Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano

We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.     

Adenohypophysis regulates cell proliferation in the gonads of the developing chick embryo

The aim of the present study was to evaluate the effect of hypophysectomy on cell proliferation in the left ovary and the left testis of 8- to 14-day-old chick embryos. Hypophysectomy was performed by the partial decapitation technique. At 44-46 h of incubation, chick embryo heads were sectioned at the mesencephalic level and the prosencephalic region removed. Embryos were further incubated until 8-14 days of development. Cell division was evaluated by bromodeoxyuridine (BrdU) incorporation and by counting the total number of somatic and germ cells in the gonads. The ovary displayed an exponential increase in the number of somatic and germ cells and a higher rate of BrdU incorporation compared to the testis. BrdU incorporation was reduced in the ovary of hypophysectomized embryos at 9-14 days of incubation, while in the testis, the reduction was significant at 14 days of development. Changes in the total number of somatic and germ cells further suggest that the absence of hypophysis affects the growth of the ovary earlier than the growth of the testis. Reduction in the number of somatic and germ cells after hypophysectomy in the ovary was reversed by a hypophyseal graft on the chorioallantoic membrane. The adenohypophysis regulates, probably through gonadotropic hormones, proliferation of somatic and germ cells in the gonads during chick embryo development.     

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G₁/G₀ arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours. Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G₁/G₀ arrest. Lastly, in XX germ cells we observed a down‐regulation of genes involved in both G₁‐ and G₂‐phases of the cell cycle consistent with their entry into meiosis. Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.     

Cross talk between germ cells and gonadal somatic cells is critical for sex differentiation of the gonads in the teleost fish, medaka (Oryzias latipes)

To evaluate the possible role of germ cells on sex differentiation of the gonads in vertebrates, the teleost fish, medaka ( Oryzias latipes ), was used to generate a gonad without germ cells. The germ cell-deficient medaka reveals multiple effects of germ cells on the process of sex differentiation. The previously isolated mutant medaka, hotei , with the excessive number of germ cells may support the contention that the proliferation of germ cells is related to feminization of the gonad. Futhermore, we show that two modes of proliferation for either maintenance of germ cells or commitment to gametogenesis are important components of the sex differentiation of medaka developing gonads. An intimate cross talk between germ cells and gonadal somatic cells during the sex differentiation will be discussed.     

Moderate and severe malnutrition alters proliferation of spleen cells in rats

Objectives

Previous studies have shown alterations in bone marrow cell proliferation in malnourished rats, during lactation. The objective of this study was to determine in vivo effects of moderate and severe malnutrition on spleen cell proliferation in 21‐day‐old rat pups.

Materials and methods

Spleen cell proliferation was determined following administration of bromodeoxyuridine (BrdUrd) over a time course of 2, 4, 6 and 8 h. Incorporation of BrdUrd was detected using FITC‐conjugated anti‐BrdUrd monoclonal antibodies and total DNA content was detected and evaluated using propidium iodide using flow cytometry.

Results

Proportions of cells in S and G₂/M were reduced in the rats with moderate (MN2^nd) and severe (MN3^rd) malnutrition. BrdUrd incorporation was lower in both groups of malnourished rat. In cells of MN2nd individuals, length of G₁ became shorter, while length of S‐phase increased. In contrast, fraction of cells in proliferation was significantly lower in both groups of malnourished rat, with MN3rd group having lowest percentage of cell population growth. In this study, severe malnutrition did not significantly affect duration of phases of the cell cycle, although fractions of proliferating cells were dramatically reduced.

Conclusion

Moderate malnutrition increased time of cells in DNA synthesis and time of total cell cycle and severe malnutrition reduced growth fraction of spleen cells in malnourished rats during lactation.

     

Polyamine synthesis inhibition induces S phase cell cycle arrest in vascular smooth muscle cells

Polyamines are important for cell growth and proliferation and they are formed from arginine and ornithine via arginase and ornithine decarboxylase (ODC). Arginine may alternatively be metabolised to NO via NO synthase. Here we study if vascular smooth muscle cell proliferation can be reversed by polyamine synthesis inhibitors and investigate their mechanism of action. Cell proliferation was assessed in cultured vascular smooth muscle A7r5 cells and in endothelium-denuded rat arterial rings by measuring [³H]-thymidine incorporation and by cell counting. Cell cycle phase distribution was determined by flow cytometry and polyamines by HPLC. Protein expression was determined by Western blotting. The ODC inhibitor DFMO (1–10 mM) reduced polyamine concentration and attenuated proliferation in A7r5 cells and rat tail artery. DFMO accumulated cells in S phase of the cell cycle and reduced cyclin A expression. DFMO had no effect on cell viability and apoptosis as assessed by fluorescence microscopy. Polyamine concentration and cellular proliferation were not affected by the arginase inhibitor NOHA (100–200 μM) and the NO synthase inhibitor l-NAME (100 μM). Lack of effect of NOHA was reflected by absence of arginase expression. Polyamine synthesis inhibition attenuates vascular smooth muscle cell proliferation by reducing DNA synthesis and accumulation of cells in S phase, and may be a useful approach to prevent vascular smooth muscle cell proliferation in cardiovascular diseases.     

Vitamin C promotes the proliferation of human adipose-derived stem cells via p53-p21 pathway

Although adipose-derived stem cells (ADSCs) have demonstrated a promising potential for the applications of cell-based therapy and regenerative medicine, excessive reactive oxygen species (ROS) are harmful to ADSCs cell survival and proliferation. Vitamin C is an important antioxidant, and is often added into culture media as an essential micronutrient. However, its roles on the proliferation of human ADSCs have not been studied. Therefore, in this study, human ADSCs were isolated, and detected by flow cytometry for the analysis of their cell surface antigens. Cell proliferation and cell cycle progression were measured with cell counting kit-8 assay and flow cytometry, respectively. Western blotting was used to detect the expression levels of cyclin E1, p53, p21, and CDK2 proteins. The effect of vitamin C pretreatment on the production of hydrogen peroxide (H₂O₂)-mediated ROS in the ADSCs was evaluated by flow cytometry. Our results indicated that vitamin C treatment significantly increased cell proliferation, and changed the cell cycle distribution of ADSCs by decreasing the percentage of G₁ phase, and concurrently increased the percentage of S and G₂/M phase. Western blot analysis indicated that vitamin C treatment up-regulated the expression levels of cyclin E1 and CDK2, but down-regulated p53 and p21 proteins expression, which contributed to cell proliferation and cell cycle progression. Vitamin C pretreatment significantly reduced the production of H₂O₂-induced ROS in the ADSCs. These findings suggest that vitamin C can promote the proliferation and cell cycle progression in the ADSCs possibly through regulation of p53-p21 signal pathway.     

Regulation of the female mouse germ cell cycle during entry into meiosis

During mouse embryonic development germ cells proliferate extensively until they commit to the male or female pathway and arrest in mitosis or meiosis respectively. Whilst the transition of female germ cells exiting the mitotic cell cycle and entering meiosis is well defined histologically, the essential cell cycle proteins involved in this process have remained unresolved. Using flow cytometry we have examined the entry of female germ cells into meiosis, their termination of DNA synthesis and entry into prophase I. Analysis of key G₂/M cell cycle proteins revealed that entry into meiosis and cell cycle exit at G₂/M involves repression of G₂/M promoting Cyclin B1, coincident upregulation of G₂/M repressing Cyclin B3 and robust establishment of the ATM/CHK2 pathway. By contrast we show that the ATR/CHK1 pathway is activated in male and female germ cells. This data indicates that an important G₂/M surveillance mechanism operates during germ cell proliferation and that passage into meiotic G₂/M involves the combined repression of G₂/M through Cyclin B3 and activation of the key G₂/M checkpoint regulatory network modulated through ATM and CHK2. This work shows that the core regulatory machinery that controls G₂/M progression in mitotic cells is activated in female mouse germ cells as they enter meiosis.     

Quantification of somatic and spermatogenic cell proliferation in the testes of ruminants, using a proliferation marker and flow cytometry analysis

We compared 2 methods for the quantification of proliferation in somatic and spermatogenic compartments of post mortem-collected testes in cattle and roe deer. Proliferation was evaluated by estimation of the tissue polypeptid specific antigen (TPS) using an ELISA. This proliferation-specific marker was detected in homogenized cells after selective enrichment of different cell types by density gradient centrifugation. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry and analyzed for mitotic cell cycle. Somatic and spermatogenic cells were discriminated by dual-parameter flow cytometry after DNA staining with propidium iodide and selective labelling of stromatic cells with a vimentin antibody. The TPS was related to the ploidy of cells and their somatic or spermatogenic type. High concentrations of TPS were found in both species. The TPS values varied with different contents of spermatogenic and somatic cells in the fractions of the density gradient. The TPS was positively correlated with spermatogenic cells in the G2/M phase of mitotic cycle (r = 0.474; P < 0.01) and negatively correlated with somatic cells (r = -0.676; P < 0.0001) in roe deer (n = 40). Discrimination of germinative and stromatic cells in the G2-M phase showed their varying proliferation during the annual cycle in roe deer. The quantification of tetraploid spermatogenic cells allowed the calculation of an exact meiotic transformation (ratio haploid:tetraploid cells). In conclusion, TPS indicates proliferation in the germinative compartment of the testes. However, this marker provides only relative values, without information on the number and type of proliferating cells. Dual-parameter flow cytometry using specific staining for vimentin proves to be a better method for studying changing mitotic and meiotic steps during the involution and recrudescence of testes in seasonally breeding ruminants, as it relates proliferative processes directly to both spermatogenic and somatic cells.     

Transforming growth factor beta inhibits proliferation of somatic cells without influencing germ cell number in the chicken embryonic ovary

The gonadal development of chicken embryo is regulated by hormones and growth factors. Transforming growth factor beta (TGF-β) isoforms may play a critical role in the regulation of growth in chicken gonads. We have investigated the effect of the TGF-β isoforms on the number of germ and somatic cells in the ovary of the chicken embryo. Ovaries were obtained from chicken embryos at 9 days of incubation. They were organ-cultured for 72 h in groups treated with TGF-β1, TGF-β2, soluble betaglycan, TGF-β1 plus soluble betaglycan, or TGF-β2 plus soluble betaglycan, and untreated (control). TGF-β1 and TGF-β2 diminished the somatic cell number in the ovary of the chicken embryo at this age by inhibiting the proliferation of the somatic cells without increasing apoptosis. On the other hand, TGF-β1 and TGF-β2 did not affect the number of germ cells in the cultured ovary. The capacity of TGF-β1 and TGF-β2 to diminish the number of somatic cells in the ovary was blocked with soluble betaglycan, a natural TGF-β antagonist. However, changes in the location of germ cells within the ovary suggested that TGF-β promoted the migration of the germ cells from the ovarian cortex to the medulla. Thus, TGF-β affects germ and somatic cells in the ovary of the 9-day-old chicken embryo and inhibits the proliferation of somatic cells.This work was supported by DGAPA-UNAM (IN214403) and CONACYT (45030).     

Metformin facilitates the proliferation,migration, and osteogenic differentiation of periodontal ligament stem cells in vitro

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis‐related markers were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis, respectively. Metformin treatment at 10 μM did not affect PDLSC proliferation, while at 50 and 100 μM, metformin time‐dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 μM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 μM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.     

Inhibition of sphingosine kinase 1 suppresses proliferation of glioma cells under hypoxia by attenuating activity of extracellular signal-regulated kinase

Objectives: Sphingosine kinase (SphK), which is regulated by hypoxia, catalyses phosphorylation of sphingosine to produce sphingosine‐1‐phosphate, which stimulates invasiveness of gliomas. However, whether SphK is involved in proliferation of glioma cells under hypoxic conditions is not clearly understood. In this study, we have investigated the role of SphK in of proliferation glioma cells under hypoxia. Materials and methods: Effects of small interfering RNA (siRNA) on SphKs, SKI (inhibitor of SphK) and U0126 (inhibitor of ERK) on proliferation of glioma cells under hypoxia were studied using CCK‐8 assay and flow cytometry. Protein expression profiles were evaluated by Western blot analysis. Results:  SKI suppressed proliferation of glioma cells under hypoxia. Similarly, downregulation of SphKs by siRNA inhibited glioma cell proliferation, and the cell cycle was arrested in G₂/M phase when SphK1 was inhibited. In addition, inhibition of SphK1 attenuated phosphorylation of ERK in hypoxic conditions. Furthermore, U0126 markedly inhibited cell population growth and arrested cells in G₂/M as effectively as SKI. However, silencing SphK2 induced cell cycle arrest in the S phase and it showed little effect on hypoxia‐induced activation of ERK. Conclusions: SphK1 and SphK2 are involved in proliferation of glioma cells in hypoxic conditions through distinct signalling pathways. SphK1, but not SphK2, promotes cell population expansion in hypoxic conditions by activating ERK.     

Number of somatic and germ cells during early stages of gonadal development in frog larvae treated with zinc sulphate

Summary ZnSO₄ treatment of early frog tadpoles resulted, initially, in a mitotic stimulation of primordial germ cells. In later larval stages, ZnSO₄ was responsible for the atresy of gonads in which germ cell and medullary cell numbers sharply decreased. At the same time, very few germ cells entered the meiotic prophase, while the degeneration of some of them was observed. Our results are discussed in connection with previous findings about the influence of Zn on cellular proliferation.     

DNA FLOW CYTOMETRY OF ISOLATED KERATINIZED EPITHELIA: A METHODOLOGICAL STUDY BASED ON ULTRASONIC TISSUE DISAGGREGATION

Ultrasonication of keratinized, stratified, squamous epithelium, which had been separated from underlying tissue by means of acetic acid, resulted in disaggregation of all cellular layers in the epithelium, giving a suspension of single nuclei with mitoses preserved. This suspension was treated with RNAse and ethidium bromide for analysis by flow cytometry. From the resulting DNA histogram the G₁, S and G₂+ M fractions were estimated using the computer program of Fried (1976). Treatment with dithiothreitol before sonication increased the yield of nuclei in suspension and decreased the amount of debris and clumps, thereby suppressing overestimation of small S fractions. This method of preparation prior to DNA flow cytometry was useful for the study of the hamster cheek pouch epithelium and of normal and pathological human epidermis.     

Proliferation of germ cells during gonadal sex differentiation in medaka: Insights from germ cell-depleted mutant zenzai

The proliferation of germ cells becomes sexually dimorphic during gonadal sex differentiation, although the underlying dynamics of this are not well understood in vertebrates. By tracing GFP-labeled germ cells in vivo and analyzing the germ cell-depleted mutant, zenzai, we show that the proliferation and differentiation of germ cells are regulated in a sexually dimorphic manner in the teleost fish medaka. In the undifferentiated gonads, germ cells resume proliferation by slow intermittent division (type I), producing isolated daughter cells. While germ cells in the male gonads continue this mode of proliferation, some germ cell fractions in the female gonads initiate two to four rounds of continuous division (type II), forming cysts of four, eight, or sixteen cells, which subsequently enter meiosis synchronously. Thus, female germ cells become differentiated much earlier than do male germ cells. In the zenzai mutant, a defect in slow intermittent division eventually leads to the depletion of germ cells in the adult gonads in both sexes, despite the fact that cyst-forming division is unaffected. This argues that slow intermittent division is essential for the maintenance of germ cells. The proliferation and differentiation of germ cells are thus important components of gonadal sex differentiation in vertebrates.     

Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells

Abstract. Objectives: Previously, we have found that the ClC‐3 chloride channel is involved in endothelin‐1 (ET‐1)‐induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC‐3 in cell cycle progression/distribution and the underlying mechanisms of proliferation. Materials and methods: Small interference RNA (siRNA) is used to silence ClC‐3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively. Results: ET‐1‐induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC‐3 protein. Silence of ClC‐3 by siRNA inhibited expression of ClC‐3 protein, prevented an increase in BrdU incorporation and cell number induced by ET‐1. Silence of ClC‐3 also caused cell cycle arrest in G₀/G₁ phase and prevented the cells’ progression from G₁ to S phase. Knockdown of ClC‐3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin‐dependent kinase inhibitors (CDKIs) p27^KIP and p21^CIP expression. Furthermore, ClC‐3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase‐3β (GSK‐3β) induced by ET‐1. Conclusion: Silence of ClC‐3 protein effectively suppressed phosphorylation of the Akt/GSK‐3β signal pathway, resulting in down‐regulation of cyclin D1 and cyclin E, and up‐regulation of p27^KIP and p21^CIP. In these BASMCs, integrated effects lead to cell cycle G₁/S arrest and inhibition of cell proliferation.     

Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells

Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H₂O₂ for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H₂O₂‐induced apoptosis. Resveratrol also prevented H₂O₂‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H₂O₂‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Do germ line cells in Allacma fusca (Insecta, Collembola, Symphypleona) have a higher metabolic rate than somatic cells.

Stereological analysis of the ultrastructure of primordial germ cells (PGCs) and the somatic (ectoderm) cells in two developmental stages of embryos and freshly hatched juveniles of Allacma fusca have shown great differences in mitochondria volume density (vd) between the two types of cells. In younger embryos (migration phase of the PGCs) the vd of mitochondria in the cytoplasm of the PGCs is 74.64% higher than in the ectoderm cells. In older embryos, (PGCs in the gonads) the vd of mitochondria is 123% higher than the corresponding value for the somatic cells cytoplasm. In the juvenile the vd of mitochondria in the ectoderm cells grows twice but is still only 2/3 of the value for the PGCs. On the basis of papers describing a direct relationship between stereological and physiological results the authors conclude that the metabolism of the primordial germ cells during embryonic development of Allacma fusca is much higher than that of the somatic ones. If the above conclusion is correct, the results presented here confirm the "disposable soma theory" (Kirkwood & Holliday 1979).