Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells

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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.     

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.     

Follicular cells versus oocytes: cell population dynamics in the developing ovary

The aim of the current paper is to evaluate the correlation of germ and follicular cells kinetics during ovarian morphogenesis. Thus, immunohistochemical detection of PCNA and Ki-67 proteins has been examined using PC10 (Dako) and NCL-Ki-67 (Novocastra) antibodies in the developing ovaries of Wistar rat embryos and neonates [14.5, 18.5, 20.5days post-coitum (dpc), birth (day 0), 1, 3, 5, 7day post-partum (dpp)]. Estimation of reactive/total cell ratio, per cell type (germ and follicular cells) and visual field was achieved using the Image Pro Plus Software. The statistical interpretation of the results has shown that, before birth, using the PCNA antibody, the percentage of labeled/total germ cells (labeling index, LI) increases from 71.19% at 14.5dpc to 75.66% at 18.5dpc. It then decreases to 73.26% at 20.5dpc. At birth, the labeling index drops significantly (28.57%). Immediately after birth, the percentage of labeled/total germ cells increases, reaching 43.58% at 1dpp. Subsequently, a further decrease in the percentage of reactive cells is observed resulting to a maximum drop of the LI at 7dpp (18.41%). Using the Ki-67 antibody, the percentage of labeled/total germ cells is generally lower although the fluctuation is similar with that observed using the first marker of cell proliferation. Using the PCNA antibody, the LI of follicular cells in the developing ovary, increases from 0.70% (at 14.5dpc) to 28.94% (at 18.5dpc) and then drops to 18.03% (at 20.5dpc). At birth, the percentage of reactive follicular cells, reaches 27.66% and remains high thereafter. Similar results are obtained using the Ki-67 antibody. In conclusion, follicular cell reaction ratio, using both antibodies (PCNA and Ki-67), increases continuously throughout the examined period with a maximum value at 7dpp, suggesting a kinetics profile similar to that observed for Sertoli cells in the testis. In all age groups, PCNA labeling is more intense than Ki-67, a result that may be attributed to selective staining at different periods of the cell cycle.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Immunohistochemical visualization of insulin receptors in formalin-fixed bovine ovaries post mortem and in granulosa cells collected in vivo

Insulin is crucial for granulosa cell (GC) function, follicle growth and ovulation in cows; low insulin levels increase the risk for anoestrus. Apart from insulin concentration, alterations in the insulin receptor (IR) density on GC may affect follicular growth and steroidogenesis. Data about the IR protein distribution in the bovine follicle are scarce. Therefore, we aimed to develop a quantifiable staining method for IR protein on histological sections of bovine follicles in different developmental stages, and to apply this technique on GC obtained in living cows.In a first experiment, bovine ovaries were collected post mortem, formalin fixed, routinely processed, and stained with monoclonal murine IR-antibodies, peroxidase-labeled goat anti-mouse antibodies, and substrate chromogen. Based on their diameter, follicles were morphologically classified as small antral (SAF; n = 141), dominant (DF; n = 28) or subordinate (SF; n = 8); DF and SF were further classified as healthy or atretic based on the ratio of estrogen and progesterone concentrations in their follicular fluid. Using specialized software, the proportion of pixels displaying a positive staining signal was computed as a measure for IR density in three selected follicular regions: GC, theca (T) and stroma (STR). Results were analyzed in an ANOVA model with follicle type, region and health status as fixed factors. In SAF, DF, and SF, IR density was notably higher in GC than T or STR; the latter two displayed very low or no IR presence. The IR density in SAF was stronger than in DF and tended to be stronger than in SF. Staining intensity was not altered in atretic compared to healthy follicles. In corpus luteum, cumulus-oocyte complexes and pre-antral follicles, no IR could be detected. In a second experiment, GC samples were collected from 20 live cows on 30 and 70 d post partum by transvaginal follicular fluid aspiration, projected on glass slides, and stained using the protocol described above. Most samples yielded sufficient GC and IR was clearly visualized. However, objective quantification of the staining signal was impeded by extensive variation in the arrangement and density of GC and the amount of cellular debris on the slides.Altogether, strong IR presence in GC, most notably in SAF, suggests acquisition of IR as a key event in early follicle growth. Furthermore, we have developed a quantifiable staining technique for bovine follicles that may be applicable for GC obtained in live cows, although this method requires further standardization.     

Sphingolipids in apoptosis, survival and regeneration in the nervous system

Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.     

Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine “irregular” patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.     

Peroxisome deficiency-induced ER stress and SREBP-2 pathway activation in the liver of newborn PEX2 knock-out mice

Disruption of the Pex2 gene leads to peroxisome deficiency and widespread metabolic dysfunction. We previously demonstrated that peroxisomes are critical for maintaining cholesterol homeostasis, using peroxisome-deficient Pex2^−/− mice on a hybrid Swiss Webster × 129S6/SvEv (SW/129) genetic background. Peroxisome deficiency activates hepatic endoplasmic reticulum (ER) stress pathways, leading to dysregulation of the endogenous sterol response mechanism. Herein, we demonstrate a more profound dysregulation of cholesterol homeostasis in newborn Pex2^−/− mice congenic on a 129S6/SvEv (129) genetic background, and substantial differences between newborn versus postnatal Pex2^−/− mice in factors that activate ER stress. These differences extend to relationships between activation of genes regulated by SREBP-2 versus PPARα. The SREBP-2 pathway is induced in neonatal Pex2^−/− livers from 129 and SW/129 strains, despite normal hepatic cholesterol levels. ER stress markers are increased in newborn 129 Pex2^−/− livers, which occurs in the absence of hepatic steatosis or accumulation of peroxins in the ER. Moreover, the induction of SREBP-2 and ER stress pathways is independent of PPARα activation in livers of newborn 129 and SW/129 Pex2^−/− mice. Two-week-old wild-type mice treated with the peroxisome proliferator WY-14,643 show strong induction of PPARα-regulated genes and decreased expression of SREBP-2 and its target genes, further demonstrating that SREBP-2 pathway induction is not dependent on PPARα activation. Lastly, there is no activation of either SREBP-2 or ER stress pathways in kidney and lung of newborn Pex2^−/− mice, suggesting a parallel induction of these pathways in peroxisome-deficient mice. These findings establish novel associations between SREBP-2, ER stress and PPARα pathway inductions.