Seasonal mammals use the photoperiodic variation in the nocturnal production of the pineal hormone melatonin to synchronize their reproductive activity with seasons. In rodents, the (SD) short day profile of melatonin secretion has long been proven to inhibit reproductive activity. Lately, we demonstrated that melatonin regulates the expression of the hypothalamic peptides kisspeptins (Kp) and RFamide-related peptide-3 (RFRP-3), recently discovered as potent regulators of gonadotropin-releasing hormone (GnRH) neuron activity. In the male Syrian hamster, Kp expression in the arcuate nucleus is down-regulated by melatonin independently of the inhibitory feedback of testosterone. A central or peripheral administration of Kp induces an increase in pituitary gonadotropins and gonadal hormone secretion, but most importantly a chronic infusion of the peptide reactivates the photo-inhibited reproductive axis of Syrian hamsters kept in SD conditions. RFRP-3 expression in the dorsomedial hypothalamus is also strongly inhibited by melatonin in a SD photoperiod. Although RFRP-3 is usually considered as an inhibitory component of the gonadotropic axis, a central acute administration of RFRP-3 in the male Syrian hamster induces a marked increase in gonadotropin secretion and testosterone production. Furthermore, a chronic central infusion of RFRP-3 in SD-adapted hamsters reactivates the reproductive axis, in the same manner as Kp. Both Kp and RFRP-3 neurons project onto GnRH neurons and both neuropeptides regulate GnRH neuron activity. In addition, central RFRP-3 infusion was associated with a significant increase in arcuate Kp expression. However, the actual sites of action of both peptides in the Syrian hamster brain are still unknown. Altogether our findings indicate that Kp and RFRP neurons are pivotal relays for the seasonal regulation of reproduction, and also suggest that RFRP neurons might be the primary target of the melatoninergic message. Living on earth imposes daily and annual changes in light, temperature, and humidity, which in turn cause cyclic changes in food availability. Organisms therefore must anticipate these predictable changes and adapt their biological functions to ensure their survival. Specifically, most mammals restrict their fertility to a particular time of the year to ensure that the birth and weaning of the offspring occur during the most favorable season. Although various environmental factors are important to consider, a majority of mammalian species use the highly predictable annual variations in light duration (or photoperiod) to establish the time of year and adapt their reproductive activity accordingly.The photoperiod is converted into neuroendocrine signals via a dedicated photoneuroendocrine pathway, which involves the master biological clock located in the suprachiasmatic nuclei and other hypothalamic nuclei, which in turn synchronize various biological activities with the time of the day and year  and RFRPs, we chose to develop a backwards approach to delineate how melatonin may synchronize reproduction in seasonal rodents.In mammals, early studies on the regulation of the reproductive axis have emphasized the pivotal role of the pulsatile release of gonadotropin-releasing hormone (GnRH), a decapeptide synthesized by a small population of neurons located in the rostral hypothalamus, mainly the preoptic area (POA), and released principally into the pericapillary space of the pituitary portal system at the median eminence  precursor further processed into Kp fragments of smaller sizes (from 54 aa in humans or 52 aa in rodents down to 10 aa). All fragments share the same 10 amino acid C-terminal sequence with a final amidated Arg-Phe-(or Tyr in rat and mice)-NH2 motif, a hallmark of the RFamide super family of peptides , and a more rostral area, the anteroventral periventricular nucleus  and negative (in ARC) central feedback effects of sex steroids , the spontaneous sexual reactivation was associated with an increase in Kiss1 mRNA in the ARC, indicating that Kiss1 expression also escapes melatonin’s inhibitory signal in photorefractory animals  according to the protocol of Chomczynski and Sacchi . PCR amplified products of the predicted size (263-bp) were purified and ligated into the pCR2.1 cloning vector (Invitrogen). Identity of the clone was confirmed by sequencing (AGOWA) and the obtained sequence was deposited in GenBank (accession number GQ872419). The Syrian hamster Kiss1r cloned sequence shares 94 and 92% identity with the corresponding sequence of the known Kiss1r genes of mice and rats, respectively.For d Sacchi  and reveIn situ hybridization with both sense and antisense [35S]UTP-labeled Kiss1r riboprobes was performed on 20 μm thick frozen hamster brain sections containing the main part of the diencephalon. Tissue sections were submitted to prehybridization treatments before an overnight hybridization of the riboprobe at 54°C in a 2X SSC/50% formamide hybridization medium. The posthybridization consisted in an RNAse (Sigma) treatment and a final high stringency wash in 0.01X SSC at 56°C to eliminate most of the non-specific labeling. The hybridized sections were subsequently exposed to X-ray films for 10 days and scanned for quantitative analysis.Kiss1r mRNA, the ARC in rats, and rhesus monkeys directly on the kisspeptin system via melatonin has not been demonstrated so far. Importantly, Kp infusion during the anestrous season caused ovulation in more than 80% of treated animals whereas <20% of control animals ovulated, indicating that in sheep like in hamsters, Kp are a critical component of the seasonal regulation of reproductive activity  and inhibitory (in the ARC) feedback effect of sex steroids. In the Siberian hamster, sex steroid feedback appears more potent than that of melatonin to control Kp expression. In the sheep, as opposed to hamsters, Kp expression is inhibited in LD when animals are sexually quiescent. To complicate matters some more, metabolic factors like leptin or food resources, also influenced by the seasons, impact on Kp expression  gene is expressed in neurons located in the mediobasal hypothalamus and it encodes a precursor that produces two peptides, RFRP-1 and RFRP-3  motif were shown to regulate reproductive activity in birds and mammals  and peripheral (pituitary gonadotropes) sites of action (Clarke et al., Kiss1 expression the ARC.In an attempt to evaluate whether the photoperiodic variation in RFRP expression is critical for the seasonal timing of reproductive activity, we tested the effect of chronic RFRP-3 administration in SD-adapted male Syrian hamsters. Actually, we observed that a 5-week icv infusion of a low dose of RFRP-3 was able to reactivate testicular function, to levels comparable to those observed in LD-adapted hamsters (Ancel et al., Taken together, these studies indicate that the SD conditions, via an increased secretion of melatonin, inhibit RFRP expression in the mediobasal hypothalamus of various seasonal breeders, irrespective of their seasonal physiology. Of interest however, the effects of RFRP-3 diverge among seasonal breeders, being stimulatory in LD breeders like hamsters and inhibitory in SD breeders like sheep. Therefore, it is tempting to speculate that RFRP neurons may play a key role in discriminating between LD and SD breeders. In order to fully understand the role of RFRP-3 in the seasonal control of reproduction, further experiments will have to be carried out in other species with particular attention to the animal gender.Both Kp and RFRP-3 regulate reproductive activity mainly via an action on GnRH neurons. Clearly, Kp and RFRP-3 act independently on the gonadotropic axis since RF9; a putative RFRP-3 antagonist is capable of increasing LH in mice devoid of Kiss1r (García-Galiano et al., In a variety of mammalian species both Kp and RFRP fibers project onto GnRH neurons and both Kiss1r and GPR147 receptors are expressed in GnRH neurons. According to studies and species, about 80–90% of GnRH neurons are targeted by Kp-ir fibers, express Kiss1r, and respond to a direct action of Kp (Irwig et al., On the other hand, Kp-ir fibers (Clarkson et al., Gpr147 in the Syrian hamster hypothalamus and design highly selective RFamide receptor ligands.In our early experiment in the Syrian hamster, we did not check whether the stimulatory effect of Kp on the reproductive axis (Revel et al., Several studies have now reported that Kp and RFRP-3 are regulated by photoperiod/melatonin and play a critical role in the regulation of seasonal reproduction. Kp are always very potent activators of the gonadotropic axis, ultimately able to reactivate reproduction of photoinhibed animals, either LD or SD breeders. Importantly, variation in Kp expression appears to depend on the species’ seasonal physiology, probably as a results of Kp sensitivity to a large array of endocrine and metabolic signals, particularly melatonin, leptin, and sex steroids, which all are critical for the regulation of seasonal breeding. In contrast, RFRP-3 expression appears to be reproducibly down-regulated in SD in the various species studied up to date. In Syrian and Siberian hamsters, the SD inhibition is driven by melatonin and independent of sex steroid feedback. Although RFRP-3 is considered an inhibitor of gonadotropin secretion in rats, mice, and sheep, it is able to stimulate the gonadotropic axis in male Syrian and Siberian hamsters. Furthermore, a chronic infusion of the peptide in SD-adapted hamsters restores an LD profile of both ARC Kp expression and reproductive activity. From these findings, we propose a working model for the seasonal regulation of reproduction in the male Syrian hamster (Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The only criterion which is commonly used to evaluate oocyte quality is its morphology. There is a mass of oocytes in the in vitro fertilization programme which are not fertilized in spite of normal morphology. In the past, several attempts focused on oocyte gene expression profiling by different approaches. The results elucidated groups of genes related to the human oocyte. It was confirmed that some factors, such as oocyte in vitro maturation, are detectable at the molecular level of human oocytes and their polar bodies in terms of gene expression profile. Furthermore, the first genetic evaluations of oocyte-like cells developed in vitro from human stem cells of different origin were performed showing that these cells express some genes related to oocytes. All these findings provide some new knowledge and clearer insights into oocyte quality and oogenesis that might be introduced into clinical practice in the future.The quality of the human oocyte determines the success of fertilization and affects the consequent embryo development, pregnancy and birth; it therefore serves as a basis for human reproduction and fertility. The possibility to evaluate oocyte quality in the  This is mainly due to a lack of reliable criteria on oocyte quality and the selection of embryos with sufficient developmental potential. The quality of oocytes obtained under ovarian stimulation for in vitro fertilization varies considerably and depends on several factors. A large proportion of oocytes are capable of being fertilized, but only approximately half of fertilized oocytes fully complete their preimplantation development and still fewer implant [in vitro from different types of stem cells. These include human embryonic stem cells (hESCs) [in vivo and in vitro. A viable quality human oocyte is a prerequisite for successful fertilization, implantation, and development of a new human being. Although an enormous development of techniques of till low . A mass  implant . More st (hESCs) –7, human (hESCs) , 7, puta (hESCs) –10, and  (hESCs) . Therefoμm when mature. The oocyte is rich in cytoplasm that contains yolk granules to support the cell's growth, maturation, and the early development after fertilization. In vivo it is produced in the ovarian follicle, a functional unit of the ovary, during the process of oogenesis/folliculogenesis [primary oocyte. The diploid primary oocyte, however, does not complete meiosis I but is stopped at the first meiotic prophase stage, called dictyate. At this stage the oocyte nucleus is called the germinal vesicle (GV), as this stage refers to the GV stage of maturity. GV oocytes are localized within the primordial follicle, also consisting of a flattened and condensed layer of surrounding mesenchymal granulosa cells. By the end of the fetal period, all primary oocytes have formed and are stopped at the dictyate stage. Although meiosis was arrested, the dictyate chromosomes continue to synthesize large amounts of mRNA and rRNA, which are later used to generate a bulk of essential proteins needed for oocyte maturation and further development of the fertilized oocyte and embryo [primary oocytes are maintained for years, until puberty (menarche). Upon puberty the primary oocytes finish meiosis I, and a primary oocyte divides into two daughter cells: a haploid secondary oocyte and an extruded nonfunctional polar body. During the menstrual cycle only a few of primary oocytes are recruited, and only one matures and is ovulated. When this secondary oocyte enters meiosis (meiosis II), it does not finish but is arrested again and held at the metaphase II (MII) stage until fertilization. The MII-stage oocyte has the potential to be fertilized. When the oocyte is fertilized, the process of meiosis is terminated and the second polar body is extruded. During the menstrual cycle only 15–20 early antral follicles/oocytes are recruited and only one dominant follicle matures fully and is ovulated. Each oocyte develops and matures in the functional unit of the ovary—the follicle. The primary oocytes grow and mature during the development and maturation of primordial, primary and secondary follicles, whereas the secondary oocytes develop along with the tertiary and preovulating Graafian follicle. This process is regulated by hormones and other substances.An oocyte is a female germ cell involved in reproduction. It is one of the largest cells in the body with a diameter of at least 100 ogenesis . The oogd embryo . Then thin vitro fertilization programme oocytes are retrieved after hormonal stimulation of patient's ovaries and by ultrasound guided aspiration. Oocytes at different stages of maturity can be retrieved. Most oocytes are mature metaphase II (MII) oocytes, while a relatively small proportion of oocytes are immature metaphase I (MI) oocytes and prophase I (PI) or germinal-vesicle- (GV-) stage oocytes. The MII oocytes are characterized by a round shape, zona pellucida, and an extruded polar body; MI oocytes by a round shape, zona pellucida, and the absence of a polar body; GV-stage oocytes by a round shape, zona pellucida, and the presence of a germinal vesicle  complexes and then translated when needed ; additioThe first gene expression analyses in human oocytes were performed by reverse transcription-polymerase chain reaction , and chromatin modification is in agreement with the oocyte need to remodel the sperm chromatin after fertilization. The human oocyte is proposed to be transcriptionally silent at the MII stage of maturity but is very active in transcription and translation during the growth phase and must be prepared to initiate transcription during embryonic genome activation at the 4- to 8-cell embryo stage. The authors concluded that further understanding of the biological role of genes up-regulated in mature human oocytes may extend the knowledge on the meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. Moreover, Assou et al. found 1,514 genes which were up-regulated in human oocytes in addition to already known genes, such as DAZL, BMP15, or GDF9. Among them were the meiosis-related genes PTTG3 (securing) and AURKC (Aurora kinase), previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14 and IL4, as well as transcription factors, including OTX2, SOX15, and SOX30 [Using microarray analysis, Kocabas et al. analyzed the transcriptome of non-fertilized mature MII oocytes from the rogramme . They annd SOX30 . The mosFurther attempts were made to optimize a microarray-based approach for deriving representative gene expression profiles of human oocytes. Since the human oocyte is a low RNA template sample which contains approximately 55 to 100 pg of polyA+ mRNA , 21, it in vitro fertilization programme can also be successfully analyzed by the Biomark Real-Time Quantitative PCR (qPCR) System (Fluidigm). This methodology enables qualitative analyses of single cells after preamplification. The advantage of the methodology is that many genes can be analyzed at the same time. In our preliminary work 19 single human oocytes at different stages of maturity , and 4 mature MII oocytes) were analyzed on 56 genes related to pluripotent stem cells and oocytes by the Fluidigm system after the preamplification procedure. The analyzed genes were selected to elucidate the relation of human oocytes to hESCs and to better understand the molecular status of oocyte-like cells developed in vitro from stem cells of different origins, including pluripotent hESCs and iPSCs. The inventoried TaqMan assays (Applied Biosystem) were pooled to a final concentration of 0.2× for each assay. The cells to be analyzed were harvested directly into RT-PreAmp Master Mix. The harvested cells were immediately frozen and stored at −80°C. Cell lysis and sequence-specific reverse transcription were performed at 50°C for 15 minutes. The reverse transcriptase was inactivated by heating to 95°C for 2 minutes. Subsequently, cDNA went through limited sequence-specific amplification by denaturing at 95°C for 15 seconds and annealing and amplification at 60°C for four minutes for 14 cycles. These pre-amplified products were diluted 5-fold prior to analysis with the Universal PCR Master Mix and inventoried with TaqMan gene expression assays in 96.96 Dynamic Arrays on a BioMark System. Ct values obtained from the BioMark System were transferred to the GenEx software (MultiD) to analyze the gene expressions. The results confirmed that a small proportion—three outstanding oocytes (two immature MI oocytes and one in vitro matured oocyte IVM)—differed at the molecular level from other oocytes and did not cluster with them, as revealed by heatmap, hierarchical clusterings, and principal component analysis  and small ubiquitin-like modifier isoforms  in four single MII-stage oocytes, two 4-cell and three 8-cell embryos using the reverse transcription-polymerase chain reaction [Wells et al. quantified the expression of nine different genes y RT-PCR , as can reaction . The mRNin vitro fertilization programme. In the study of Li et al. the cDNA microarray from single oocytes and 4- and 8-cell embryos were used to elucidate the differential expression profiles [Using microarray technology Dobson et al. analyzed gene expression in single human oocytes and preimplantation embryos to elucidate the gene expression profile during human preimplantation development from the oocyte to the 8-cell stage embryos . The resprofiles . In oocyNOBOX gene in human ovarian follicles and oocytes and presented the first cDNA cloning and transcript expression analysis of the human NOBOX gene using RT-PCR [ NOBOX is a homeobox gene that is preferentially expressed in the oocytes and ovarian follicles and was first identified in the mouse. When analyzing adult human tissues, they found that the expression of this gene is limited to the ovary, testis, and pancreas. The NOBOX gene expression within the ovary was oocyte-specific and was found from the primordial stage of ovarian follicles through to the mature MII oocytes. Additionally, Huntriss et al. analyzed the expression profiles of 14 additional homeobox genes during human oogenesis and early development. Using RT-PCR they found that the expression of HOXA10 was limited to primordial and early primary follicles. The gene HOXB7 was expressed from primordial and early primary stage follicles to the GV-stage oocytes. The homeobox genes gastrulation brain homeobox 1 (GBX1) and HOXA7 were predominately expressed by GV oocytes and HOXA1 and HEX by MII oocytes. It was concluded that the homeobox gene transcripts detected in ovarian follicles and oocytes were distinct from those expressed in human embryos at the blastocyst stage  and in granulosa cells . Huntriss et al. studied the expression of the g RT-PCR , as can TMEFF2): the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). The genes TMEFF2  and OPHN1 were among the most expressed genes overall. In oocytes from primordial/primary follicles the mitochondria-related gene DC48 was also strongly expressed. In the oocytes from the primordial/primary follicles the genes related to ovarian steroidogenesis were not expressed or were only weakly expressed. In terms of steroid receptors the androgen receptor (AR), estrogen receptor 2 (ER2), and the progesterone receptor membrane component 1 + 2 were expressed at a low level, while there was no expression of the follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR). Among transcription factors several genes from the POU and Forkhead families were expressed in oocytes from early follicles: POUF3F2 (OCT-3), POU4F2, POU2F1 (OCT-1), POU5F1 (OCT-4), and FOXO1, FOXO3, FOXR1, FOXP1. Among TGFβ family of growth factors and other growth factors were several genes which were not expressed: bone morphogenetic protein (BMP15), anti-Mullerian hormone (AMH), AMH receptor 2 (AMRH2), activin/inhibin subunits, insulin-like growth factor I (IGFI), platelet-derived growth factor (PDGF) and its receptor, and epidermal growth factor (EGF) and its receptor (EGFR) were not expressed in appreciable levels, while in contrast a number of BMP receptors (BMP1A and BMP1B), activin receptors , IGFI receptor, fibroblast-growth factor (FGF)-9, FGF14, FGF receptor 4, and transforming growth factor alpha (αTGF) were consistently expressed. In oocytes from early follicles several genes of signal transduction were expressed, such as genes for enzymes phosphatidylinositol 3-kinase (PI3K), PI4K, and PTEN that regulate the levels of phosphatidylinositol 3,4,5-triphosphate and genes for phosphodiesterases  and for the mitogen-activated protein kinase I (MAPKI). In oocytes from primordial follicles some oocyte or germline-specific genes were found to be expressed, such as the factor in the germline alpha (FIGLA), zona pellucida glycoproteins ZP1, ZP2, ZP3, ZP4, spermatogenesis and oogenesis-specific basic helix-loop-helix I (SOLHI), SOLH2, the newborn ovary homeobox (NOBOX) gene, the maternal embryonic leucine zipper kinase (MELK), and oocyte-specific gene (MATER). The authors proposed a technique of laser capture microdissection combined with transcriptome analysis of the rare material of human oocytes from the early follicle stages as an important tool to gain better understanding of early human folliculogenesis and oogenesis. These results indicated some genes which might be related to the oocyte maturation process.Markholt et al. have isolated pure populations of oocytes from the early follicles at different stages of maturity—primordial, intermediate, and primary follicles—by laser capture microdissection methodology and analyzed them by whole-genome microarray analysis . The micin vivo equivalent or whether their properties mostly reflect their artificial culture environment. Some authors have proposed that they might be related to the germinal lineage [in vitro fertilization methodology and regenerative medicine. Human mature MII-stage oocytes and hESCs have some common features: both are able to achieve cell reprogramming towards pluripotency in different ways . The comparison of the transcriptome of these two types of cells may highlight some genes that are involved in the pluripotency initiation. Zhang et al. confirmed the expression of distinct sets of developmentally regulated genes that are expressed by both the human oocytes and hESCs [in vitro fertilization programme, hESCs, and human foreskin fibroblasts. In their study 10,183 genes were expressed in human GV oocytes, and 45% of these genes were still unclassified by their biological function. Four oocyte-specific genes—MATER, ZAR1, NPM2, and FIGLA—were for the first time detected in human GV-stage oocytes. Additionally, the components of 4 signaling pathways—MOS-MPF, TGF-β, Wnt, and Notch—were also found to be expressed in human GV oocytes. Distinct sets of genes were detected by comparison of gene expression profiles between human GV oocytes, hESCs, and human foreskin fibroblasts, and these gene sets may be involved in the processes of oocyte maturation and early embryonic development. Furthermore, Assou et al. compared the gene expression profile of mature MII oocytes from the in vitro fertilization programme and hESCs to that of somatic tissues [METAP2, SHMT2, etc.), nucleoside synthesis , DNA repair including mismatch repair (MSH2 and MSH6) or base excision repair (UNG and PCNA), main components of the cell cycle regulatory machinery , regulators of the topologic state of DNA (TOP1 and TOP2A) and components of the mitotic spindle assembly checkpoint , genes related to pluripotency , large chromatin remodeling network genes , 18 different zinc finger transcription factors, including ZNF84, and several genes which are still poorly understood, such as KLHL7, MRS2, or the selenophosphate synthetase 1 (SEPHS1). Additionally, a large set of genes was also found to encode proteins involved in the ubiquitination and proteasome pathways. After differentiation of hESCs into embryoid bodies, the transcription of these gene pathways was found to decline. These data indicated the important relation of oocytes to hESCs but also some important oocyte-specific differences. The real origin of hESCs is still a matter of debate. Thus far it has been believed that they most closely resemble pluripotent primitive ectoderm cells derived from the blastocyst inner cell mass. However, differences between ESCs and primitive ectoderm cells have opened the question whether ESCs really have an  lineage . Early hnd hESCs . The mai tissues . They idin vitro development of hESC-derived germ-like cells and that they may play an important role in human oogenesis. There were also several successful attempts to culture oocyte-like cells (OLCs) from hESCs –7, hiPSCVASA, DAZL, GDF3, GDF9, MLH1, SCP1, PUM1, PUM2, and POU5F1  and late germ cells and meiosis , as can be seen in in vitro from stem cells might pass through the process of meiosis and finally differentiated in haploid cells. Medrano et al.  found thin vitro developed OLCs expressed some oocyte-specific genes [The OLCs and parthenogenetic-like structures were developed from the putative human ovarian stem cells from the ovarian surface epithelium of women with no naturally present oocytes and follicles—postmenopausal women and women with premature ovarian failure—as reported by our group –10. Thesic genes , 9 and aμm and zona-pellucida- (ZP-) like structures were observed. They were trying to elucidate if the bone morphogenetic protein 15 (BMP15) gene was activated during the differentiation of human amniotic fluid stem cells into OLCs. When OLCs were analyzed by RT-PCR, BMP15 was activated from approximately day 10 of cell differentiation. Additionally, the green-fluorescent-protein- (GFP-) BMP15 was transfected into the differentiating human amniotic stem cells, and its expression was positive in the OLCs. The RT-PCR analysis showed that the oocyte-specific genes, such as ZP1, ZP2, ZP3, and c-kit, were expressed in the differentiating hAFSCs, and the immunofluorescence assay confirmed that the ZP2 was detected in the OLCs. Using quantitative RT-PCR they found that that ZP2 and ZP3 expressions were significantly elevated in the differentiating stem cells. They concluded that the BMP15 could be used as an important marker of oogenesis to follow the differentiation of amniotic fluid stem cells and other stem cells into the OLCs.Recently, Cheng et al. successfully developed stem cells from human amniotic fluid (hAFSCs) into OLCs . After 1μm, and some of them expressed a zona-pellucida-like structure. In spite of some promising new knowledge, the existing results of genetic analyses showed that the OCLs developed in vitro are still far from real, fully competent human oocytes. They mostly failed to progress through the whole process of meiosis in spite of the complex in vitro maturation medium; therefore, the transplantation of these cells to mature them in vivo was proposed as a more reliable approach which may lead to potential clinical practice in the future [in vitro would be of great importance for the treatment of severe ovarian infertility . Yet, in general, the OLCs which developed in vitro from human stem cells have not been genetically analyzed in greater detail, including the microarrays. This reveals the need to analyze the OLCs genetically and epigenetically in more detail in comparison with mature oocytes from the in vitro fertilization programme to evaluate their quality and safety in the future. At present the most important task is to better elucidate the expression of oocyte-specific genes  as a feeder layer . The ooce future . Developenes see . BRCA1 and 2, ATM, TP53, RB1, BUB1, MAD2, APC, and ACTB was analyzed. The expression of ACTB and MAD2 was present in all samples. Gene RB1 was down-regulated in oocytes, while the genes BUB1, BRCA1 and 2, and MAD2 were down-regulated in cumulus cells. The expression of BRCA1 and 2 was absent in cumulus cells, while the expression of TP53 was absent in MI oocytes and RB1 expression in MII oocytes. They identified new potential regulators and marker genes involved in the human in vivo oocyte maturation, such as BARD1, RBL2, RBBP7, BUB3, or BUB1B  and the highest number in GV oocytes . On the other hand, in MII oocytes 444 genes were overexpressed, while in GV oocytes only 104 genes were overexpressed; in the transient MI oocytes only 4 genes were overexpressed. The expression variations between GV, MI, and MII oocytes were low. Similarly, Wells and Patrizio [in vivo matured MII oocytes the lowest number. Most of the differences between GV-stage oocytes and other stages were the consequence of a reduction in the number of mRNA transcripts occurring during progression to MII. This suggested that relatively little new gene expression occurred after the GV stage of maturity, with degradation of mRNA transcripts leading to the differences in transcript numbers observed at later stages of maturity in spite of the fact that the post-GV oocytes were not completely quiescent in terms of gene transcription, as revealed by the detection of some transcripts absent at the GV stage. Differently expressed genes were classified according to their molecular functions and biological processes. In in vivo matured MII oocytes. For example, a number of genes for storage proteins displayed up-regulation in GV oocytes compared with in vivo matured MII oocytes.The maturity of the human oocyte is one of the crucial features to sustaining a successful pregnancy to birth. It is known that immature human oocytes at the GV stage and MI stage of maturity cannot be fertilized. Human oocyte maturity is related to nuclear and cytoplasm maturity. Gasca et al. analyzed a pool of immature oocytes (20 GV oocytes in 7 patients and 20 MI oocytes in 7 patients), 37 non-fertilized mature MII oocytes, and cumulus cells by microarrays [or BUB1B . In the turation . The expytes see . FurtherPatrizio  found thIn vitro maturation of human oocytes is an attractive strategy for in vitro fertilization treatment, especially in women with polycystic ovarian syndrome (PCOS) and with a risk of ovarian hyperstimulation after gonadotropin administration for oocyte retrieval. There is also a proportion of immature oocytes which are discarded in daily medical practice because of their immaturity. However, current in vitro maturation protocols unfortunately produce oocytes with a poor clinical outcome shown by poor embryo development and early pregnancy loss [in vitro matured oocytes. Wells and Patrizio analyzed in vivo matured oocytes at GV or MII stages of maturity and compared the gene expression profiles between MII oocytes matured in vivo and in vitro [in vitro fertilization programme were analyzed for more than 29,000 genes with RNA amplification and microarray technology. It was found that GV oocytes expressed 12,219 genes, in vivo matured MII oocytes 9,735, and in vitro matured MII oocytes 8,510. It is interesting that in vivo matured MII and in vitro matured MII oocytes shared very similar patterns of gene expressions, but they also noted some significant differences (see DAZL) and genes related to meiosis  were also among genes which were up-regulated in oocytes matured in vitro. In in vivo versus in vitro. Some immature GV oocyte patterns of gene expression still persisted in MII oocytes matured in vitro. Although the in vitro matured MII oocytes closely resemble the in vivo matured oocytes, especially for genes related to nuclear maturity, there were several genes related to cytoplasmic functions which were expressed in an immature manner. It seems that the cytoplasmic maturation may be a crucial point regarding the worse clinical outcome obtained by in vitro matured MII oocytes. In vivo matured MII oocytes also expressed some genes associated with cellular storage and homeostasis differently. ncy loss . This isin vitro . The nonnces see . It is iin vitro in comparison with in vivo matured oocytes was elucidated [in vitro than those matured in vivo, and 162 of them were expressed at 10-fold or greater levels in oocytes matured in vitro  and cell cycle regulation  that may lead to a reduction in the oocyte developmental competence, while in vitrified oocytes many genes of the ubiquitination pathway were down-regulated, including those of the ubiquitin-specific peptidase family and subunits of the 26S proteasome. Such inhibition of the oocyte degradation machinery might stabilize the maternal protein content which is necessary for oocyte developmental competence. They concluded that the low pregnancy rates achieved by frozen-thawed human MII oocytes could be explained by the alterations of the oocyte gene expression profile. Oocytes matured otherapy –41  and fertile control patients diagnosed with tubal or male infertility [in vitro fertilization programme may express some molecular abnormalities at the gene expression level, especially those related to the failure of MII oocyte activation. The authors proposed the microarray approach to improve the clinical therapeutic treatment and to offer informed counseling to the patients about alternate therapeutic solutions. These studies need to be performed with a higher number of oocytes to make a real conclusion. Not only were the procedures of oocyte cryopreservation confirmed to be related to the changed oocyte gene expression profile, but so was the total fertilization failure (no fertilization of oocytes) in the ertility . Transcrin vitro fertilization programme some improved methods are needed to reliably evaluate oocyte quality prior to fertilization and to choose the right embryos to implant. Recent evaluation of oocyte quality is based only on the evaluation of morphology, but this is very limited, and also oocytes with high-grade morphology sometimes do not fertilize or develop into the embryo. mRNA and proteins produced during the oogenesis support embryonic development until the zygotic transition. Polar body gene expression profiling might be an important safe tool to verify the molecular status of in vivo or in vitro matured oocytes before the in vitro fertilization procedure. The first polar body is extruded from a mature oocyte before fertilization and can be biopsied without damaging the oocyte. The polar body transcriptome was proposed as a potential tool for the evaluation of oocyte quality in the in vitro fertilization programme [in vitro fertilization programme is present and detectable in a single polar body prior to fertilization [BCL2L10, ODC1, DPPA3, PADI6, DDX4, AGO2, DROSHA, GAPDH, EIF6, PABP, DICER); interestingly, the oocyte-specific gene H1FOO was not expressed in any single polar body. Transcripts that were present in greater abundance in the single oocytes were also detected in qPCR replicates from single polar bodies. They concluded that preamplification of cDNA synthesized without RNA isolation can facilitate the quantitative detection of mRNA in single human polar bodies. This study was followed by the study by Reich et al. [in vitro fertilization and might become a very important safe tool to evaluate the molecular status of in vitro matured oocytes and improve the clinical outcome of oocyte in vitro maturation in the future.In the rogramme , 31. Klalization . In theiin vitro fertilization programme [SMAD2 and activin B1 receptor). It was concluded that these genes may be associated with the ageing process and decreased fertility. Advanced female age may be also related to oocyte aneuploidy . Fragouli et al. confirmed the link between mRNA transcript numbers in oocytes and female age [It is known that female fertility and development competence of human oocytes decline with increasing female age and that the proportion of oocytes with genetic abnormalities increased with female ageing. Steuerwald et al. found that the global gene expression profiles in human oocytes are related to female age . Their rrogramme . Based omale age . They coin vitro maturation procedure, it is more appropriate to be applied in younger women because of a higher risk of oocyte genetic abnormalities in older women. Increased female age may be an important contraindication for the oocyte in vitro maturation procedure. When talking about the oocyte in vitro oogenesis in women with no naturally present oocytes in the future. All these new findings provide some new knowledge and better insights into human oocyte quality and oogenesis that might be introduced in clinical practice and might also lead to successful


In addition, PTEN deficient dendritic cells showed reduced activation of p38 MAP-kinase and increased inhibitory phosphorylation of GSK3β in vitro. Dendritic cell and macrophage phenotypic maturation and migration to lymph nodes as well as collagen specific T and B cell activation was comparable in wt and myeloid specific PTEN-/-. However, analysing the impact of myeloid specific PTEN deficiency on T cell polarization, we found a significant reduction of a Th17 type of immune response characterized by reduced production of IL-17 and IL-22. Moreover, there was an increase in IL-4 production and higher numbers of regulatory T cells myeloid specific PTEN-/-. In contrast, myeloid specific PTEN deficiency did not affect serum transfer arthritis, which is independent of the adaptive immune system and solely depends on innate effector functions. These data demonstrate that the presence of PTEN in myeloid cells is required for the development of systemic autoimmunity. Deletion of PTEN in myeloid cells inhibits the development of CIA and EAE by preventing the generation of a pathogenic Th17 type of immune response.Autoreactive T cells are a central element in many systemic autoimmune diseases. The generation of these pathogenic T cells is instructed by antigen presenting cells. However, signalling pathways in APC that drive autoimmunity are not completely understood. Here we show that that conditional deletion of PTEN in myeloid cells are almost completely protected from the development of two prototypic model autoimmune diseases, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE). Myeloid specific deletion of PTEN lead to a significant reduction of cytokines pivotal for the induction of systemic autoimmunity such as IL-23 and IL-6


For many years, adipose tissue was considered as an inert energy storage organ that accumulates and stores triacylglycerols during energy excess and releases fatty acids in times of systemic energy need. However, over the last two decades adipose tissue depots have been established as highly active endocrine and metabolically important organs that modulate energy expenditure and glucose homeostasis. In rodents, brown adipose tissue plays an essential role in non-shivering thermogenesis and in energy dissipation that can serve to protect against diet-induced obesity. White adipose tissue collectively referred too as either subcutaneous or visceral adipose tissue is responsible for the secretion of an array of signaling molecules, termed adipokines. These adipokines function as classic circulating hormones to communicate with other organs including brain, liver, muscle, the immune system, and adipose tissue itself. The dysregulation of adipokines has been implicated in obesity, type 2 diabetes, and cardiovascular disease. Recently, inflammatory responses in adipose tissue have been shown as a major mechanism to induce peripheral tissue insulin resistance. Although leptin and adiponectin regulate feeding behavior and energy expenditure, these adipokines are also involved in the regulation of inflammatory responses. Adipose tissue secretes various pro- and anti-inflammatory adipokines to modulate inflammation and insulin resistance. In obese humans and rodent models, the expression of pro-inflammatory adipokines is enhanced to induce insulin resistance. Collectively, these findings have suggested that obesity-induced insulin resistance may result, at least in part, from an imbalance in the expression of pro- and anti-inflammatory adipokines. Thus we will review the recent progress regarding the physiological and molecular functions of adipokines in the obesity-induced inflammation and insulin resistance with perspectives on future directions. Excess nutrition and sedentary lifestyle induce excessive lipid accumulation in adipose and peripheral tissues resulting in obesity. Obesity has become a pandemic health problem in which more than 60% of American adults are overweight or obese and is closely associated with metabolic diseases such as insulin resistance, type 2 diabetes (T2D), hypertension, non-alcoholic fatty liver disease, and polycystic ovarian diseases , glycogen storage , and inhibits gluconeogenesis and glycogenolysis (liver). Insulin also increases lipogenesis in hepatocytes and adipocytes and diminishes adipocyte free fatty acid generation from triacylglycerols (lipolysis)  mutations are very rare but at least more than 30 INSR mutations have been shown to mediate insulin receptor dysfunction, and these mutations may induce insulin resistance with polygenic defects in its downstream signaling , anti-inflammatory IL-10 producer, are unique cell population that suppresses inflammation, and Treg cells are decreased in HFD-induced and genetically modified obese mice resulting in insulin resistance  colitis and experimental autoimmune encephalomyelitis (EAE)  , which regulate food intake. Leptin expressed from obese gene is identified by positional cloning and has been shown to regulate feeding behavior through the hypothalamic regulation in central nervous system  and its proximal Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signaling pathway. Leptin activates monocytes and macrophages to produce pro-inflammatory IL-6, TNF-α, and IL-12 , and leptin indeed induces inflammatory responses through the long isoform of the leptin receptor b , 10 kDa polypeptide with 114 amino acids in rodents, is identified as an inducer of pulmonary inflammation  and CCR5 are typical chemokine and chemokine receptor, respectively that mediate inflammatory responses and are significantly enhanced in obese rodents and humans. Accordingly CCR2, the receptor of CCL2, deficient mice show attenuated macrophage infiltration, inflammation, and insulin resistance , and ANGPTL2 expression is enhanced in obese humans and rodents . Thus SFRP5 that is highly expressed in adipocytes of mouse white adipose tissues prevents the binding of WNT proteins to its receptors. WNT proteins especially WNT5a is closely linked inflammatory responses. The expression of SFRP5 is decreased, but the expression of WNT5a, an antagonizing target of SFRP5, is increased in white adipose tissues of obese rodents and humans suggesting that SFRP5 might have potential to attenuate inflammatory effect of WNT5a in adipose tissues. Accordingly, HFD fed SFRP5 deficient mice Visceral adipose tissue-derived serine protease inhibitor is identified from visceral white adipose tissues of Otsuka Ling-Evans Tokushima fatty (OLETF) rat as an insulin sensitizing adipokine because vaspin suppresses the expression of pro-inflammatory adipokines such as resistin, leptin, and TNF-α  that is responsible for generating the adipose tissue inflammatory cascade. It has been suggested that adipocyte released chemokines such as CCL2 is responsible for the initiation of pro-inflammatory macrophage infiltration (Weisberg et al., In any case, another unresolved issue is the mechanisms by which adipose tissue inflammation results in liver and skeletal muscle insulin resistance. Several studies have also observed the local liver and skeletal muscle expression of pro-inflammatory cytokines and activation of inflammatory cells (Odegaard and Chawla, It is becoming clear that targeting the pro-inflammatory pathway may provide a novel therapeutic approach to prevent insulin resistance, particularly in obesity-induced insulin resistance. For example, although early efforts to block TNF-α failed to show efficacy, more recently the use of salicylate to reduce inflammation by inhibiting IKKβ signaling was found to improve insulin sensitivity in animal models and humans (Goldfine et al., In summary, adipose tissue has multiple integrative functions serving as energy storage organ that can provide fuel for energy production in times of external nutrient shortage. However, over the past two decades adipocytes have become established as bona fide professional endocrine cells that integrates whole body energy status with eating behavior, energy expenditure, and insulin sensitivity. Moreover, adipose tissue has become a central node for driving local and systemic sterile inflammation that is a key element in obesity-induced insulin resistance. Although many adipokines have been identified and well studied, the identification and functional studies of new adipokines and their control of integrative physiologic responses are essential to understand pathophysiological mechanisms of obesity-induced metabolic diseases.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Proper regulation of gene expression is essential for cellular viability, and the integrity of chromatin is a fundamental factor in maintaining proper gene regulation. In cancer cells, this precise regulation is disrupted and aberrant gene expression ensues - a hallmark of the disease. In many forms of hematological malignancy, aberrant expression, activation or deletion of a critical enzymatic regulator of chromatin modification underlies the disease. One such regulator is MMSET (multiple myeloma SET domain) protein, a histone methyltransferase, that is rearranged and joined with the immunoglobulin locus associated with t(4:14) due to aberrant class switch DNA recombination in approximately 20% of multiple myeloma cases and is associated with particularly poor prognosis. Our lab has previously shown that an increase in MMSET expression results in global histone modification changes and up-regulation of hundreds of genes. We believe that these changes are due to radical reprogramming of chromatin, including repositioning of nucleosomes in MMSET-overexpressed cells. Mechanistic insight into epigenetic changes during MMSET-mediated gene expression will provide deeper understanding of disease progression in multiple myeloma, and potentially other similar malignancies.To begin understanding the changes in chromatin structure when MMSET is overexpressed, we generated a genome wide nucleosome map in cells expressing high and low levels of MMSET. Initial analysis of this mapping reveals changes in nucleosome positioning in promoters where gene expression and histone modifications had been identified to be significantly altered in cells expressing high and low levels of MMSET. Generally, overexpression of MMSET results in an increase in H3K36 dimethylation, a histone modification associated with actively transcribed genes, and a concomitant genome-wide loss of H3K27 trimethylation, a histone modification associated with repressed genes. Indeed, in cells expressing high levels of MMSET, genes that have been identified to have an increase in expression and an increase in H3K36 dimethylation have a distinct change in nucleosome positioning compared to the cells expressing low levels of MMSET. Further, differences in nucleosome positioning can be detected in promoter regions with increased H3K27 trimethylation and decreased gene expression. We are currently examining whether these changes in nucleosome positioning are due to the presence of MMSET and require MMSET histone methyltransferase activity. Through these and related studies, our aim is to determine how MMSET behaves in multiple myeloma through a likely radical reprogramming of chromatin. We believe that mechanistic insight into MMSET-mediated gene expression changes will provide significant avenues for potential therapeutic strategies, as MMSET overexpression has been implicated in myeloma and other solid tumors. Moreover, our work will provide insight into mechanisms that may be shared amongst various diseases that are initiated through epigenetic misregulation.


As a chemokine coreceptor antagonist, maraviroc (MVC) inhibits HIV infection by preventing viral entry into the host cell. As this effect is extracellular, we hypothesized that virus might be returned to plasma upon antagonism of CCR5 by MVC and that this thereby influences measurement of plasma viral load by qRT-PCR, in contrast to other drug classes that act intracellularly.4-107 copies of viral RNA/ml. Cells were centrifuged and supernatant viral load was measured by qRT-PCR for viral RNA at various times from shortly after infection through 48 hours after infection.PM-1 and TZM/bl cells were infected with titrations of a CCR5 tropic reference virus in the presence of inhibitory concentrations of MVC (500nM), efavirenz (EFV) (500nm), and raltegravir (RAL) (1µM). Viral inoculation varied between 10At the highest inoculum used, the amounts of viral RNA detected in culture supernatants following treatment with MVC were consistently higher by ~ 0.5 log copies HIV-1 RNA/ml than found with untreated cells despite no detectable infection in the presence of MVC as revealed by production of p24 Ag in the case of PM-1 cells and expression of luciferase in the case of TZM/bl cells. At lower levels of inoculum, the results were consistent although less striking, but there was always a significant difference in regard to the use of MVC versus other drugs. In contrast, the results obtained with EFV and RAL resembled those obtained in the absence of drug, despite no detectable infection.These results suggest in a tissue culture model that MVC can return virus to plasma, where it may contribute to viral load despite complete inhibition of infection. In contrast, for drugs that act at an intracellular level, cells can absorb residual virus before such virus it is inhibited by the compound. Consequently, such virus will not contribute to viral load. In view of the fact that viral production by infected cells is an ongoing process, these findings imply that the true effectiveness of MVC is currently underestimated when viral load is used as a sole indicator of clinical success. These data may also be relevant for other classes of entry inhibitors.


Women who have an inherited mutation in the BRCA1 or BRCA2 genes have a substantial increased lifetime risk of developing epithelial ovarian cancer (EOC), and epidemiological factors related to parity, ovulation, and hormone regulation have a dramatic effect on the risk in both BRCA mutation carriers and non-carriers. The most common and most aggressive histotype of EOC, high-grade serous carcinoma (HGSC), is also the histotype associated with germline BRCA mutations. In recent years, evidence has emerged indicating that the likely tissue of origin of HGSC is the fallopian tube. We have reviewed, what is known about the fallopian tube in BRCA mutation carriers at both the transcriptional and translational aspect of their biology. We propose that changes of the transcriptome in BRCA heterozygotes reflect an altered response to the ovulatory stresses from the microenvironment, which may include the post-ovulation inflammatory response and altered reproductive hormone physiology. In 2013, about 22,240 women in the United States would have been diagnosed with invasive epithelial ovarian cancer (EOC) and an estimated 14,000 women with EOC would have died , and somHigh-grade serous carcinoma is the predominant histotype associated with hereditary breast-ovarian cancer , 7. WomeMolecularly, the breast cancer susceptibility genes (BRCA) BRCA1 and BRCA2 can sense DNA damage and are involved in DNA repair via interactions with RAD51 –25; thesBreast cancer susceptibility gene mutation carriers develop cancers in hormonally regulated tissues, most frequently in breast and ovarian/tubal tissues, but a unifying mechanism of early malignant transformation in these tissues is not known. The BRCA associated carcinomas share some common features including a high-grade phenotype, frequent mutations of TP53, and other copy number landscape features like Cyclin-E amplification and deletion of Rb . AlteredThe mechanisms underlying malignant transformation in these estrogen responsive tissues are poorly understood, but likely involve loss of heterozygosity of the remaining wild type BRCA allele  in additIn normal cells of mutation carriers, only one allele is mutated, and BRCA1 function is presumed to be intact. This may however not be true, as evidence in support of BRCA1 haploinsufficiency accumulates. For example, in normal human mammary epithelial cells from BRCA1 heterozygotes, DNA homologous repair is suppressed . BRCA1 hFor many years, in the absence of a reproducible histological precursor lesion of HGSC, the cell of origin was presumed to be the ovarian surface epithelium (OSE), a modified type of mesothelium. Detailed histo-pathological examination of tubal epithelia (FTE) in the genetically high-risk population undergoing risk-reducing surgery has led to the discovery of putative cancer precursor lesions in the fallopian tube, some of which, i.e., the p53 signature – described as a string of 10–12 histologically normal secretory (non-ciliated) cells expressing the TP53 protein with a low proliferation rate (Ki67) , are fouOccult invasive carcinoma and serous tubal intraepithelial carcinomas (STICs) were identified in the fallopian tubes of mutation carriers undergoing risk-reducing surgery, with an incidence of about 4–6% for occult cancers , 54, 55.in situ carcinomas, because in at least some cases while tumor cells do not invade underlying stroma, they can detach, and because of the accessibility of the ipsilateral ovary and other peritoneal surfaces to the tubal fimbria, cells may implant and establish tumor growth in other sites. Currently, little evidence exists that patients with only a diagnosis of STIC require adjuvant therapy  and luteal (post-ovulatory phase) at the time of surgery , 71. We + and CD8+ lymphocytes and CD68+ macrophages) and (2) the proliferation status of FTE cells in both BRCA mutation carriers and population control. This study although not exhaustive, revealed that independent of BRCA mutation status: (1) macrophages were more prevalent in the luteal phase than the follicular phase of the ovarian cycle and (2) proliferation in FTE cells is predominantly an effect of the follicular phase rather than BRCA mutation status in histologically normal tissue , the anatomical region of highest risk and the ampulla for: (1) the presence of immune infiltrates (CD3ges) and  the prolMost women who develop sporadic cases of EOC are peri- or post-menopausal with a mean age of 58 years ; howeverThe influence of sex hormones on tubal/ovarian malignant transformation is not well understood, but seems likely that the BRCA1/2 associated changes in reproductive hormones and their receptors play a role in tumor formation, in addition to the alterations in DNA damage repair. BRCA1/2 mutation carriers do not have menopause at an early age . Higher The role of estrogen and progesterone in early malignant transformation in the FTE is not yet well understood. Estrogen mediates its action primarily through the estrogen receptor (ERα and ERβ). Estrogen stimulates the expression of a number of genes that promote cell proliferation, motility/invasion, and inhibition of apoptosis namely: IL6, TGF-α, EGF, PI3K/Akt, IFG-1, and Bcl-2 (which is predominantly expressed in secretory FTE) . The estDuring ovulation, there is a surge of estrogen released into the FTE microenvironment with release of follicular fluid, which contains high estrogen levels. This effect might be exacerbated  in falloA decrease in the transcription and translation (by immunohistochemistry) of PR-A and PR-B were observed in the luteal phase of both BRCA mutation carriers and population control . PR geneEpidemiological data indicate that HGSC risk is closely linked to the events of ovulation, and these risk factors and protective factors for the most part are true for both sporadic and hereditary HGSC. In addition, evidence suggests that the risk for EOC increases during the pre-menopausal years, and that menopause is protective against ovarian cancer . The rolIn conclusion, there are many epidemiological studies linking ovulation, parity, and hormonal use to the development of EOC. About 60% or more of HGSC demonstrate a BRCAness profile predominantly through a dysfunctional homologous recombinant pathway, which synergizes with the ubiquitousness of the p53 mutations found amongst these tumors. In the normal fallopian tube of BRCA mutation carriers, transcription profiles reveal predominant differences in DNA damage and inflammation pathways. Interestingly and may be not surprisingly, FTE-BRCA samples are transcriptionally indistinguishable from FTE-normal samples when transcription profiles undergo unsupervised hierarchical clustering . InsteadThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Skeletal muscle possesses a remarkable ability for regeneration and can go through rapid repair following muscle injury. This regeneration depends on the activity and contributions of muscle satellite cells, which are located between the sarcolemma of myofibers and the basal lamina . Upon muDuring myogenesis, these satellite cells can be distinguished based on Pax7 and MyoD expression; both are essential myogenic transcription factors. Pax7+MyoD− cells are in a quiescent state, Pax7+MyoD+ cells are proliferating, and Pax7−MyoD+ cells are undergoing differentiation ,4. In adCurrently, there are two mechanisms that attempt to define the maintenance of the muscle satellite cell pool in adult muscle. The first mechanism suggests that satellite cells are a functionally heterogeneous population while the other states that satellite cells are capable of self-renewal to generate a stem cell pool after cell division. Previous studies have proposed that satellite cells are composed of two different populations: One population consists of myogenic satellite cells, which possess myogenic differentiation potential. The other population is an undifferentiated subgroup which retains the satellite stem cell profile ,11,12. TA small population of satellite cells, possessing a potential of “stemness”, is capable of maintenance of the number of satellite cells as proved by transplantation experiments. This small cell niche also produces myoblasts during muscle regeneration , indicatMyoD belongs to the myogenic basic helix-loop-helix (bHLH) transcription factors that play essential roles in myogenic specification, differentiation and maintenance during muscle development and regeneration . MyoD spSix1, which belongs to Six homeoprotein family and is expressed in quiescent and activated satellite cells, is reported to regulate skeletal muscle regeneration as well as satellite cell self-renewal . ExpressWhile several transcription factors are expected to be key elements in sustaining the satellite cell pool, the position of satellite cells in contact with muscle fibers is also expected to hold a comparable level of importance in management of the satellite stem cell niche. A recent paper demonstrates that increased levels of FGF2, typically a stimulator of satellite cell activation, induces loss of satellite cells through decreased Spry1, the downstream target of FGF2, in aged muscle fiber. These results indicate that age-related changes of FGF signaling in the stem cell niche influence altered stem cell maintenance and function . In addiIn relation to Notch signaling, a recent paper demonstrates interesting results in which the depletion of the satellite cell pool in RBP-J mutant mice, a key mediator of Notch signaling,, is recovered by the deletion of MyoD . In thisIn addition to cell commitment, recent reports indicate that physiological conditions in satellite cells may influence the regulation of self-renewal. Liu and colleagues have proposed that hypoxia does not affect myoblast proliferation but instead promotes satellite cell self-renewal by up-regulating Pax7 . In factThus, accumulating observations demonstrate that maintenance of muscle satellite cells is intricately involved in several biological and physiological processes. Although the idea in which satellite cells are essential for muscle regeneration is widely accepted, the mechanism of cell fate determination, whether satellite cells differentiate into myocytes or maintain as stem cells, is still controversial. In addition, it is of great interest to the field to define the number of satellite cells in adult and aged muscles in addition to what distinct niches regulate satellite cell number and heterogeneity. In order to reveal these mysteries, further exploration that approaches the identity of fundamental mechanisms in satellite cells must be conducted. Recent studies report that satellite cells are divided predominantly by asymmetric template DNA segregation, generating daughter cells which retain stem cell potential and myogenic committed cells . In addi


Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, we identified and analyzed novel enhancers that drive midline and/or tracheal expression and compared them to previously characterized enhancers. In addition, we tested expression of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.Within the  Drosophila embryo: the central nervous system (CNS) midline and the trachea. In Drosophila, Single-minded (Sim) is the master control gene of CNS midline cells The genes expressed within a particular cell type control its developmental fate and physiological potential. Early in development, master control genes play pivotal roles in controlling cell fate and most master control genes are transcription factors that promote their own expression as well as a variety of downstream target genes. Each target gene, in turn, contributes to tissue development by regulating cellular processes, such as 1) morphology 2) interactions with surrounding cells through signaling, 3) cell divisions and/or 4) the expression of additional genes. To understand how genes are differentially regulated within tissues, we compare the development and gene expression of two tissues in the Drosophila embryogenesis. CNS midline cells are specified early in embryogenesis when sim is activated prior to gastrulation, in a single row of cells sandwiched in between the mesoderm and ectoderm on each side of the embryo; cells called the mesectoderm Netrin (Net) A and B to attract axons to cross the midline slit to prevent recrossing roundabout (robo), the receptor for slitcommissureless (comm) to temporarily prevent robo localization at the growth cone, allowing them to cross The embryonic midline and trachea differ in many ways and the following is a brief summary and comparison of the development of these tissues during Drosophila, the trachea are a network of air-filled tubes constructed during embryogenesis that function in gas exchange  and trh are activated by JAK/STAT signaling Decapentaplegic (Dpp) and Epidermal Growth Factor (EGF) signaling limit the embryonic dorsal and ventral boundaries of the trachea, while wingless (wg) restricts the location of trachea within each segment fibroblast growth factor (FGF), called breathless (btl) in flies branchless, which stimulates and guides branch formation Drosophila hypoxia inducible factor, also guides later growth and branching of the trachea, driven, in part, by oxygen demands of tissues In Notch, FGF, EGF, engrailed, wg and hedgehog (hh)The functions and morphology of midline and tracheal cells differ, yet certain aspects of their embryonic development are similar. Both cell types are derived from the ectoderm  and project long cellular extensions to form specialized contacts with many different cell types To compare regulatory functions of Sim/Tgo and Trh/Tgo during fly development, we selected genes expressed in the midline, trachea or both tissues, identified enhancers that control the expression of each gene and compared them to previously identified midline and tracheal enhancers. To test the importance of previously identified sequence motifs, we generated synthetic reporters that contain the CME combined with binding sites for other factors expressed in the midline or trachea. To further analyze these enhancer sets, we searched for novel motifs common to both, as well as motifs unique to either midline or tracheal genes. The results identify sequence contexts, both proximal and distal to the CME, which promote midline and/or tracheal expression.Drosophila melanogaster genomic sequences encompassing select genes expressed in the midline and trachea were compared across the 12 sequenced Drosophila genomes 67 Drosophila melanogasteryw strain. These fragments were either cloned into the pSTBlue1 intermediary vector and then into the pHstinger vector pCR8/GW/TOPO (Invitrogen) and transferred into pMintgate using the Gateway system pHstinger constructs using standard procedures and three independent lines analyzed for each GFP reporter gene. pMintgate constructs were injected into the φC31 genomic destination site attP2 (68A1-B2) as previously described CG33275. The CG33275 ML577 fragment was generated by first digesting the CG33275 ML 2544:GFP construct in pSTBlue with BglII, re-ligating it and then subcloning the remaining 577 bp fragment into pHstinger. The CG33275 ML 1312 fragment was generated from the CG33275 ML2544:GFP construct using KpnI/SwaI digestion and blunt end ligation, which removed 1232 bp from the original 2544 bp construct  GFP , mouse anti-GFP , rabbit anti-dys (1∶400) odd-skipped ; and anti-reversed polarity , anti-engrailed (1∶1), and 2A12 (1∶5) MAbs . Secondary antibodies were used at 1∶200 and included anti-rat-Alexa568, anti-mouse-Alexa568, anti-rabbit-Alexa405 and anti-rabbit-Alexa488 (Molecular Probes). Images were obtained on a Zeiss 710 in the Cellular and Molecular Imaging Facility at NCSU.Embryos were collected and labeled with antibodies as previously described sim, Toll, slitrhomboid (rho)btlVvlroughest (rst)wrappergliolectin, organic anion transporter protein26f, liprin γlinkCG33275, NetB, comm, escargot (esg) and Ectoderm3 (Ect3). The following previously identified tracheal enhancers were included: trh, Vvl earlyVvl autoregulatoryCG13196, CG15252, dysbtlrholinkCG33275, NetB, liprin γ, esg and moody.MEME Drosophila development. The seven genes studied include three genes that encode axon guidance and synaptic proteins: liprin γ, comm and Net B; a gene in the EGFR signaling pathway, CG33275; a G protein coupled receptor, moody; a cell death gene, Ectoderm 3 (Ect3), and finally, the esg transcription factors. Several of these contain large introns and are separated from other genes by large intergenic regions and, therefore, to facilitate the identification of midline and tracheal enhancers, we searched for sequences conserved in a relatively large number of the sequenced Drosophila species GFP within the pHstinger or Mintgate enhancer tester vectors and generating transgenic fly lines. In certain cases, we also identified a minimal region capable of driving tissue specific expression. The composition and expression patterns of the identified enhancers are briefly summarized below.To understand how diverse genes are transcriptionally regulated in the midline, trachea or both tissues, we identified and compared enhancers of seven genes that are expressed during CG33275 capable of driving high levels of GFP in midline glia and a separate and distinct tracheal enhancer downstream of the midline enhancer  and the smaller CG33275 TRH1297:GFP reporter gene drove the same tracheal expression pattern (CG33275 TRH1228:GFP reporter was not expressed in trachea or midline cells (not shown), whereas the CG33275 TRH265:GFP reporter was restricted to tracheal fusion cells , whereas the liprin γ 182:GFP reporter is expressed at high levels in all the trachea  the diverse tracheal expression pattern of the larger liprin γ reporter genes, suggest that this region may only drive tracheal expression when isolated from surrounding sequences. Because multiple copies of the CME within a reporter gene, can drive expression in both the midline and trachea (see below), one function of sequences flanking the CME within enhancers is to limit expression of the gene to certain cell types. In summary, these experiments further define the minimal sequences needed for expression in the CNS midline within the previously identified liprin γ enhancer Liprin proteins interact with tyrosine phosphatases to regulate synapse formation. 3141:GFP  and lipr1781:GFP  reporterbranches . Only thon cells . The lip trachea . In addisegments . TherefoNetA and NetB are signaling molecules secreted by midline glia that attract axons to cross the midline and also function in glial migration Net797:GFP reporter identifies a midline and tracheal enhancer located between NetA and NetB , comm693:GFP  enhancers are expressed in lateral CNS glia. moody608:GFP drove expression in the fat body (not shown), but is not expressed in the trachea , the identified midline enhancer is found just upstream of Ect3 as well as within the first intron of Tk (Tachykinin (Tk) is a neuropeptide hormone expressed at high levels during 18–24 hours of embryogenesis, early larval stages and in the adult male Ect3 gene, because only Ect3, and not Tk, is expressed in the embryonic midline Ect3 3194:GFP reporter contains the region bordered by the first exon of Tk on the 5′ end and the Ect3 transcription start site on the 3′ end  the Ect3 2311:GFP  E 517:GFP  reporter 572:GFP ’-F’ repo1071:GFP ’-L’ repoCG33275 and esg each contain adjacent, separable midline and tracheal enhancers; whereas liprin γ, Net and comm each contain one enhancer that drove expression in both the midline and trachea. The moody enhancer drove expression only in the trachea and the Ect3 enhancer drove expression only in the midline. Despite providing expression in overlapping cell types, each enhancer drove a unique expression pattern within the midline and trachea. Next, these enhancers, together with previously reported enhancers discovered by several groups, were combined to search for overrepresented motifs that may correspond to binding sites for transcription factors that activate or repress genes in the midline and/or trachea.In summary, ten enhancers were identified: six of the enhancers drove expression in the midline, seven in the trachea and three in both the midline and trachea . CG33275A longterm goal is to use the midline and trachea as models to study how transcription factors combine with cell type specific co-factors to regulate unique gene sets, and, in this way, dictate development of unique tissues. Including the ten enhancers identified here, nineteen different midline enhancers and nineteen tracheal enhancers have been identified. To identify sequences that promote or inhibit CME utilization in either the midline or trachea, we analyzed the enhancers in two different ways. First, we searched sequences directly flanking the CME within defined enhancers to determine if these sequences could predict whether a particular CME is utilized by Sim/Tgo or Trh/Tgo and secondly, we searched the smallest region sufficient to drive expression in a tissue for reiterated motifs that may help restrict or promote gene expression in the midline and trachea.ACGTGC, TACGTGA or TACGTGC (CME underlined), while the sequences AACGTGG, GACGTGT, TACGTGG were not found in any of the enhancers, suggesting that Sim/Tgo and Trh/Tgo may not bind these sequences ACGTG(A/C/T) and enhancers that function in both the midline and trachea, most often contain the consensus (A/T)ACGTGC ACGTGC . Therefo-galactosidase or GFP, is sufficient to drive reporter expression in both the midline and trachea wrapper (synth 1–4 and synth 6) or Toll midline enhancers . We chose these particular sequences because both had been tested previously within synthetic reporter genes and drove different patterns of expression. The CME and flanking sequences found in the wrapper enhancer, when multimerized four times, drives expression only in the midline Toll enhancer drives expression in both the midline and trachea Enhancers are modular and contain multiple binding sites for many activators and repressors that work together in large multi-protein complexes to regulate transcription in different cell types. Nevertheless, individual binding sites of a limited number of transcription factors are sufficient to drive expression in certain tissues, particularly when present in more than one copy. Relevant to this study, four copies of the CME fused to βwrapper enhancer was tested using two approaches. First, the 70 bp minimal wrapper enhancer was divided into two sections and tested independently: synth 1 contained sequences 7–53 and synth 2 contained sequences 28–70 of the wrapper minimal enhancer. Both of these constructs contain the single CME and flanking sequences found in the wrapper enhancer and both of these multimerized reporters were expressed in the midline, but not the trachea  to TAAAA within synth 4, had only a small effect on the midline expression pattern of GFP  as the Sim/Tgo binding site ACGTG C; GCGTGR and future experiments are needed to determine their importance within the various enhancers. Our results indicated that the CME context favored within midline and tracheal enhancers as well as enhancers active in both tissues, was very similar  if changing the sequence, TACGTGC, to AACGTGC within an enhancer that drives expression in both the midline and trachea, will restrict expression to only the trachea. Sequences proximal to the CME likely affect the affinity of either Sim/Tgo and/or Trh/Tgo heterodimers to the DNA, but binding sites for additional factors that interact cooperatively to stabilize an entire transcription complex are needed for high levels of expression within a particular cell. Moreover, recent experiments indicate that enhancers containing multiple CMEs are activated earlier in the embryonic midline than enhancers containing only one CME Results from both endogenous enhancers and the synthetic reporter genes confirm the importance of the proximal sequences in limiting expression to either the midline or trachea. While the proximal context of the CME plays a role, additional sequences clearly combine with the CME to ultimately determine if an enhancer is functional in the midline or trachea. Taken together, these results indicate that proximal motifs combine with additional sequences not only to determine whether or not a gene is expressed in the midline or trachea, but also to determine which cellular subtypes express the gene and when it is activated within a tissue. Future experiments will reveal if 1) changing the sequence, AVvl enhancer that drove expression in both the midline and trachea Vvltrh autoregulatory enhancer 4) the link enhancer, after its sole CME has been destroyed dys tracheal enhancer CG15252, 7) a tracheal enhancer of CG13196, and 8) the 517 bp Ect3 midline enhancer described here  a 517 bp autoregulatory bed here . These sbed here . These rcis-regulatory sequences that control the expression of genes within the midline and trachea, they do not address why many genes are expressed in both of these tissues and regulated by related PAS heterodimers. It is predominantly genes expressed in the CNS midline glia, rather than the midline neurons, that are also expressed in tracheal cells. PAS proteins perform diverse functions across all biological kingdoms and most characterized members function as environmental sensors Drosophila, a stage when Sim and Trh may function more similarly. For instance, in adult flies, both glia and trachea provide support and energy to neurons and trh is expressed in the CNS late in embryogenesis and throughout the remainder of the fly’s life. In the adult fly brain, tracheal development is guided by glial cells, and ablating glia causes the trachea to branch more extensively within this tissue Drosophila PAS proteins that interact with Tgo are expressed in the trachea, including Trh, Dys and Similar (the fly version of HIF-1α), and Sim likely descended from a common ancestral gene. Developmental functions of Sim and Trh may have arisen later than their adult functions and common ancestral functions of these two tissues in the adult may explain why many enhancers drive expression in both the midline and trachea and why other midline and tracheal enhancers are closely linked. Further dissecting the similarities and differences in gene regulation within the CNS midline and trachea will reveal novel molecular mechanisms used to construct these tissues during development. Additional experiments are also needed to understand how signaling pathways combine with Sim and Trh to regulate genes in midline glia and trachea, not only in embryos, but also in larvae and adults, under different environmental conditions.While these experiments focus on the Table S1List of PCR primers used to generate fragments of the CG33275, esg, liprin γ, Netrin, comm, moody and Ect3 genes that were tested for their ability to drive midline and tracheal transcription. Restriction sites introduced for cloning purposes are indicated in lower case.(DOC)Click here for additional data file.Table S2List of PCR primers used to generate the synthetic reporter genes tested for their ability to drive midline and tracheal transcription. The 1Toll and Sox synthetic reporters have been previously reported (DOC)Click here for additional data file.Table S31the name of the enhancer, 2the tissue that expressed For each enhancer, GFP3,4PCR primers used to generate the enhancers derived from  driven by the enhancer, and esg and Netrin genes are listed. Restriction sites introduced for cloning purposes are indicated in lower case.(DOC)Click here for additional data file.Table S41the midline, 2the trachea or 3both tissues.Listed is the immediate context of CMEs found within enhancers that drive expression in  For each CME (ACGTG), 4the gene where it is found, 5the number of CMEs within each enhancer and the 6seven bp sequence of the site are shown. 7The total number of CMEs found within the enhancers examined is indicated at the bottom of the table.(DOC)Click here for additional data file.Table S51the midline, 2the trachea or 3both tissues.Listed is the immediate context of GCGTG motifs found within identified enhancers that drive expression in  For each GCGTG motif, 4the gene where it is found, 5the number found within each enhancer and the 6seven bp sequence of the site are shown. 7The total number of CMEs found within the enhancers examined is indicated at the bottom of the table.(DOC)Click here for additional data file.