Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots

This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1). © 2008 Elsevier Inc. All rights reserved

Chemicals Facilitating Reprogramming: Targeting the SAM Binding Site to Identify Novel Methyltransferase Inhibitors

© 2014 John Wiley & Sons, Ltd. Reprogramming of somatic cells to a pluripotent state has been achieved by viral-mediated transduction of defined transcription factors. In order to achieve the goal of clinical application, it is necessary to overcome a variety of limitations, including poor reprogramming efficiencies and viral integration. One strategy is to identify small-molecule inhibitors that can improve reprogramming efficiency or replace defined transcription factors. Several reports have demonstrated that modulation of chromatin-modifying enzymes can significantly improve reprogramming efficiency. Key enzymes include DNA and histone methyltransferases, which utilize the cofactor S-adenosyl methionine (SAM) to transfer a methyl group. In this chapter, we review our efforts to identify SAM analogues by virtual screening

The influence of estradiol and progesterone on the concentrations of uterine oxytocin receptors and plasma PGFM in response to oxytocin in ovariectomized gilts

Peripubertal gilts ($n = 25$) were treated with corn oil (CO) or ovarian steroids, one month following an ovariectomy. The first day of treatment was assigned as the first day of the experiment. The gilts received: Group (Gr) I ($n = 4$) – CO (2 ml$\cdot$day$^{-1}$ from 1st to 12th day), Gr II ($n = 4$) and Gr III ($n = 4$) – progesterone (P$_4$; 10 to 100 mg$\cdot$day$^{-1}$ from 1st to 12th day), Gr IV ($n = 5$) – estradiol benzoate (EB; 400 $\mu$g$\cdot$day$^{-1}$ from 1st to 3rd day), Gr V ($n = 4$) and Gr VI ($n = 4$) – EB + P$_4$ (EB 400 $\mu$g$\cdot$day$^{-1}$ from 1st to 3rd day, 20 $\mu$g$\cdot$day$^{-1}$ at 6th and 9th day, 50 $\mu$g at 12th day plus P$_4$ 10 to 100 mg from 4th to 15th day). All gilts were injected with oxytocin (OT; 20 IU; i.v.) on the following days of the experiment: 13th (Gr I and Gr II), 15th (Gr III and Gr IV), 16th (Gr V) and 18th (Gr VI). Concentrations of the PGF$_{2\alpha}$ metabolite – PGFM were determined in blood samples, collected from 30 min before to 120 min after OT injection. Baseline PGFM concentrations (30 min before OT) differed among treatment groups and were the highest in Gr V and Gr VI ($P < 0.01$ vs. other groups). The magnitude of the PGFM response to OT increased only in four of the five gilts of Gr IV and in three of the four gilts of Gr VI, and it was higher ($P = 0.009$) in Gr VI than in Gr IV. In the remaining groups, PGFM concentrations did not increase above the baseline in response to OT. The day after OT injection, oxytocin receptors (OTR) were found in the uterine tissues of all animals studied. The lowest OTR concentrations were in Gr I – 75.5 $\pm$ 11.2 fmol$\cdot$mg protein$^{-1}$ and the highest in Gr IV – 712.9 $\pm$ 86.7 fmol$\cdot$mg protein$^{-1}$; ($P < 0.05$ vs. other groups). The values of K$_{\rm d}$ of OTR differed among groups ($P < 0.001$) and ranged from 1.62 $\pm$ 0.44 nM in Gr I to 12. 08 $\pm$ 1.9 nM in Gr VI. A positive correlation ($r = 0.54$; $P < 0.01$) between plasma E$_2$ and uterine OTR concentrations was observed. In conclusion, E$_2$ and P$_4$ are involved in both PGF$_{2\alpha}$ synthesis/secretion and OTR formation, however, full PGF$_{2\alpha}$ response to OT does not develop before puberty. Estrogens are evident stimulators of uterine OTR synthesis in gilts

Static Balance in Female Artistic Gymnasts and Non-Training Girls

Sports activities can constitute a factor in improving postural control. The aim of this study is to compare static balance in the tandem stance between female artistic gymnasts and non-training girls. This was performed with and without visual control, as well as in a position with open eyes, on a 1.25 m high platform. Two groups of thirty girls participated in the study. The first group consisted of gymnasts (mean age 9.50 ± 1.20 years, body height 133.50 ± 11.40 cm, and body mass 28.90 ± 6.90 kg), while the second group comprised non-training girls (mean age 9.80 ± 1.20 years, body height 142.60 ± 10.90 cm, and body mass 37.40 ± 9.70 kg). Balance measurements were taken using the CQ Stab 2P two-plate stability platform. In the measurements conducted with the subjects’ eyes closed, the gymnasts obtained a lower value for displacement regarding the maximal centre of pressure (COP) in the anterior–posterior direction and a higher frequency of COP displacement (p p < 0.001). Differences in the values of the variables characterising the static balance of gymnasts and non-training girls were revealed only in conditions of increasing difficulty concerning the motor task (increasing the height of the position and/or without eye control)

Diet-induced obesity in stem cell antigen-1 KO mice

Stem Cell Antigen-1 (Sca-1) is a member of the lymphocyte-activated protein 6 family and has served as a marker for the identification of stem cells in various tissues, including fat depots. In vitro and in vivo studies suggest the possible involvement of Sca-1 in adipogenic differentiation and link Sca-1 antigenicity with adipocyte progenitors. Previously, we showed that Sca-1-enriched populations of ear mesenchymal stem cells possess enhanced capacity to differentiate into adipocytes. Additionally, we determined the natural frequency and localization of Sca-1-positive progenitor/stem cells in brown and white fat in situ. The present study addressed the question whether Sca-1 deficiency alters the white adipose tissue response to a high-saturated-fat diet. Our results show that Sca-1 null mice (Sca-1(-/-)) fed high-fat diet developed obesity equally well as wild-type mice, suggesting either an indirect in vivo effect of Sca-1 or a compensatory response to Sca-1 deficiency. However, contrary to wild-type mice, high fat diet-fed Sca-1(-/-) mice showed no alterations in serum adipocytokines. The data lead to the conclusion that Sca-1 is either redundant or a nonessential marker of adipose progenitor/stem cells. Nevertheless, since Sca-1-deficient mice displayed elevated blood glucose at fasting and exhibited glucose intolerance and insulin resistance, Sca-1 has subtle effects on adipose function. Thus, the Sca-1-deficient mice may provide a useful model for metabolic studies

Silencing histone deacetylase-specific isoforms enhances expression of pluripotency genes in bovine fibroblasts

Histone deacetylases (HDACs) catalyze deacetylation of histones that results in altered transcriptional activity. Inhibitors of HDACs have been shown to induce transcriptional changes that contribute positively to reprogramming somatic cells either by nuclear transfer or inducing a pluripotent state. However, the exact molecular mechanisms whereby HDAC inhibitors function and the specificity of the HDAC isoforms in cell reprogramming are not yet fully understood. Herein, we report the ability of individual isoform-specific HDACs to modulate endogenous expression of pluripotency-associated genes in bovine somatic cells. This in vitro study showed that a transient selective depletion of HDACs resulted in elevated mRNA levels of Oct-4, Sox2, and Nanog. In particular, we found that inhibition of specific HDAC isoforms using small interfering (si) RNA significantly increased expression of Nanog, a key factor required for totipotency induced by somatic cell nuclear transfer and for maintaining pluripotency in embryonic and induced pluripotent stem cells. Our study suggests that this gene might be the most susceptible to HDAC activity inhibition. Moreover, a regulatory role of the class III HDAC, SIRT3, on an Oct4-Sox2-Nanog transcriptional network was revealed. We observed the upregulation of pluripotency-related genes by depletion of SIRT3. SIRT3 is localized to mitochondria and is associated with energy metabolism processes, suggesting metabolic changes may be linked to reprogramming in bovine fibroblasts. In conclusion, we show that targeting selective HDACs can potentially be useful to enhance reprogramming and that sirtuins may play a pivotal role in somatic cell reprogramming by upregulating an Oct4-Sox2-Nanog transcriptional network. Dedifferentiating donor somatic cells by upregulating developmentally important genes through specific knockdown of epigenetic targets, in particular HDACs, may provide a path to improving livestock cloning and the in vitro production of pluripotent cells. © Mary Ann Liebert, Inc

Silencing Histone Deacetylase–Specific Isoforms Enhances Expression of Pluripotency Genes in Bovine Fibroblasts

Histone deacetylases (HDACs) catalyze deacetylation of histones that results in altered transcriptional activity. Inhibitors of HDACs have been shown to induce transcriptional changes that contribute positively to reprogramming somatic cells either by nuclear transfer or inducing a pluripotent state. However, the exact molecular mechanisms whereby HDAC inhibitors function and the specificity of the HDAC isoforms in cell reprogramming are not yet fully understood. Herein, we report the ability of individual isoform-specific HDACs to modulate endogenous expression of pluripotency-associated genes in bovine somatic cells. This in vitro study showed that a transient selective depletion of HDACs resulted in elevated mRNA levels of Oct-4, Sox2, and Nanog. In particular, we found that inhibition of specific HDAC isoforms using small interfering (si) RNA significantly increased expression of Nanog, a key factor required for totipotency induced by somatic cell nuclear transfer and for maintaining pluripotency in embryonic and induced pluripotent stem cells. Our study suggests that this gene might be the most susceptible to HDAC activity inhibition. Moreover, a regulatory role of the class III HDAC, SIRT3, on an Oct4–Sox2–Nanog transcriptional network was revealed. We observed the upregulation of pluripotency-related genes by depletion of SIRT3. SIRT3 is localized to mitochondria and is associated with energy metabolism processes, suggesting metabolic changes may be linked to reprogramming in bovine fibroblasts. In conclusion, we show that targeting selective HDACs can potentially be useful to enhance reprogramming and that sirtuins may play a pivotal role in somatic cell reprogramming by upregulating an Oct4–Sox2–Nanog transcriptional network. Dedifferentiating donor somatic cells by upregulating developmentally important genes through specific knockdown of epigenetic targets, in particular HDACs, may provide a path to improving livestock cloning and the in vitro production of pluripotent cells