + bFGF, suggesting that cells responsive to bFGF were very labile in the primary culture condition. This result is in contrast with cells pretreated with bFGF, which could survive for up to 5 days in the absence of bFGF or EGF, and then were capable of efficiently forming colonies in response to EGF. Basic FGF was also able to support survival of EGF‐responsive neural precursors from both ventral and dorsal mesencephalon. The population requiring bFGF to form colonies in response to EGF was identified at different developmental stages (11.5–15.5 dpc), with higher contribution to the total number of neural precursors cells detected (EGF‐responsive plus bFGF‐responsive) at early stages and in the dorsal region. We show that the differentiation effect of bFGF resulted in the appearance of the mRNA coding for the EGF receptor. Our data suggest that bFGF‐responsive neural precursors are the source of EGF‐responsive neural precursors. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 14–27, 1999     

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress,BAX, and caspase 2

Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function. The work was supported by grants P50 DK45179 and DK53072.     

Galectin-3 stimulates preadipocyte proliferation and is up-regulated in growing adipose tissue

Objective: Some cytokines and mediators of inflammation can alter adiposity through their effects on adipocyte number. To probe the molecular basis of obesity, this study determined whether galectin‐3 was present in adipose tissue and investigated its effects on fat cell number. Research Methods and Procedures: In the first study, obesity‐prone C57BL/6J mice were fed with high‐fat (58%) diet. Epididymal fat pads were collected at Day 0, Day 60, and Day 120 after the start of high‐fat feeding. Results: Levels of adipocyte galectin‐3 protein, determined using Western blot analysis, increased as the mice became obese. Galectin‐3 mRNA and protein were then detected in human adipose tissue, primarily in the preadipocyte fraction. It was found that recombinant human galectin‐3 stimulated proliferation of primary cultured preadipocytes as well as DNA synthesis through lectin‐carbohydrate interaction. Discussion: Galectin‐3, which has been known to play a versatile role especially in immune cells, might play a role also in adipose tissue and be associated with the pathophysiology of obesity.     

Odorant receptor proteins in olfactory axons and in cells of the cribriform mesenchyme may contribute to fasciculation and sorting of nerve fibers

Odorant receptors (ORs) have been shown to be present not only in the chemosensory cilia of the olfactory sensory neurons, but also in their axon terminals. This observation has emphasized the notion that the receptor protein may contribute to the precise receptor-specific targeting of olfactory axons in the olfactory bulb. This concept implies a particularly important role for the axonal receptor protein during the onset and early phase of the wiring process during development. In the present study, we have demonstrated, by means of specific antibodies, that, as early as mouse embryonic day E12, the OR protein can be visualized in outgrowing axonal processes of the olfactory epithelium and in cells located in the cribriform mesenchyme. On their trajectory from the olfactory epithelium through the cribriform mesenchyme toward the forebrain, axons with strong OR immunoreactivity have only been seen in the dorsal part of the mesenchyme where they traverse the region of OR-positive cells. Upon visualization by specific antibodies, these cells have been revealed to have long protrusions extending along the surface of nerve fascicles. They are often located at bifurcations where two small axon fascicles merge to form a stronger bundle. Within this region, fascicles coalesce forming a coherent nerve. Moreover, within the now compact nerve bundle, axons visualized by the OR-specific antibody are no longer distributed evenly but are segregated from other axonal populations within the nerve. These findings suggest that OR proteins in the membrane of axonal processes and of cells in the cribriform mesenchyme are involved in crucial processes such as fasciculation and the sorting of outgrowing axons, both of which are fundamental for the initiation and establishment of the precise wiring of the olfactory system during early development. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 495).     

Localization patterns of fibroblast growth factor 1 and its receptors FGFR1 and FGFR2 in postnatal mouse retina

Fibroblast growth factors (FGFs) exert basic functions both during embryonic development and in the adult. The expression of FGFs and their receptors has been reported in mammalian retinas, although information on the organization of the FGF system is still incomplete. Here, we report a detailed double-label immunohistochemical investigation of the localization patterns of FGF1 and its receptors FGFR1 and FGFR2 in adult and early postnatal mouse retinas. In adult retinas, FGF1 is localized to ganglion cells, horizontal cells, and photoreceptor inner and outer segments. FGFR1 is found in ganglion cells and Müller cells, whereas FGFR2 is primarily located in ganglion cells, the nuclei of Müller cells, and glycine-containing amacrine cells. During postnatal development, the patterns of FGF1, FGFR1, and FGFR2 immunostaining are similar to those in the adult, although transient FGF1-expressing cells have been detected in the proximal inner nuclear layer before eye opening. These patterns are consistent with a major involvement of FGF1, FGFR1, and FGFR2 in ganglion cell maturation (during development) and survival (in the adult). Moreover, FGF1 may affect amacrine cell development, whereas Müller cells appear to be regulated via both FGFR1 and FGFR2 throughout postnatal life. In immature retinas, large numbers of amacrine cells, including those containing calbindin and glycine, display both FGF1 and FGFR2 immunoreactivities in their nuclei, suggesting an action of FGF1 on FGFR2 leading to the maturation of these amacrine cells during a restricted period of postnatal development. This work was supported by funding from the Italian Ministry of Education.     

Neutrophil depletion retards endometrial repair in a mouse model

The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair. This work was funded by the National Health and Medical Research Council of Australia (#143798, #241000) and by an Australian Postgraduate Scholarship to T.K.     

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells

Background

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs.

Methods

Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide.

Results

We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs.

Conclusions

Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state.

General significance

These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy.     

Allograft inflammatory factor-1/Ionized calcium-binding adapter molecule 1 is specifically expressed by most subpopulations of macrophages and spermatids in testis

Ionized calcium-binding adapter molecule 1 (Iba1) is a 147-amino-acid calcium-binding protein widely in use as a marker for microglia. It has actin-crosslinking activity and is involved in aspects of motility-associated rearrangement of the actin cytoskeleton. The Iba1 gene and protein are identical to allograft inflammatory factor-1 (AIF-1), a protein involved in various aspects of inflammation, which was investigated independently from Iba1. Although regarded to be monocyte/macrophage-specific, expression by germ cells in testis showed that AIF-1/Iba1 is not exclusively expressed by cells of the monocyte/macrophage lineage. Furthermore, AIF-1 was found in cells not belonging to the monocyte/macrophage lineage under pathological conditions. Here, the distribution of AIF-1/Iba1 in the normal mouse has been examined, by immunohistochemistry, to determine whether AIF-1/Iba1 expression is confined to macrophages and spermatids. Spermatids are the only cells not belonging to the monocyte/macrophage lineage found to express AIF-1/Iba1 in the normal mouse, by this method. This study has not demonstrated AIF-1/Iba1 expression in dendritic cells, although this protein might be expressed by subsets of dendritic cells. AIF-1/Iba1 can be regarded a “pan-macrophage marker” because, except for alveolar macrophages, all subpopulations of macrophages examined express AIF-1/Iba1.     

Effects of strain and age on hepatic gene expression profiles in murine models of HFE-associated hereditary hemochromatosis

Hereditary hemochromatosis is an iron overload disorder most commonly caused by a defect in the HFE gene. While the genetic defect is highly prevalent, the majority of individuals do not develop clinically significant iron overload, suggesting the importance of genetic modifiers. Murine hfe knockout models have demonstrated that strain background has a strong effect on the severity of iron loading. We noted that hepatic iron loading in hfe−/− mice occurs primarily over the first postnatal weeks (loading phase) followed by a timeframe of relatively static iron concentrations (plateau phase). We thus evaluated the effects of background strain and of age on hepatic gene expression in Hfe knockout mice (hfe−/−). Hepatic gene expression profiles were examined using cDNA microarrays in 4- and 8-week-old hfe−/− and wild-type mice on two different genetic backgrounds, C57BL/6J (C57) and AKR/J (AKR). Genes differentially regulated in all hfe−/− mice groups, compared with wild-type mice, including those involved in cell survival, stress and damage responses and lipid metabolism. AKR strain-specific changes in lipid metabolism genes and C57 strain-specific changes in cell adhesion and extracellular matrix protein genes were detected in hfe−/− mice. Mouse strain and age are each significantly associated with hepatic gene expression profiles in hfe−/− mice. These affects may underlie or reflect differences in iron loading in these mice.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0443-1) contains supplementary material, which is available to authorized users.     

Uptake and utilization of human polymorphonuclear leukocyte granule myeloperoxidase by mouse peritoneal macrophages

Summary Functional myeloperoxidase contained in granules of polymorphonuclear neutrophil leukocytes or in fixed whole cells can be endocytosed by mouse peritoneal macrophages. Acquired myeloperoxidase was distributed in what we considered to be the secondary lysosomal system and, following a phagocytic stimulation, was delivered to newly formed phagosomes containing the targets.     

神经干细胞体外增殖分化的钙成像研究

神经干细胞具有广阔的应用前景,但对于其增殖和分化的内源机制、外部环境信号还并不十分了解。研究表明,钙信号很可能在其中起到了调控作用。利用钙离子成像技术,观察神经干细胞的单细胞体外增殖和分化过程,记录了在细胞分裂过程中钙信号变化的曲线。发现细胞增殖和分化过程中都会产生钙浓度的变化,但在细胞分裂后期两者钙信号的模式却存在差别。实验结果提示,胞内钙水平的波动只是细胞增殖的伴随产物,但却是细胞分化的必要条件。由此提出钙信号对神经干细胞分化调控机制的假设,并指出其对今后研究的意义。     

Distribution of TRPC4 in developing and adult murine brain

The transient receptor potential (TRP) superfamily comprises of a group of non-selective cation channels that have been implicated in both receptor and store-operated channel functions. The family of classical TRPs (TRPCs) consists of seven members (TRPC1-7), with TRPC4 possibly playing a role in neuronal signaling. We have examined the distribution pattern of TRPC4 mRNA and protein in the developing and postnatal murine brain by using in situ hybridization, Western blotting, and immunocytochemistry. Expression of TRPC4 mRNA starts at embryonic day 14.5 (E14.5) in the developing septal area and cerebellar anlagen. At E16.5, prominent expression is additionally seen in the hippocampal formation and cortical plate. High densities of cells expressing TRPC4 mRNA occur in the adult olfactory bulb and hippocampus, whereas the cortex and septum display lower densities of cells positive for TRPC4 mRNA. Analysis of the adult hippocampal formation has revealed TRPC4 immunoreactivity in hippocampal areas CA1 to CA3 and in the dentate gyrus. Functions consistent with this spatially restricted pattern of expression remain to be revealed.     

Comparison of murine dental follicle precursor and retinal progenitor cells after neural differentiation in vitro

Human dental stem or precursor cells can differentiate into multiple cell types like adipocytes, osteoblasts or chondrocytes. Recently, a number of different human dental stem cell lines were differentiated into neurons. This makes dental stem cells interesting as possible cell-based therapeutics for neural degenerative diseases. To test this hypothesis, we have investigated the neural differentiation potential of murine dental follicle precursor cells (mDFPCs). The mDFPC cell line was newly established without cell immortalization. After differentiation, neural cell marker expression in mDFPCs was checked and compared with that of murine retinal progenitor cells (mRPCs). Differentiated mDFPCs became neuron-like cells with small cell bodies and long/branching neurites, similar to differentiated mRPCs. However, mRPCs showed more complete neural differentiation. Furthermore, 5% of the differentiated mDFPCs and 37% of the differentiated mRPCs were positive for the glia cell marker GFAP (glial fibrillary acidic protein). The data presents new evidence of neural differentiation of mDFPCs, but only a small percentage of mDFPCs differentiated into glia cells, unlike mRPCs.     

Tissue- and species-specific expression patterns of class I,III, and IV Adh and Aldh1 mRNAs in rodent embryos

Alcohol and aldehyde dehydrogenases (ADHs and ALDHs) may be of interest in the pathology of Parkinson's disease (PD) because of their role in protection against toxins and in retinoid metabolism, which is required for growth and development of the mesencephalic dopamine system. In the present study, the spatial and temporal expression patterns of Adh1, Adh3, Adh4, and Aldh1 mRNAs in embryonic C57BL/6 mice (E9.5-E19.5) and Sprague-Dawley rats (E12.5-P0) have been investigated by using radioactive oligonucleotide in situ hybridization. High expression of Aldh1 mRNA was found in the developing mesencephalic dopamine neurons of both mice and rats. Expression of Adh1 and Adh4 mRNAs was observed in adrenal cortex and olfactory epithelium in mice. Additionally, Adh1 was expressed in epidermis, liver, conjunctival, and intestinal epithelium. In rat embryos, expression was less extensive, with Adh1 mRNA being found in liver and intestines. Adh3 expression was ubiquitous in both mouse and rat embryos, suggesting a housekeeping function of the gene. Consistent with previous studies in adult rats and mice, our data suggest that Adh3 is the only ADH class present in rodent brain. Adh and Aldh gene activity in mouse and rat embryos indicate the possible involvement of the respective enzymes in retinoid metabolism and participation in defense against toxic insults, including those that may be involved in the pathogenesis of PD. This work was supported by grants from the Swedish Research Council, the Swedish Parkinson Foundation, the Swedish Brain Foundation, Karolinska Institutet funds, AstraZeneca, and the US Public Health Service.