Primordial Germ Cell Specification and Migration

PubMed Central

Marlow, Florence

2015-01-01

Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration. PMID:26918157

The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification.

PubMed

Fresques, Tara; Swartz, Steven Zachary; Juliano, Celina; Morino, Yoshiaki; Kikuchi, Mani; Akasaka, Koji; Wada, Hiroshi; Yajima, Mamiko; Wessel, Gary M

2016-07-01

Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32-128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution. © 2016 Wiley Periodicals, Inc.

Functions of Huntingtin in Germ Layer Specification and Organogenesis

PubMed Central

Nguyen, Giang D.; Molero, Aldrin E.; Gokhan, Solen; Mehler, Mark F.

2013-01-01

Huntington’s disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities. PMID:23967334

The zebrafish tailbud contains two independent populations of midline progenitor cells that maintain long-term germ layer plasticity and differentiate in response to local signaling cues

PubMed Central

Row, Richard H.; Tsotras, Steve R.; Goto, Hana; Martin, Benjamin L.

2016-01-01

Vertebrate body axis formation depends on a population of bipotential neuromesodermal cells along the posterior wall of the tailbud that make a germ layer decision after gastrulation to form spinal cord and mesoderm. Despite exhibiting germ layer plasticity, these cells never give rise to midline tissues of the notochord, floor plate and dorsal endoderm, raising the question of whether midline tissues also arise from basal posterior progenitors after gastrulation. We show in zebrafish that local posterior signals specify germ layer fate in two basal tailbud midline progenitor populations. Wnt signaling induces notochord within a population of notochord/floor plate bipotential cells through negative transcriptional regulation of sox2. Notch signaling, required for hypochord induction during gastrulation, continues to act in the tailbud to specify hypochord from a notochord/hypochord bipotential cell population. Our results lend strong support to a continuous allocation model of midline tissue formation in zebrafish, and provide an embryological basis for zebrafish and mouse bifurcated notochord phenotypes as well as the rare human congenital split notochord syndrome. We demonstrate developmental equivalency between the tailbud progenitor cell populations. Midline progenitors can be transfated from notochord to somite fate after gastrulation by ectopic expression of msgn1, a master regulator of paraxial mesoderm fate, or if transplanted into the bipotential progenitors that normally give rise to somites. Our results indicate that the entire non-epidermal posterior body is derived from discrete, basal tailbud cell populations. These cells remain receptive to extracellular cues after gastrulation and continue to make basic germ layer decisions. PMID:26674311

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

PubMed

Antonucci, Ivana; Di Pietro, Roberta; Alfonsi, Melissa; Centurione, Maria Antonietta; Centurione, Lucia; Sancilio, Silvia; Pelagatti, Francesca; D'Amico, Maria Angela; Di Baldassarre, Angela; Piattelli, Adriano; Tetè, Stefano; Palka, Giandomenico; Borlongan, Cesar V; Stuppia, Liborio

2014-01-01

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.

PubMed

Makoolati, Zohreh; Movahedin, Mansoureh; Forouzandeh-Moghadam, Mehdi

2016-12-01

An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs. © 2016 The Author(s).

Identification of a putative germ plasm in the amphipod Parhyale hawaiensis

PubMed Central

2013-01-01

Background Specification of the germ line is an essential event during the embryonic development of sexually reproducing animals, as germ line cells are uniquely capable of giving rise to the next generation. Animal germ cells arise through either inheritance of a specialized, maternally supplied cytoplasm called 'germ plasm’ or though inductive signaling by somatic cells. Our understanding of germ cell determination is based largely on a small number of model organisms. To better understand the evolution of germ cell specification, we are investigating this process in the amphipod crustacean Parhyale hawaiensis. Experimental evidence from previous studies demonstrated that Parhyale germ cells are specified through inheritance of a maternally supplied cytoplasmic determinant; however, this determinant has not been identified. Results Here we show that the one-cell stage Parhyale embryo has a distinct cytoplasmic region that can be identified by morphology as well as the localization of germ line-associated RNAs. Removal of this cytoplasmic region results in a loss of embryonic germ cells, supporting the hypothesis that it is required for specification of the germ line. Surprisingly, we found that removal of this distinct cytoplasm also results in aberrant somatic cell behaviors, as embryos fail to gastrulate. Conclusions Parhyale hawaiensis embryos have a specialized cytoplasm that is required for specification of the germ line. Our data provide the first functional evidence of a putative germ plasm in a crustacean and provide the basis for comparative functional analysis of germ plasm formation within non-insect arthropods. PMID:24314239

Molecular control of gut formation in the spider Parasteatoda tepidariorum.

PubMed

Feitosa, Natália Martins; Pechmann, Matthias; Schwager, Evelyn E; Tobias-Santos, Vitória; McGregor, Alistair P; Damen, Wim G M; Nunes da Fonseca, Rodrigo

2017-05-01

The development of a digestive system is an essential feature of bilaterians. Studies of the molecular control of gut formation in arthropods have been studied in detail in the fruit fly Drosophila melanogaster. However, little is known in other arthropods, especially in noninsect arthropods. To better understand the evolution of arthropod alimentary system, we investigate the molecular control of gut development in the spider Parasteatoda tepidariorum (Pt), the primary chelicerate model species for developmental studies. Orthologs of the ectodermal genes Pt-wingless (Pt-wg) and Pt-hedgehog (Pt-hh), of the endodermal genes, Pt-serpent (Pt-srp) and Pt-hepatocyte-nuclear factor-4 (Pt-hnf4) and of the mesodermal gene Pt-twist (Pt-twi) are expressed in the same germ layers during spider gut development as in D. melanogaster. Thus, our expression data suggest that the downstream molecular components involved in gut development in arthropods are conserved. However, Pt-forkhead (Pt-fkh) expression and function in spiders is considerably different from its D. melanogaster ortholog. Pt-fkh is expressed before gastrulation in a cell population that gives rise to endodermal and mesodermal precursors, suggesting a possible role for this factor in specification of both germ layers. To test this hypothesis, we knocked down Pt-fkh via RNA interference. Pt-fkh RNAi embryos not only fail to develop a proper gut, but also lack the mesodermal Pt-twi expressing cells. Thus, in spiders Pt-fkh specifies endodermal and mesodermal germ layers. We discuss the implications of these findings for the evolution and development of gut formation in Ecdysozoans. © 2017 Wiley Periodicals, Inc.

Methods to study maternal regulation of germ cell specification in zebrafish

PubMed Central

Kaufman, O.H.; Marlow, F.L.

2016-01-01

The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4–5 h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology. PMID:27312489

BMP4 density gradient in disk-shaped confinement

NASA Astrophysics Data System (ADS)

Bozorgui, Behnaz; Teimouri, Hamid; Kolomeisky, Anatoly B.

We present a quantitative model that explains the scaling of BMP4 gradients during gastrulation and the recent experimental observation that geometric confinement of human embryonic stem cells is sufficient to recapitulate much of germ layer patterning. Based on a assumption that BMP4 diffusion rate is much smaller than the diffusion rate of it's inhibitor molecules, our results confirm that the length-scale which defines germ layer territories does not depend on system size.

Human Stem Cells Can Differentiate in Post-implantation Mouse Embryos.

PubMed

Tam, Patrick P L

2016-01-07

The potency of human pluripotent stem cells (hPSCs) to differentiate into germ layer derivatives is conventionally assessed by teratoma induction and in vitro differentiation. In this issue of Cell Stem Cell, Mascetti and Pedersen (2016) demonstrate that the human-mouse post-implantation chimera offers an efficient avenue to test the germ layer differentiation potential of hPSCs in mouse embryos ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Intermolecular Interactions of Homologs of Germ Plasm Components in Mammalian Germ Cells

PubMed Central

Fox, Mark S.; Clark, Amander T.; El Majdoubi, Mohammed; Vigne, Jean-Louis; Urano, Jun; Hostetler, Chris E.; Griswold, Michael D.; Weiner, Richard I.; Pera, Renee A. Reijo

2007-01-01

In some species such as flies, worms, frogs, and fish the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically-distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration, that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells. PMID:16996493

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

PubMed

Plouhinec, Jean-Louis; Medina-Ruiz, Sofía; Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B; Harland, Richard M; Monsoro-Burq, Anne H

2017-10-01

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates

PubMed Central

Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B.; Harland, Richard M.

2017-01-01

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research. PMID:29049289

Tissue-Specific Methylation of Long Interspersed Nucleotide Element-1 of Homo Sapiens (L1Hs) During Human Embryogenesis and Roles in Neural Tube Defects.

PubMed

Wang, L; Chang, S; Guan, J; Shangguan, S; Lu, X; Wang, Z; Wu, L; Zou, J; Zhao, H; Bao, Y; Qiu, Z; Niu, B; Zhang, T

2015-01-01

Epigenetic regulation of long interspersed nucleotide element-1 (LINE-1) retrotransposition events plays crucial roles during early development. Previously we showed that LINE-1 hypomethylation in neuronal tissues is associated with pathogenesis of neural tube defect (NTD). Herein, we further evaluated LINE-1 Homo sapiens (L1Hs) methylation in tissues derived from three germ layers of stillborn NTD fetuses, to define patterns of tissue specific methylation and site-specific hypomethylation at CpG sites within an L1Hs promoter region. Stable, tissue-specific L1Hs methylation patterns throughout three germ layer lineages of the fetus, placenta, and maternal peripheral blood were observed. Samples from maternal peripheral blood exhibited the highest level of L1Hs methylation (64.95%) and that from placenta showed the lowest (26.82%). Between samples from NTDs and controls, decrease in L1Hs methylation was only significant in NTD-affected brain tissue at 7.35%, especially in females (8.98%). L1Hs hypomethylation in NTDs was also associated with a significant increase in expression level of an L1Hs-encoded transcript in females (r = -0.846, p = 0.004). This could be due to genomic DNA instability and alternation in chromatins accessibility resulted from abnormal L1Hs hypomethylation, as showed in this study with HCT-15 cells treated with methylation inhibitor 5-Aza.

Evaluation of the Adult Goldfish Brain as a Model for the Study of Progenitor Cells

DTIC Science & Technology

2007-08-27

embryo [34]. ESCs are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm, and they are...postnatal brain is their functional and anatomical destiny . Based on many reports investigating neurogenesis, the majority of newly produced cells...Homeodomain-bearing transcriptional factor. Expression is specific to early embryos and pluripotential stem cells. Key molecule involved in the

Micro-PIXE mapping of mineral distribution in mature grain of two pearl millet cultivars

NASA Astrophysics Data System (ADS)

Minnis-Ndimba, R.; Kruger, J.; Taylor, J. R. N.; Mtshali, C.; Pineda-Vargas, C. A.

2015-11-01

Micro-proton-induced X-ray emission (micro-PIXE) was used to map the distribution of several nutritionally important minerals found in the grain tissue of two cultivars of pearl millet (Pennisetum glaucum (L.) R. Br.). The distribution maps revealed that the predominant localisation of minerals was within the germ (consisting of the scutellum and embryo) and the outer grain layers (specifically the pericarp and aleurone); whilst the bulk of the endosperm tissue featured relatively low concentrations of the surveyed minerals. Within the germ, the scutellum was revealed as a major storage tissue for P and K, whilst Ca, Mn and Zn were more prominent within the embryo. Fe was revealed to have a distinctive distribution pattern, confined to the dorsal end of the scutellum; but was also highly concentrated in the outer grain layers. Interestingly, the hilar region was also revealed as a site of high accumulation of minerals, particularly for S, Ca, Mn, Fe and Zn, which may be part of a defensive strategy against infection or damage. Differences between the two cultivars, in terms of the bulk Fe and P content obtained via inductively coupled plasma optical emission spectrometry (ICP-OES), concurred with the average concentration data determined from the analysis of micro-PIXE spectra specifically extracted from the endosperm tissue.

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

PubMed Central

Hassoun, Romia; Schwartz, Peter; Rath, Detlef; Viebahn, Christoph; Männer, Jörg

2010-01-01

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human-like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two-step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron-dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam-like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid-somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations. PMID:20874819

Stochastic Cell Fate Progression in Embryonic Stem Cells

NASA Astrophysics Data System (ADS)

Zou, Ling-Nan; Doyle, Adele; Jang, Sumin; Ramanathan, Sharad

2013-03-01

Studies on the directed differentiation of embryonic stem (ES) cells suggest that some early developmental decisions may be stochastic in nature. To identify the sources of this stochasticity, we analyzed the heterogeneous expression of key transcription factors in single ES cells as they adopt distinct germ layer fates. We find that under sufficiently stringent signaling conditions, the choice of lineage is unambiguous. ES cells flow into differentiated fates via diverging paths, defined by sequences of transitional states that exhibit characteristic co-expression of multiple transcription factors. These transitional states have distinct responses to morphogenic stimuli; by sequential exposure to multiple signaling conditions, ES cells are steered towards specific fates. However, the rate at which cells travel down a developmental path is stochastic: cells exposed to the same signaling condition for the same amount of time can populate different states along the same path. The heterogeneity of cell states seen in our experiments therefore does not reflect the stochastic selection of germ layer fates, but the stochastic rate of progression along a chosen developmental path. Supported in part by the Jane Coffin Childs Fund

Germ layers, the neural crest and emergent organization in development and evolution.

PubMed

Hall, Brian K

2018-04-10

Discovered in chick embryos by Wilhelm His in 1868 and named the neural crest by Arthur Milnes Marshall in 1879, the neural crest cells that arise from the neural folds have since been shown to differentiate into almost two dozen vertebrate cell types and to have played major roles in the evolution of such vertebrate features as bone, jaws, teeth, visceral (pharyngeal) arches, and sense organs. I discuss the discovery that ectodermal neural crest gave rise to mesenchyme and the controversy generated by that finding; the germ layer theory maintained that only mesoderm could give rise to mesenchyme. A second topic of discussion is germ layers (including the neural crest) as emergent levels of organization in animal development and evolution that facilitated major developmental and evolutionary change. The third topic is gene networks, gene co-option, and the evolution of gene-signaling pathways as key to developmental and evolutionary transitions associated with the origin and evolution of the neural crest and neural crest cells. © 2018 Wiley Periodicals, Inc.

Pluripotency factors in embryonic stem cells regulate differentiation into germ layers.

PubMed

Thomson, Matt; Liu, Siyuan John; Zou, Ling-Nan; Smith, Zack; Meissner, Alexander; Ramanathan, Sharad

2011-06-10

Cell fate decisions are fundamental for development, but we do not know how transcriptional networks reorganize during the transition from a pluripotent to a differentiated cell state. Here, we asked how mouse embryonic stem cells (ESCs) leave the pluripotent state and choose between germ layer fates. By analyzing the dynamics of the transcriptional circuit that maintains pluripotency, we found that Oct4 and Sox2, proteins that maintain ESC identity, also orchestrate germ layer fate selection. Oct4 suppresses neural ectodermal differentiation and promotes mesendodermal differentiation; Sox2 inhibits mesendodermal differentiation and promotes neural ectodermal differentiation. Differentiation signals continuously and asymmetrically modulate Oct4 and Sox2 protein levels, altering their binding pattern in the genome, and leading to cell fate choice. The same factors that maintain pluripotency thus also integrate external signals and control lineage selection. Our study provides a framework for understanding how complex transcription factor networks control cell fate decisions in progenitor cells. Copyright © 2011 Elsevier Inc. All rights reserved.

Germ tube-specific antigens of Candida albicans cell walls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundstrom, P.R.

1986-01-01

Studies were performed to characterize the surface differences between blastospores and germ tubes of the pathogenic, dimorphic yeast, Candida albicans, and to identify components of yeast cells responsible for these differences. Investigation of surfaces differences of the two growth forms was facilitated by the production of rabbit antiserum prepared against Formalin-treated yeast possessing germ tubes. To prepare antiserum specific for germ tubes, this serum was adsorbed with stationary phase blastospores. Whereas the unadsorbed antiserum reacted with both blastospore and germ tube forms by immunofluorescence and Enzyme-Linked Immunosorbent Assay, the adsorbed antiserum did not react with blastospores but detected germ tube-specificmore » antigens in hyphal forms. The differences between blastospores and germ tubes of Candida albicans, were further studied by comparing enzymatic digests of cell walls of both growth forms in radiolabeled organisms. Organisms were labeled either on the surface with /sup 125/I, or metabolically with (/sup 35/S) methionine or (/sup 3/H) mannose. Three-surface-located components (as shown by antibody adsorption and elution experiments) were precipitated from Zymolase digests. All three components were mannoproteins as shown by their ability to bind Concanavalin A, and to be labeled in protein labeling procedures, and two of these (200,000 and 155,000 molecular weight) were germ tube specific, as shown by their ability to be precipitated by germ tube-specific antiserum. Monoclonal antibodies were prepared to C. albicans, using blastospores bearing germ tubes as immunogen.« less

Convergent evolution of germ granule nucleators: A hypothesis.

PubMed

Kulkarni, Arpita; Extavour, Cassandra G

2017-10-01

Germ cells have been considered "the ultimate stem cell" because they alone, during normal development of sexually reproducing organisms, are able to give rise to all organismal cell types. Morphological descriptions of a specialized cytoplasm termed 'germ plasm' and associated electron dense ribonucleoprotein (RNP) structures called 'germ granules' within germ cells date back as early as the 1800s. Both germ plasm and germ granules are implicated in germ line specification across metazoans. However, at a molecular level, little is currently understood about the molecular mechanisms that assemble these entities in germ cells. The discovery that in some animals, the gene products of a small number of lineage-specific genes initiate the assembly (also termed nucleation) of germ granules and/or germ plasm is the first step towards facilitating a better understanding of these complex biological processes. Here, we draw on research spanning over 100years that supports the hypothesis that these nucleator genes may have evolved convergently, allowing them to perform analogous roles across animal lineages. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Functional differentiation of human pluripotent stem cells on a chip.

PubMed

Giobbe, Giovanni G; Michielin, Federica; Luni, Camilla; Giulitti, Stefano; Martewicz, Sebastian; Dupont, Sirio; Floreani, Annarosa; Elvassore, Nicola

2015-07-01

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.

Molecular biological features of male germ cell differentiation

PubMed Central

HIROSE, MIKA; TOKUHIRO, KEIZO; TAINAKA, HITOSHI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

2007-01-01

Somatic cell differentiation is required throughout the life of a multicellular organism to maintain homeostasis. In contrast, germ cells have only one specific function; to preserve the species by conveying the parental genes to the next generation. Recent studies of the development and molecular biology of the male germ cell have identified many genes, or isoforms, that are specifically expressed in the male germ cell. In the present review, we consider the unique features of male germ cell differentiation. (Reprod Med Biol 2007; 6: 1–9) PMID:29699260

Expression of uncharacterized male germ cell-specific genes and discovery of novel sperm-tail proteins in mice.

PubMed

Kwon, Jun Tae; Ham, Sera; Jeon, Suyeon; Kim, Youil; Oh, Seungmin; Cho, Chunghee

2017-01-01

The identification and characterization of germ cell-specific genes are essential if we hope to comprehensively understand the mechanisms of spermatogenesis and fertilization. Here, we searched the mouse UniGene databases and identified 13 novel genes as being putatively testis-specific or -predominant. Our in silico and in vitro analyses revealed that the expressions of these genes are testis- and germ cell-specific, and that they are regulated in a stage-specific manner during spermatogenesis. We generated antibodies against the proteins encoded by seven of the genes to facilitate their characterization in male germ cells. Immunoblotting and immunofluorescence analyses revealed that one of these proteins was expressed only in testicular germ cells, three were expressed in both testicular germ cells and testicular sperm, and the remaining three were expressed in sperm of the testicular stages and in mature sperm from the epididymis. Further analysis of the latter three proteins showed that they were all associated with cytoskeletal structures in the sperm flagellum. Among them, MORN5, which is predicted to contain three MORN motifs, is conserved between mouse and human sperm. In conclusion, we herein identify 13 authentic genes with male germ cell-specific expression, and provide comprehensive information about these genes and their encoded products. Our finding will facilitate future investigations into the functional roles of these novel genes in spermatogenesis and sperm functions.

Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification.

PubMed

Oh, Denise; Houston, Douglas W

2017-12-15

The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development. Copyright © 2017 Elsevier Inc. All rights reserved.

Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum

PubMed Central

2014-01-01

Background Germline specification in some animals is driven by the maternally inherited germ plasm during early embryogenesis (inheritance mode), whereas in others it is induced by signals from neighboring cells in mid or late development (induction mode). In the Metazoa, the induction mode appears as a more prevalent and ancestral condition; the inheritance mode is therefore derived. However, regarding germline specification in organisms with asexual and sexual reproduction it has not been clear whether both strategies are used, one for each reproductive phase, or if just one strategy is used for both phases. Previously we have demonstrated that specification of germ cells in the asexual viviparous pea aphid depends on a preformed germ plasm. In this study, we extended this work to investigate how germ cells were specified in the sexual oviparous embryos, aiming to understand whether or not developmental plasticity of germline specification exists in the pea aphid. Results We employed Apvas1, a Drosophila vasa ortholog in the pea aphid, as a germline marker to examine whether germ plasm is preformed during oviparous development, as has already been seen in the viviparous embryos. During oogenesis, Apvas1 mRNA and ApVas1 protein were both evenly distributed. After fertilization, uniform expression of Apvas1 remained in the egg but posterior localization of ApVas1 occurred from the fifth nuclear cycle onward. Posterior co-localization of Apvas1/ApVas1 was first identified in the syncytial blastoderm undergoing cellularization, and later we could detect specific expression of Apvas1/ApVas1 in the morphologically identifiable germ cells of mature embryos. This suggests that Apvas1/ApVas1-positive cells are primordial germ cells and posterior localization of ApVas1 prior to cellularization positions the preformed germ plasm. Conclusions We conclude that both asexual and sexual pea aphids rely on the preformed germ plasm to specify germ cells and that developmental plasticity of germline specification, unlike axis patterning, occurs in neither of the two aphid reproductive phases. Consequently, the maternal inheritance mode implicated by a preformed germ plasm in the oviparous pea aphid becomes a non-canonical case in the Hemimetabola, where so far the zygotic induction mode prevails in most other studied insects. PMID:24855557

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 4: intercellular bridges, mitochondria, nuclear envelope, apoptosis, ubiquitination, membrane/voltage-gated channels, methylation/acetylation, and transcription factors.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic. Copyright 2009 Wiley-Liss, Inc.

DAZL is essential for stress granule formation implicated in germ cell survival upon heat stress.

PubMed

Kim, Byunghyuk; Cooke, Howard J; Rhee, Kunsoo

2012-02-01

Mammalian male germ cells should be maintained below body temperature for proper development. Here, we investigated how male germ cells respond to heat stress. A short exposure of mouse testes to core body temperature induced phosphorylation of eIF2α and the formation of stress granules (SGs) in male germ cells. We observed that DAZL, a germ cell-specific translational regulator, was translocated to SGs upon heat stress. Furthermore, SG assembly activity was significantly diminished in the early male germ cells of Dazl-knockout mice. The DAZL-containing SGs played a protective role against heat stress-induced apoptosis by the sequestration of specific signaling molecules, such as RACK1, and the subsequent blockage of the apoptotic MAPK pathway. Based on these results, we propose that DAZL is an essential component of the SGs, which prevent male germ cells from undergoing apoptosis upon heat stress.

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

PubMed

Gordeeva, O F; Nikonova, T M; Lifantseva, N V

2009-01-01

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

Germ Tube Growth Inhibitor from Cronartium comandrae Aeciospores

PubMed Central

Eppstein, Deborah A.; Tainter, F. H.

1979-01-01

Two compounds showing self-inhibitory action during germination of aeciospores of the comandra blister rust fungus (Cronartium comandrae Pk.) were extracted from these aeciospores by shaking with 0.2 M NH4HCO3 (pH 7.8) for 4 h. One of these, the germination self inhibitor (D. A. Eppstein and F. H. Tainter, Phytopathology 66:1395-1397, 1976), was removed from the ammonium bicarbonate buffer by using chloroform. The water layer which remained contained a substance which, at ca. 10−4 M concentration, had no apparent effect on germ tube emergence but which inhibited normal germ tube growth. Linear germ tube growth ceased or a dendritic or vesicular pattern of growth resulted, depending on the concentration of inhibitor added to extracted germinating spores. The germ tube growth inhibitor appears to be a peptide with a molecular weight of ca. 2,000. Images PMID:16345335

bmp15l, figla, smc1bl, and larp6l are preferentially expressed in germ cells in Atlantic salmon (Salmo salar L.).

PubMed

Kleppe, Lene; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Andersson, Eva; Juanchich, Amélie; Wargelius, Anna

2017-01-01

Atlantic salmon is a valuable commercial aquaculture species that would benefit economically and environmentally by controlling precocious puberty and preventing escapees from reproducing with wild populations. One solution to both these challenges is the production of sterile individuals by inhibiting the formation of germ cells, but achieving this requires more information on the specific factors that control germ cell formation. Here, we identified and characterized novel factors that are preferentially expressed in Atlantic salmon germ cells by screening for gonad-specific genes using available adult multi-tissue transcriptomes. We excluded genes with expression in tissues other than gonads based on quantity of reads, and then a subset of genes was selected for verification in a multi-tissue PCR screen. Four gonad-specific genes (bmp15l, figla, smc1bl, and larp6l) were chosen for further characterization, namely: germ cell specificity, investigated by comparing mRNA abundance in wild-type and germ cell-free gonads by quantitative real-time PCR, and cellular location, visualized by in situ hybridization. All four genes were expressed in both testis and ovary, and preferentially within the germ cells of both sexes. These genes may be essential players in salmon germ cell development, and could be important for future studies aiming to understand and control reproduction. Mol. Reprod. Dev. 84: 76-87, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

β-Catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii

PubMed Central

Darras, Sébastien; Gerhart, John; Terasaki, Mark; Kirschner, Marc; Lowe, Christopher J.

2011-01-01

The canonical Wnt/β-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of β-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by β-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into β-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of β-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for β-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by β-catenin should be distinguished and carefully re-examined. PMID:21303849

Antagonistic BMP–cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm

PubMed Central

Wijesena, Naveen; Simmons, David K.

2017-01-01

Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis. Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral–aboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue. PMID:28652368

The Xenopus Maternal-to-Zygotic Transition from the Perspective of the Germline.

PubMed

Yang, Jing; Aguero, Tristan; King, Mary Lou

2015-01-01

In Xenopus, the germline is specified by the inheritance of germ-plasm components synthesized at the beginning of oogenesis. Only the cells in the early embryo that receive germ plasm, the primordial germ cells (PGCs), are competent to give rise to the gametes. Thus, germ-plasm components continue the totipotent potential exhibited by the oocyte into the developing embryo at a time when most cells are preprogrammed for somatic differentiation as dictated by localized maternal determinants. When zygotic transcription begins at the mid-blastula transition, the maternally set program for somatic differentiation is realized. At this time, genetic control is ceded to the zygotic genome, and developmental potential gradually becomes more restricted within the primary germ layers. PGCs are a notable exception to this paradigm and remain transcriptionally silent until the late gastrula. How the germ-cell lineage retains full potential while somatic cells become fate restricted is a tale of translational repression, selective degradation of somatic maternal determinants, and delayed activation of zygotic transcription. © 2015 Elsevier Inc. All rights reserved.

In vitro gamete derivation from pluripotent stem cells: progress and perspective.

PubMed

Nagano, Makoto C

2007-04-01

Germ cells constitute a highly specialized cell population that is indispensable for the continuation and evolution of the species. Recently, several research groups have shown that these unique cells can be produced in vitro from pluripotent stem cells. Furthermore, live births of offspring using induced germ cells have been reported in one study. These results suggest that it may be possible to investigate germ cell development ex vivo and to establish novel reproductive technologies. To this end, it is critical to assess if gamete induction processes in vitro faithfully recapitulate normal germ cell development in vivo. Here, this issue is discussed with a focus on the germ line specification and the sex-specific development of pre- and postnatal germ cells. The aim of this paper is to concisely summarize the past progress and to present some future issues for the investigation into in vitro gamete production from pluripotent stem cells.

nanos function is essential for development and regeneration of planarian germ cells.

PubMed

Wang, Yuying; Zayas, Ricardo M; Guo, Tingxia; Newmark, Phillip A

2007-04-03

Germ cells are required for the successful propagation of sexually reproducing species. Understanding the mechanisms by which these cells are specified and how their totipotency is established and maintained has important biomedical and evolutionary implications. Freshwater planarians serve as fascinating models for studying these questions. They can regenerate germ cells from fragments of adult tissues that lack reproductive structures, suggesting that inductive signaling is involved in planarian germ cell specification. To study the development and regeneration of planarian germ cells, we have functionally characterized an ortholog of nanos, a gene required for germ cell development in diverse organisms, from Schmidtea mediterranea. In the hermaphroditic strain of this species, Smed-nanos mRNA is detected in developing, regenerating, and mature ovaries and testes. However, it is not detected in the vast majority of newly hatched planarians or in small tissue fragments that will ultimately regenerate germ cells, consistent with an epigenetic origin of germ cells. We show that Smed-nanos RNA interference (RNAi) results in failure to develop, regenerate, or maintain gonads in sexual planarians. Unexpectedly, Smed-nanos mRNA is also detected in presumptive testes primordia of asexual individuals that reproduce strictly by fission. These presumptive germ cells are lost after Smed-nanos RNAi, suggesting that asexual planarians specify germ cells, but their differentiation is blocked downstream of Smed-nanos function. Our results reveal a conserved function of nanos in germ cell development in planarians and suggest that these animals will serve as useful models for dissecting the molecular basis of epigenetic germ cell specification.

Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis.

PubMed

Aguero, Tristan; Zhou, Yi; Kloc, Malgorzata; Chang, Patrick; Houliston, Evelyn; King, Mary Lou

2016-03-01

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1 , localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos -3'UTR. Importantly, Hermes/Rbpms specifically binds nanos , but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1 . One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA .

Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis

PubMed Central

Aguero, Tristan; Zhou, Yi; Kloc, Malgorzata; Chang, Patrick; Houliston, Evelyn; King, Mary Lou

2016-01-01

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm that assembles within the mitochondrial cloud or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1, localize to the germ plasm. nanos1 has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell (PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm. Previous work indicates that nanos accumulates within the germ plasm through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3′UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1 RNA in the nucleus of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1. One such factor may be hnRNP I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus that facilitates selection of germline RNAs for germ plasm localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA. PMID:26998427

Dasatinib, Ifosfamide, Carboplatin, and Etoposide in Treating Young Patients With Metastatic or Recurrent Malignant Solid Tumors

ClinicalTrials.gov

2018-02-09

Brain and Central Nervous System Tumors; Childhood Germ Cell Tumor; Extragonadal Germ Cell Tumor; Kidney Cancer; Liver Cancer; Lymphoma; Neuroblastoma; Ovarian Cancer; Sarcoma; Testicular Germ Cell Tumor; Unspecified Childhood Solid Tumor, Protocol Specific

Bone Morphogenetic Protein (BMP) signaling in animal reproductive system development and function.

PubMed

Lochab, Amaneet K; Extavour, Cassandra G

2017-07-15

In multicellular organisms, the specification, maintenance, and transmission of the germ cell lineage to subsequent generations are critical processes that ensure species survival. A number of studies suggest that the Bone Morphogenetic Protein (BMP) pathway plays multiple roles in this cell lineage. We wished to use a comparative framework to examine the role of BMP signaling in regulating these processes, to determine if patterns would emerge that might shed light on the evolution of molecular mechanisms that may play germ cell-specific or other reproductive roles across species. To this end, here we review evidence to date from the literature supporting a role for BMP signaling in reproductive processes across Metazoa. We focus on germ line-specific processes, and separately consider somatic reproductive processes. We find that from primordial germ cell (PGC) induction to maintenance of PGC identity and gametogenesis, BMP signaling regulates these processes throughout embryonic development and adult life in multiple deuterostome and protostome clades. In well-studied model organisms, functional genetic evidence suggests that BMP signaling is required in the germ line across all life stages, with the exception of PGC specification in species that do not use inductive signaling to induce germ cell formation. The current evidence is consistent with the hypothesis that BMP signaling is ancestral in bilaterian inductive PGC specification. While BMP4 appears to be the most broadly employed ligand for the reproductive processes considered herein, we also noted evidence for sex-specific usage of different BMP ligands. In gametogenesis, BMP6 and BMP15 seem to have roles restricted to oogenesis, while BMP8 is restricted to spermatogenesis. We hypothesize that a BMP-based mechanism may have been recruited early in metazoan evolution to specify the germ line, and was subsequently co-opted for use in other germ line-specific and somatic reproductive processes. We suggest that if future studies assessing the function of the BMP pathway across extant species were to include a reproductive focus, that we would be likely to find continued evidence in favor of an ancient association between BMP signaling and the reproductive cell lineage in animals. Copyright © 2017 Elsevier Inc. All rights reserved.

Epigenome regulation during germ cell specification and development from pluripotent stem cells.

PubMed

Kurimoto, Kazuki; Saitou, Mitinori

2018-06-13

Germ cells undergo epigenome reprogramming for proper development of the next generation. The realization of germ cell derivation from human and mouse pluripotent stem cells offers unprecedented opportunity for investigation of germline development. Primordial germ cells reconstituted in vitro (PGC-like cells [PGCLCs]) show progressive dilution of genomic DNA methylation, tightly linked with chromatin remodeling, during their specification. PGCLCs can be further expanded by plane culture, allowing maintenance of the gene-expression profiles of early PGCs and continuance of the DNA methylation erasure, thereby establishing an epigenetic `blank slate'. PGCLCs undergo further epigenome regulation to acquire the male or female fates. These findings will provide a foundation for basic germ cell biology and for in-depth evaluations of in vitro gametogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development without germ cells: the role of the germ line in zebrafish sex differentiation.

PubMed

Slanchev, Krasimir; Stebler, Jürg; de la Cueva-Méndez, Guillermo; Raz, Erez

2005-03-15

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin-antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males.

Development without germ cells: The role of the germ line in zebrafish sex differentiation

PubMed Central

Slanchev, Krasimir; Stebler, Jürg; de la Cueva-Méndez, Guillermo; Raz, Erez

2005-01-01

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin–antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males. PMID:15728735

Generation of germ cells in vitro in the era of induced pluripotent stem cells.

PubMed

Imamura, Masanori; Hikabe, Orie; Lin, Zachary Yu-Ching; Okano, Hideyuki

2014-01-01

Induced pluripotent stem cells (iPSCs) are stem cells that can be artificially generated via "cellular reprogramming" using gene transduction in somatic cells. iPSCs have enormous potential in stem-cell biology as they can give rise to numerous cell lineages, including the three germ layers. An evaluation of germ-line competency by blastocyst injection or tetraploid complementation, however, is critical for determining the developmental potential of mouse iPSCs towards germ cells. Recent studies have demonstrated that primordial germ cells obtained by the in vitro differentiation of iPSCs produce functional gametes as well as healthy offspring. These findings illustrate not only that iPSCs are developmentally similar to embryonic stem cells (ESCs), but also that somatic cells from adult tissues can produce gametes in vitro, that is, if they are reprogrammed into iPSCs. In this review, we discuss past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells, with an emphasis on ESCs and iPSCs. While this field of research is still at a stage of infancy, it holds great promises for investigating the mechanisms of germ-cell development, especially in humans, and for advancing reproductive and developmental engineering technologies in the future. © 2013 Wiley Periodicals, Inc.

Ca2+ signalling and early embryonic patterning during zebrafish development.

PubMed

Webb, Sarah E; Miller, Andrew L

2007-09-01

1. It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern-forming events during early vertebrate development. 2. With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time-window includes most of the major pattern-forming events of early development, which transform a single-cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3. Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4. The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern-forming signalling pathways known to modulate early developmental events.

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

PubMed Central

Lanza, Denise G.; Dawson, Emily P.; Rao, Priya; Heaney, Jason D.

2016-01-01

ABSTRACT Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis. PMID:26901436

Towards Natural Transition in Compressible Boundary Layers

DTIC Science & Technology

2016-06-29

AFRL-AFOSR-CL-TR-2016-0011 Towards natural transition in compressible boundary layers Marcello Faraco de Medeiros FUNDACAO PARA O INCREMENTO DA...to 29-03-2016 Towards natural transition in compressible boundary layers FA9550-11-1-0354 Marcello A. Faraco de Medeiros Germán Andrés Gaviria...unlimited. 109 Final report Towards natural transition in compressible boundary layers Principal Investigator: Marcello Augusto Faraco de Medeiros

Black carp vasa identifies embryonic and gonadal germ cells.

PubMed

Xue, Ting; Yu, Miao; Pan, Qihua; Wang, Yizhou; Fang, Jian; Li, Lingyu; Deng, Yu; Chen, Kai; Wang, Qian; Chen, Tiansheng

2017-07-01

Identification of molecular markers is an essential step in the study of germ cells. Vasa is an RNA helicase and a well-known germ cell marker that plays a crucial role in germ cell development. Here, we identified the Vasa homolog termed Mpvasa as the first germ cell marker in black carp (Mylopharyngodon piceus). First, a 2819-bp full-length Mpvasa complementary DNA (cDNA) was cloned by PCR using degenerated primers of conserved sequences and gene-specific primers. The Mpvasa cDNA sequence encodes a 637-amino acid protein that contains eight conserved characteristic motifs of the DEAD box protein family, and shares high identity to grass carp (81%) and zebrafish (74%) vasa homologs. Second, Mpvasa expression was restricted to the gonad in adulthood by RT-PCR and Western blot analysis. The dynamic patterns of temporal-spatial expression of Mpvasa during gametogenesis were examined by in situ hybridization, and Mpvasa transcripts were strictly detected in gonadal germ cells throughout oogenesis, predominantly in immature oocytes (stage I, II, and III oocytes). Third, Mpvasa transcripts were highly detected in unfertilized eggs and early embryos, and the signal indicated a dynamic migration of the primordial germ cells during embryogenesis, suggesting that Mpvasa transcripts were maternally inherited and specifically distributed in germ cells. Taken together, these results demonstrated that Mpvasa is an applicable molecular marker for identification of gonadal and embryonic germ cells, which facilitates the isolation and utilization of germ cells in black carp.

A role for Lin28 in primordial germ cell development and germ cell malignancy

PubMed Central

West, Jason A.; Viswanathan, Srinivas R.; Yabuuchi, Akiko; Cunniff, Kerianne; Takeuchi, Ayumu; Park, In-Hyun; Sero, Julia E.; Zhu, Hao; Perez-Atayde, Antonio; Frazier, A. Lindsay; Surani, M. Azim; Daley, George Q.

2009-01-01

The rarity and inaccessibility of the earliest primordial germ cells (PGCs) in the mouse embryo thwarts efforts to investigate molecular mechanisms of germ cell specification. Stella marks the minute founder population of the germ lineage1,2. Here we differentiate mouse embryonic stem cells (ESCs) carrying a Stella transgenic reporter into putative PGCs in vitro. The Stella+ cells possess a transcriptional profile similar to embryo-derived PGCs, and like their counterparts in vivo, lose imprints in a time-dependent manner. Using inhibitory RNAs to screen candidate genes for effects on the development of Stella+ cells in vitro, we discovered that Lin28, a negative regulator of let-7 microRNA processing3-6, is essential for proper PGC development. We further show that Blimp1, a let-7 target and a master regulator of PGC specification7-9, can rescue the effect of Lin28-deficiency during PGC development, thereby establishing a mechanism of action for Lin28 during PGC specification. Over-expression of Lin28 promotes formation of Stella+ cells in vitro and PGCs in chimeric embryos, and is associated with human germ cell tumours. The differentiation of putative PGCs from ESCs in vitro recapitulates the early stages of gamete development in vivo, and provides an accessible system for discovering novel genes involved in germ cell development and malignancy. PMID:19578360

Ectoplasmic specialization, a testis-specific cell-cell actin-based adherens junction type: is this a potential target for male contraceptive development?

PubMed

Lee, Nikki P Y; Cheng, C Yan

2004-01-01

The seminiferous tubule of the mammalian testis is largely composed of Sertoli and germ cells, which coordinate with Leydig cells in the interstitium and perform two major physiological functions, namely spermatogenesis and steroidogenesis respectively. Each tubule is morphologically divided into (i) the seminiferous epithelium composing Sertoli and germ cells, and (ii) the basement membrane (a modified form of extracellular matrix); underneath this lies the collagen fibril network, the myoid cell layer, and the lymphatic vessel, which collectively constitute the tunica propia. In the seminiferous epithelium, of rodent testes each type A1 spermatogonium (diploid, 2n) differentiates into 256 elongated spermatids (haploid, 1n) during spermatogenesis. Additionally, developing germ cells must migrate progressively from the basal to the luminal edge of the adluminal compartment so that fully developed spermatids can be released into the lumen at spermiation. Without this timely event of cell movement, spermatogenesis cannot reach completion and infertility will result. Yet developing round elongating/elongated spermatids must remain attached to the epithelium via a specialized Sertoli-germ cell actin-based adherens junction (AJ) type known as ectoplasmic specialization (ES), which is crucial not only for cell attachment but also for spermatid movement and orientation in the epithelium. However, the biochemical composition and molecular architecture of the protein complexes that constitute the ES have only recently been studied. Furthermore, the signalling pathways that regulate ES dynamics are virtually unknown. This review highlights recent advances in these two areas of research. It is expected that, if adequately expanded, these studies should yield new insights into the development of novel contraceptives targeted to perturb ES function in the testis. The potential to specifically target the ES may also mean that contraceptive action could be achieved without perturbing the hypothalamic-pituitary-testicular axis.

GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

EPA Science Inventory

Chemical mutagenesis test results ave not revealed evidence of germ-line specific mutagens. owever, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend u...

A new glycosylated dihydrophaseic acid from cacao germs (Theobroma cacao L.).

PubMed

Sannohe, Yumiko; Gomi, Shuichi; Murata, Takashi; Ohyama, Makoto; Yonekura, Kumiko; Kanegae, Minoru; Koga, Jinichiro

2011-01-01

Cacao beans are composed of cacao nibs and germs. Although numerous chemical and physiological studies on cacao nib compounds have been reported, there is little information on cacao germ compounds. We therefore analyzed an extract from the cacao germ, and found two compounds that were specific to the germ. One of these two compounds was identified as the new glycosylated abscisic acid metabolite, dihydrophaseic acid-4'-O-6″-(β-ribofuranosyl)-β-glucopyranoside, and the other as the known compound, dihydrophaseic acid-4'-O-β-D-glucopyranoside.

Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation

PubMed Central

Kee, Kehkooi; Angeles, Vanessa T; Flores, Martha; Nguyen, Ha Nam; Pera, Renee A Reijo

2009-01-01

The leading cause of infertility in men and women is quantitative and qualitative defects in human germ cell (oocyte and sperm) development. Yet, it has not been possible to examine the unique developmental genetics of human germ cell formation and differentiation due to inaccessibility of germ cells during fetal development. Although several studies have shown that germ cells can be differentiated from mouse and human embryonic stem cells, human germ cells differentiated in these studies generally did not develop beyond the earliest stages1-8. Here we used a germ cell reporter to quantitate and isolate primordial germ cells derived from both male and female hESCs. Then, by silencing and overexpressing genes that encode germ cell-specific cytoplasmic RNA-binding proteins (not transcription factors), we modulated human germ cell formation and developmental progression. We observed that human DAZL (Deleted in AZoospermia-Like) functions in primordial germ cell formation, whereas closely-related genes, DAZ and BOULE, promote later stages of meiosis and development of haploid gametes. These results are significant to the generation of gametes for future basic science and potential clinical applications. PMID:19865085

Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice

PubMed Central

Heaney, Jason D.; Anderson, Ericka L.; Michelson, Megan V.; Zechel, Jennifer L.; Conrad, Patricia A.; Page, David C.; Nadeau, Joseph H.

2012-01-01

Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19MOLF/Ei), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation. PMID:22438569

The Formation of Germ Cell for Organizational Learning

ERIC Educational Resources Information Center

Ivaldi, Silvia; Scaratti, Giuseppe

2016-01-01

Purpose: The aim of the paper is to analyze the process of "germ cell" formation by framing it as an opportunity for promoting organizational learning and transformation. The paper aims to specifically answer two research questions: Why does the "germ cell" have a pivotal role in organization's transformation? and Which…

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 5: intercellular junctions and contacts between germs cells and Sertoli cells and their regulatory interactions, testicular cholesterol, and genes/proteins associated with more than one germ cell generation.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

In the testis, cell adhesion and junctional molecules permit specific interactions and intracellular communication between germ and Sertoli cells and apposed Sertoli cells. Among the many adhesion family of proteins, NCAM, nectin and nectin-like, catenins, and cadherens will be discussed, along with gap junctions between germ and Sertoli cells and the many members of the connexin family. The blood-testis barrier separates the haploid spermatids from blood borne elements. In the barrier, the intercellular junctions consist of many proteins such as occludin, tricellulin, and claudins. Changes in the expression of cell adhesion molecules are also an essential part of the mechanism that allows germ cells to move from the basal compartment of the seminiferous tubule to the adluminal compartment thus crossing the blood-testis barrier and well-defined proteins have been shown to assist in this process. Several structural components show interactions between germ cells to Sertoli cells such as the ectoplasmic specialization which are more closely related to Sertoli cells and tubulobulbar complexes that are processes of elongating spermatids embedded into Sertoli cells. Germ cells also modify several Sertoli functions and this also appears to be the case for residual bodies. Cholesterol plays a significant role during spermatogenesis and is essential for germ cell development. Lastly, we list genes/proteins that are expressed not only in any one specific generation of germ cells but across more than one generation. Copyright 2009 Wiley-Liss, Inc.

Male Rat Germ Cells Display Age-Dependent and Cell-Specific Susceptibility in Response to Oxidative Stress Challenges1

PubMed Central

Selvaratnam, Johanna; Paul, Catriona; Robaire, Bernard

2015-01-01

For decades male germ cells were considered unaffected by aging, due to the fact that males continue to generate sperm into old age; however, evidence indicates that germ cells from aged males are of lower quality than those of young males. The current study examines the effects of aging on pachytene spermatocytes and round spermatids, and is the first study to culture these cells in isolation for an extended period. Our objective is to determine the cell-specific responses germ cells have to aging and oxidative insult. Culturing isolated germ cells from young and aged Brown Norway rats revealed that germ cells from aged males displayed an earlier decline in viability, elevated levels of reactive oxygen species (ROS), and increased spermatocyte DNA damage, compared to young males. Furthermore, oxidative insult by prooxidant 3-morpholinosydnonimine provides insight into how spermatocytes and spermatids manage excess ROS. Genome-wide microarray analyses revealed that several transcripts for antioxidants, Sod1, Cat, and Prdxs, were up-regulated in response to ROS in germ cells from young males while being expressed at lower levels in the aged. In contrast, the expression of DNA damage repair genes Rad50 and Atm were increased in the germ cells from aged animals. Our data indicate that as germ cells undergo spermatogenesis, they adapt and respond to oxidative stress differently, depending on their phase of development, and the process of aging results in redox dysfunction. Thus, even at early stages of spermatogenesis, germ cells from aged males are unable to mount an appropriate response to manage oxidative stress. PMID:26224006

Functional anatomy of the hair follicle: The Secondary Hair Germ.

PubMed

Panteleyev, Andrey A

2018-07-01

The secondary hair germ (SHG)-a transitory structure in the lower portion of the mouse telogen hair follicle (HF)-is directly involved in anagen induction and eventual HF regrowth. Some crucial aspects of SHG functioning and ontogenetic relations with other HF parts, however, remain undefined. According to recent evidence (in contrast to previous bulge-centric views), the SHG is the primary target of anagen-inducing signalling and a source of both the outer root sheath (ORS) and ascending HF layers during the initial (morphogenetic) anagen subphase. The SHG is comprised of two functionally distinct cell populations. Its lower portion (originating from lower HF cells that survived catagen) forms all ascending HF layers, while the upper SHG (formed by bulge-derived cells) builds up the ORS. The predetermination of SHG cells to a specific morphogenetic fate contradicts their attribution to the "stem cell" category and supports SHG designation as a "germinative" or a "founder" cell population. The mechanisms of this predetermination driving transition of the SHG from "refractory" to the "competent" state during the telogen remain unknown. Functionally, the SHG serves as a barrier, protecting the quiescent bulge stem cell niche from the extensive follicular papilla/SHG signalling milieu. The formation of the SHG is a prerequisite for efficient "precommitment" of these cells and provides for easier sensing and a faster response to anagen-inducing signals. In general, the formation of the SHG is an evolutionary adaptation, which allowed the ancestors of modern Muridae to acquire a specific, highly synchronized pattern of hair cycling. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

PubMed

Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

2016-08-01

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

Unique differentiation profile of mouse embryonic stem cells in rotary and stirred tank bioreactors.

PubMed

Fridley, Krista M; Fernandez, Irina; Li, Mon-Tzu Alice; Kettlewell, Robert B; Roy, Krishnendu

2010-11-01

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (Synthecon™) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1(+) progenitors and spinner flasks generated more c-Kit(+) progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

Primordial Germ Cells in Mice

PubMed Central

Saitou, Mitinori; Yamaji, Masashi

2012-01-01

Germ cell development creates totipotency through genetic as well as epigenetic regulation of the genome function. Primordial germ cells (PGCs) are the first germ cell population established during development and are immediate precursors for both the oocytes and spermatogonia. We here summarize recent findings regarding the mechanism of PGC development in mice. We focus on the transcriptional and signaling mechanism for PGC specification, potential pluripotency, and epigenetic reprogramming in PGCs and strategies for the reconstitution of germ cell development using pluripotent stem cells in culture. Continued studies on germ cell development may lead to the generation of totipotency in vitro, which should have a profound influence on biological science as well as on medicine. PMID:23125014

Developmentally regulated expression of APG-1, a member of heat shock protein 110 family in murine male germ cells.

PubMed

Kaneko, Y; Kimura, T; Nishiyama, H; Noda, Y; Fujita, J

1997-04-07

Apg-1 encodes a heat shock protein belonging to the heat shock protein 110 family, and is inducible by a 32 degrees C to 39 degrees C heat shock. Northern blot analysis of the testis from immature and adult mice, and of the purified germ cells revealed the quantitative change of the apg-1 transcripts during germ cell development. By in situ hybridization histochemistry the expressions of the apg-1 transcripts were detected in germ cells at specific stages of development including spermatocytes and spermatids. Although heat-induction of the apg-1 transcripts was observed in W/Wv mutant testis lacking germ cells, it was not detected in wild-type testis nor in the purified germ cells. Thus, the apg-1 expression is not heat-regulated but developmentally regulated in germ cells, suggesting that APG-1 plays a role in normal development of germ cells.

The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

PubMed

Tokuhiro, Keizo; Miyagawa, Yasushi; Yamada, Shuichi; Hirose, Mika; Ohta, Hiroshi; Nishimune, Yoshitake; Tanaka, Hiromitsu

2007-03-01

Haspin is a unique protein kinase expressed predominantly in haploid male germ cells. The genomic structure of haspin (Gsg2) has revealed it to be intronless, and the entire transcription unit is in an intron of the integrin alphaE (Itgae) gene. Transcription occurs from a bidirectional promoter that also generates an alternatively spliced integrin alphaE-derived mRNA (Aed). In mice, the testis-specific alternative splicing of Aed is expressed bidirectionally downstream from the Gsg2 transcription initiation site, and a segment consisting of 26 bp transcribes both genomic DNA strands between Gsg2 and the Aed transcription initiation sites. To investigate the mechanisms for this unique gene regulation, we cloned and characterized the Gsg2 promoter region. The 193-bp genomic fragment from the 5' end of the Gsg2 and Aed genes, fused with EGFP and DsRed genes, drove the expression of both proteins in haploid germ cells of transgenic mice. This promoter element contained only a GC-rich sequence, and not the previously reported DNA sequences known to bind various transcription factors--with the exception of E2F1, TCFAP2A1 (AP2), and SP1. Here, we show that the 193-bp DNA sequence is sufficient for the specific, bidirectional, and synchronous expression in germ cells in the testis. We also demonstrate the existence of germ cell nuclear factors specifically bound to the promoter sequence. This activity may be regulated by binding to the promoter sequence with germ cell-specific nuclear complex(es) without regulation via DNA methylation.

A functional Bucky ball-GFP transgene visualizes germ plasm in living zebrafish.

PubMed

Riemer, Stephan; Bontems, Franck; Krishnakumar, Pritesh; Gömann, Jasmin; Dosch, Roland

2015-01-01

In many animals, the germline is specified by maternal RNA-granules termed germ plasm. The correct localization of germ plasm during embryogenesis is therefore crucial for the specification of germ cells. In zebrafish, we previously identified Bucky ball (Buc) as a key regulator of germ plasm formation. Here, we used a Buc antibody to describe its continuous germ plasm localization. Moreover, we generated a transgenic Buc-GFP line for live imaging, which visualizes germ plasm from its assembly during oogenesis up to the larval stages. Live imaging of Buc-GFP generated stunning movies, as they highlighted the dynamic details of germ plasm movements. Moreover, we discovered that Buc was still detected in primordial germ cells 2 days after fertilization. Interestingly, the transgene rescued buc mutants demonstrating genetically that the Buc-GFP fusion protein is functional. These results show that Buc-GFP exerts all biochemical interactions essential for germline development and highlight the potential of this line to analyze the molecular regulation of germ plasm formation. Copyright © 2015 Elsevier B.V. All rights reserved.

Maintaining Sufficient Nanos Is a Critical Function for Polar Granule Component in the Specification of Primordial Germ Cells

PubMed Central

Deshpande, Girish; Spady, Emma; Goodhouse, Joe; Schedl, Paul

2012-01-01

Primordial germ cells (PGC) are the precursors of germline stem cells. In Drosophila, PGC specification is thought to require transcriptional quiescence and three genes, polar granule component (pgc), nanos (nos), and germ cell less (gcl) function to downregulate Pol II transcription. While it is not understood how nos or gcl represses transcription, pgc does so by inhibiting the transcription elongation factor b (P-TEFb), which is responsible for phosphorylating Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of the largest Pol II subunit. In the studies reported here, we demonstrate that nos are a critical regulatory target of pgc. We show that a substantial fraction of the PGCs in pgc embryos have greatly reduced levels of Nos protein and exhibit phenotypes characteristic of nos PGCs. Lastly, restoring germ cell–specific expression of Nos is sufficient to ameliorate the pgc phenotype. PMID:23173091

Maintaining sufficient nanos is a critical function for polar granule component in the specification of primordial germ cells.

PubMed

Deshpande, Girish; Spady, Emma; Goodhouse, Joe; Schedl, Paul

2012-11-01

Primordial germ cells (PGC) are the precursors of germline stem cells. In Drosophila, PGC specification is thought to require transcriptional quiescence and three genes, polar granule component (pgc), nanos (nos), and germ cell less (gcl) function to downregulate Pol II transcription. While it is not understood how nos or gcl represses transcription, pgc does so by inhibiting the transcription elongation factor b (P-TEFb), which is responsible for phosphorylating Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of the largest Pol II subunit. In the studies reported here, we demonstrate that nos are a critical regulatory target of pgc. We show that a substantial fraction of the PGCs in pgc embryos have greatly reduced levels of Nos protein and exhibit phenotypes characteristic of nos PGCs. Lastly, restoring germ cell-specific expression of Nos is sufficient to ameliorate the pgc phenotype.

Derivation, propagation and differentiation of human embryonic stem cells.

PubMed

Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

2004-04-01

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.

A framework for the establishment of a cnidarian gene regulatory network for "endomesoderm" specification: the inputs of ß-catenin/TCF signaling.

PubMed

Röttinger, Eric; Dahlin, Paul; Martindale, Mark Q

2012-01-01

Understanding the functional relationship between intracellular factors and extracellular signals is required for reconstructing gene regulatory networks (GRN) involved in complex biological processes. One of the best-studied bilaterian GRNs describes endomesoderm specification and predicts that both mesoderm and endoderm arose from a common GRN early in animal evolution. Compelling molecular, genomic, developmental, and evolutionary evidence supports the hypothesis that the bifunctional gastrodermis of the cnidarian-bilaterian ancestor is derived from the same evolutionary precursor of both endodermal and mesodermal germ layers in all other triploblastic bilaterian animals. We have begun to establish the framework of a provisional cnidarian "endomesodermal" gene regulatory network in the sea anemone, Nematostella vectensis, by using a genome-wide microarray analysis on embryos in which the canonical Wnt/ß-catenin pathway was ectopically targeted for activation by two distinct pharmaceutical agents (lithium chloride and 1-azakenpaullone) to identify potential targets of endomesoderm specification. We characterized 51 endomesodermally expressed transcription factors and signaling molecule genes (including 18 newly identified) with fine-scale temporal (qPCR) and spatial (in situ) analysis to define distinct co-expression domains within the animal plate of the embryo and clustered genes based on their earliest zygotic expression. Finally, we determined the input of the canonical Wnt/ß-catenin pathway into the cnidarian endomesodermal GRN using morpholino and mRNA overexpression experiments to show that NvTcf/canonical Wnt signaling is required to pattern both the future endomesodermal and ectodermal domains prior to gastrulation, and that both BMP and FGF (but not Notch) pathways play important roles in germ layer specification in this animal. We show both evolutionary conserved as well as profound differences in endomesodermal GRN structure compared to bilaterians that may provide fundamental insight into how GRN subcircuits have been adopted, rewired, or co-opted in various animal lineages that give rise to specialized endomesodermal cell types.

Study of Small Ligands Which Bind Specifically to Breast Cancer Cells

DTIC Science & Technology

1997-09-01

Sepharose conjugated to three different lectins: ConA, wheat germ and lentil,. Each lectin bound many proteins in both the ECD-AP sup and the control 3T3 sup...control Lanes 13-14: Wheat germ ECD-AP Lanes 15-16: Wheat germ 3T3 control Odd lanes were eluted with a low sugar concentration; even lanes were...ECD-AP post incubation with lentil-Sepharose Lane 6: Protein remaining in pp ECD-AP post incubation with wheat germ -Sepharose Lane 8: Protein

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

PubMed Central

Yamauchi, Kaori; Hasegawa, Kouichi; Chuma, Shinichiro; Nakatsuji, Norio; Suemori, Hirofumi

2009-01-01

Background Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. Methods and Findings To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. Conclusion VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates. PMID:19399191

Sinonasal teratocarcinosarcoma: a clinical and pathological analysis.

PubMed

Yang, Shudong; Sun, Rongchao; Liang, Jiabei; Zhou, Zhiyi; Zhou, Jing; Rui, Jun

2013-02-01

The goal of this study was to assess the pathological and differential diagnoses of sinonasal teratocarcinosarcoma (SNTCS) in order to ultimately improve the diagnosis and treatment of this rare disease. Data from 2 cases of sinonasal teratocarcinosarcoma from the Wuxi People's Hospital (China) were analyzed. The clinical presentation for these patients consisted of nasal obstruction, epistaxis, and headache. On further physical examination, the presence of a polypoid mass was identified and, despite surgery and radiotherapy, both cases experienced recurrence. Histologically, the tumors showed a heterogeneous mixture of components from the 3 germ layers, primitive neuroepithelial elements, diagnostic immature squamous cell nests (clear cell nests), and various epithelial and mesenchymal components. Staining of the different germ layers corresponded with the appropriate immune markers. In case 1, the postradiotherapy resection specimen was completely dominated by a mature teratoma, with a complete absence of the corresponding adenocarcinoma and fibrosarcoma components. To date, this is the first study describing this composition within an SNTCS recurrent tumor. In summary, SNTCS is a rare tumor characterized by the presence of benign and malignant epithelial, mesenchymal, and dysembryomal components. Owing to its heterogeneous histologic appearance, adequate sampling and recognition of all SNTCS components are needed for future diagnosis. Currently, surgical removal, postoperative radiotherapy, and a histology-specific multidrug chemotherapy appear to be the best therapeutic approach. Future individualized therapy may also hold promise.

Fetal bovine serum enables cardiac differentiation of human embryonic stem cells.

PubMed

Bettiol, Esther; Sartiani, Laura; Chicha, Laurie; Krause, Karl Heinz; Cerbai, Elisabetta; Jaconi, Marisa E

2007-10-01

During development, cardiac commitment within the mesoderm requires endoderm-secreted factors. Differentiation of embryonic stem cells into the three germ layers in vitro recapitulates developmental processes and can be influenced by supplements added to culture medium. Hence, we investigated the effect of fetal bovine serum (FBS) and KnockOut serum replacement (SR) on germ layers specification and cardiac differentiation of H1 human embryonic stem cells (hESC) within embryoid bodies (EB). At the time of EB formation, FBS triggered an increased apoptosis. As assessed by quantitative PCR on 4-, 10-, and 20-day-old EB, FBS promoted a faster down-regulation of pluripotency marker Oct4 and an increased expression of endodermal (Sox17, alpha-fetoprotein, AFP) and mesodermal genes (Brachyury, CSX). While neuronal and hematopoietic differentiation occurred in both supplements, spontaneously beating cardiomyocytes were only observed in FBS. Action potential (AP) morphology of hESC-derived cardiomyocytes indicated that ventricular cells were present only after 2 months of culture. However, quantification of myosin light chain 2 ventricular (mlc2v)-positive areas revealed that mlc2v-expressing cardiomyocytes could be detected already after 2 weeks of differentiation, but not in all beating clusters. In conclusion, FBS enabled cardiac differentiation of hESC, likely in an endodermal-dependent pathway. Among cardiac cells, ventricular cardiomyocytes differentiated over time, but not as the predominant cardiac cell subtype.

Germ Cell-less Promotes Centrosome Segregation to Induce Germ Cell Formation.

PubMed

Lerit, Dorothy A; Shebelut, Conrad W; Lawlor, Kristen J; Rusan, Nasser M; Gavis, Elizabeth R; Schedl, Paul; Deshpande, Girish

2017-01-24

The primordial germ cells (PGCs) specified during embryogenesis serve as progenitors to the adult germline stem cells. In Drosophila, the proper specification and formation of PGCs require both centrosomes and germ plasm, which contains the germline determinants. Centrosomes are microtubule (MT)-organizing centers that ensure the faithful segregation of germ plasm into PGCs. To date, mechanisms that modulate centrosome behavior to engineer PGC development have remained elusive. Only one germ plasm component, Germ cell-less (Gcl), is known to play a role in PGC formation. Here, we show that Gcl engineers PGC formation by regulating centrosome dynamics. Loss of gcl leads to aberrant centrosome separation and elaboration of the astral MT network, resulting in inefficient germ plasm segregation and aborted PGC cellularization. Importantly, compromising centrosome separation alone is sufficient to mimic the gcl loss-of-function phenotypes. We conclude Gcl functions as a key regulator of centrosome separation required for proper PGC development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Drosophila germ granules are structured and contain homotypic mRNA clusters

PubMed Central

Trcek, Tatjana; Grosch, Markus; York, Andrew; Shroff, Hari; Lionnet, Timothée; Lehmann, Ruth

2015-01-01

Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into ‘homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules. PMID:26242323

Developmental characteristics of somatic cell hybrids between totipotent mouse teratocarcinoma and rat intestinal villus cells.

PubMed

van der Kamp, A W; Roza-de Jongh, E J; Houwen, R H; Magrane, G G; van Dongen, J M; Evans, M J

1984-09-01

Hybrids between mouse PCC4-azal teratocarcinoma cells and rat epithelial intestinal villus cells (PCI hybrids) are phenotypically teratocarcinoma cells. They express several teratocarcinoma-specific traits but do not express functions specific for differentiated cells. Tumour formation is partially or completely suppressed. Some of the hybrids show more extensive differentiation both in vitro and in vivo than the PCC4-azal parental line. The hybrids are capable of endoderm formation in monolayer cultures and of the formation of embryoid bodies in suspension cultures. Two of the tumour-forming hybrids generate derivatives of all three germ layers, whereas differentiation in the PCC4-azal tumours is restricted to the formation of primitive neuronal tissues.

Models of germ cell development and their application for toxicity studies

PubMed Central

Ferreira, Daniel W.; Allard, Patrick

2015-01-01

Germ cells are unique in their ability to transfer genetic information and traits from generation to generation. As such, the proper development of germ cells and the integrity of their genome are paramount to the health of organisms and the survival of species. Germ cells are also exquisitely sensitive to environmental influences although the testing of germ cell toxicity, especially in females, has proven particularly challenging. In this review, we first describe the remarkable odyssey of germ cells in mammals, with an emphasis on the female germline, from their initial specification during embryogenesis to the generation of mature gametes in adults. We also describe the current methods used in germ cell toxicity testing and their limitations in examining the complex features of mammalian germ cell development. To bypass these challenges, we propose the use of alternative model systems such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans and in vitro germ cell methods that have distinct advantages over traditional toxicity models. We discuss the benefits and limitations of each approach, their application to germ cell toxicity studies, and the need for computational approaches to maximize the usefulness of these models. Together, the inclusion of these alternative germ cell toxicity models will be invaluable for the examination of stages not easily accessible in mammals as well as the large scale, high-throughput investigation of germ cell toxicity. PMID:25821157

Germ cells in the teleost fish medaka have an inherent feminizing effect

PubMed Central

Nishimura, Toshiya; Yamada, Kazuki; Fujimori, Chika; Kikuchi, Mariko; Kawasaki, Toshihiro; Siegfried, Kellee R.; Sakai, Noriyoshi

2018-01-01

Germ cells give rise to eggs or sperm. However, recent analyses in medaka (Oryzias latipes) showed that germ cells are also important for feminization of gonads, although this novel role of germ cells has not been characterized in detail. Here, we show that the feminizing effect is inherent to germ cells and is not affected by gametogenic stages or the sexual fate of germ cells. Three medaka mutants were generated to demonstrate this effect: figlα mutants, in which follicle formation is disrupted; meioC mutants, in which germ cells are unable to commit to gametogenesis and meiosis; and dazl mutants, in which germ cells do not develop into gonocytes. All these different stages of germ cells in XX mutants have an ability to feminize the gonads, resulting in the formation of gonads with ovarian structures. In addition to normal ovarian development, we also suggest that the increased number of gonocytes is sufficient for male to female sex reversal in XY medaka. These results may genetically demonstrate that the mechanism underlying the feminizing effect of germ cells is activated before the sexual fate decision of germ cells and meiosis, probably by the time of gonocyte formation in medaka. Author summary Germ cells are the only cells that can transfer genetic materials to the next generation via the sperm or egg. However, recent analyses in teleosts revealed another essential role of germ cells: feminizing the gonads. In our study, medaka mutants in which gametogenesis was blocked at specific stages provides the novel view that the feminizing effect of germ cells occurs in parallel with other reproductive elements, such as meiosis, the sexual fate decision of germ cells, and gametogenesis. Germ cells in medaka may have a potential to feminize gonads at the moment they have developed. PMID:29596424

ABT-751 in Treating Young Patients With Refractory Solid Tumors

ClinicalTrials.gov

2012-03-14

Brain and Central Nervous System Tumors; Childhood Germ Cell Tumor; Extragonadal Germ Cell Tumor; Kidney Cancer; Liver Cancer; Neuroblastoma; Ovarian Cancer; Sarcoma; Unspecified Childhood Solid Tumor, Protocol Specific

Signaling through the TGF Beta-Activin Receptors ALK4/5/7 Regulates Testis Formation and Male Germ Cell Development

PubMed Central

Stringer, Jessica M.; van den Bergen, Jocelyn A.; Wilhelm, Dagmar; Sinclair, Andrew H.; Western, Patrick S.

2013-01-01

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development. PMID:23342175

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamano, Noriko; Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp; Watanabe-Kushima, Shoko

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were culturedmore » on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.« less

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

PubMed

Hong, Ni; Li, Mingyou; Yuan, Yongming; Wang, Tiansu; Yi, Meisheng; Xu, Hongyan; Zeng, Huaqiang; Song, Jianxing; Hong, Yunhan

2016-03-08

Primordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

NASA Astrophysics Data System (ADS)

Meng, Qing-Yuan; Akaike, Toshihiro

2013-03-01

Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

Human induced pluripotent stem cells and male infertility: an overview of current progress and perspectives

PubMed Central

Li, Zili; Zhao, Qian; Li, Honggang; Xiong, Chengliang

2018-01-01

Abstract Recently, significant progress has been made in ART for the treatment of male infertility. However, current ART has failed to help infertile patients with non-obstructive azoospermia, unless donor sperm is used. In fact, most couples wish to have their own genetically related child. Human induced pluripotent stem cells (hiPSCs) can be generated from patients’ somatic cells and in vitro derivation of functional germ cells from patient-specific iPSCs may provide new therapeutic strategies for infertile couples. The overall developmental dynamics of human primordial germ cells are similar to that in mice, but accumulating evidence suggests that there are crucial differences between human and mouse PGC specification. Unlike mouse iPSCs (miPSCs) in naive state, hiPSCs exhibit a primed pluripotency which possess less potential for the germ cell fate. Based on research in mice, male germ cells at different stages have been derived from hiPSCs with different protocols, including spontaneous differentiation, overexpression of germ cell regulators, addition of cytokines, co-culture with gonadal cells in vitro and xeno-transplantation. The aim of this review is to summarize the current advances in derivation of male germ cells from hiPSCs and raise the perspectives of hiPSCs in medical application for male infertility, as well as in basic research for male germ cell development. PMID:29315416

Temozolomide and O6-benzylguanine in Treating Children With Solid Tumors

ClinicalTrials.gov

2015-04-28

Brain and Central Nervous System Tumors; Childhood Germ Cell Tumor; Extragonadal Germ Cell Tumor; Kidney Cancer; Liver Cancer; Neuroblastoma; Ovarian Cancer; Sarcoma; Unspecified Childhood Solid Tumor, Protocol Specific

vasa and piwi are required for mitotic integrity in early embryogenesis in the spider Parasteatoda tepidariorum.

PubMed

Schwager, Evelyn E; Meng, Yue; Extavour, Cassandra G

2015-06-15

Studies in vertebrate and invertebrate model organisms on the molecular basis of primordial germ cell (PGC) specification have revealed that metazoans can specify their germ line either early in development by maternally transmitted cytoplasmic factors (inheritance), or later in development by signaling factors from neighboring tissues (induction). Regardless of the mode of PGC specification, once animal germ cells are specified, they invariably express a number of highly conserved genes. These include vasa and piwi, which can play essential roles in any or all of PGC specification, development, or gametogenesis. Although the arthropods are the most speciose animal phylum, to date there have been no functional studies of conserved germ line genes in species of the most basally branching arthropod clade, the chelicerates (which includes spiders, scorpions, and horseshoe crabs). Here we present the first such study by using molecular and functional tools to examine germ line development and the roles of vasa and piwi orthologues in the common house spider Parasteatoda (formerly Achaearanea) tepidariorum. We use transcript and protein expression patterns of Pt-vasa and Pt-piwi to show that primordial germ cells (PGCs) in the spider arise during late embryogenesis. Neither Pt-vasa nor Pt-piwi gene products are localized asymmetrically to any embryonic region before PGCs emerge as paired segmental clusters in opisthosomal segments 2-6 at late germ band stages. RNA interference studies reveal that both genes are required maternally for egg laying, mitotic progression in early embryos, and embryonic survival. Our results add to the growing body of evidence that vasa and piwi can play important roles in somatic development, and provide evidence for a previously hypothesized conserved role for vasa in cell cycle progression. Copyright © 2014 Elsevier Inc. All rights reserved.

Paediatric germ cell tumours and congenital abnormalities: a Children's Oncology Group study

PubMed Central

Johnson, K J; Ross, J A; Poynter, J N; Linabery, A M; Robison, L L; Shu, X O

2009-01-01

Methods: Maternally reported congenital abnormalities (CAs) were examined in a case–control study of 278 cases of paediatric germ cell tumours (GCTs) and 423 controls. Results and conclusions Germ cell tumours were significantly associated with cryptorchidism in males (OR=10.8, 95% CI: 2.1–55.1), but not with any other specific CA in either sex. PMID:19603020

Identification of tissues and patterning events required for distinct steps in early migration of zebrafish primordial germ cells.

PubMed

Weidinger, G; Wolke, U; Köprunner, M; Klinger, M; Raz, E

1999-12-01

In many organisms, the primordial germ cells have to migrate from the position where they are specified towards the developing gonad where they generate gametes. Extensive studies of the migration of primordial germ cells in Drosophila, mouse, chick and Xenopus have identified somatic tissues important for this process and demonstrated a role for specific molecules in directing the cells towards their target. In zebrafish, a unique situation is found in that the primordial germ cells, as marked by expression of vasa mRNA, are specified in random positions relative to the future embryonic axis. Hence, the migrating cells have to navigate towards their destination from various starting positions that differ among individual embryos. Here, we present a detailed description of the migration of the primordial germ cells during the first 24 hours of wild-type zebrafish embryonic development. We define six distinct steps of migration bringing the primordial germ cells from their random positions before gastrulation to form two cell clusters on either side of the midline by the end of the first day of development. To obtain information on the origin of the positional cues provided to the germ cells by somatic tissues during their migration, we analyzed the migration pattern in mutants, including spadetail, swirl, chordino, floating head, cloche, knypek and no isthmus. In mutants with defects in axial structures, paraxial mesoderm or dorsoventral patterning, we find that certain steps of the migration process are specifically affected. We show that the paraxial mesoderm is important for providing proper anteroposterior information to the migrating primordial germ cells and that these cells can respond to changes in the global dorsoventral coordinates. In certain mutants, we observe accumulation of ectopic cells in different regions of the embryo. These ectopic cells can retain both morphological and molecular characteristics of primordial germ cells, suggesting that, in zebrafish at the early stages tested, the vasa-expressing cells are committed to the germ cell lineage.

Transcriptome analysis reveals differentially expressed genes associated with germ cell and gonad development in the Southern bluefin tuna (Thunnus maccoyii).

PubMed

Bar, Ido; Cummins, Scott; Elizur, Abigail

2016-03-10

Controlling and managing the breeding of bluefin tuna (Thunnus spp.) in captivity is an imperative step towards obtaining a sustainable supply of these fish in aquaculture production systems. Germ cell transplantation (GCT) is an innovative technology for the production of inter-species surrogates, by transplanting undifferentiated germ cells derived from a donor species into larvae of a host species. The transplanted surrogates will then grow and mature to produce donor-derived seed, thus providing a simpler alternative to maintaining large-bodied broodstock such as the bluefin tuna. Implementation of GCT for new species requires the development of molecular tools to follow the fate of the transplanted germ cells. These tools are based on key reproductive and germ cell-specific genes. RNA-Sequencing (RNA-Seq) provides a rapid, cost-effective method for high throughput gene identification in non-model species. This study utilized RNA-Seq to identify key genes expressed in the gonads of Southern bluefin tuna (Thunnus maccoyii, SBT) and their specific expression patterns in male and female gonad cells. Key genes involved in the reproductive molecular pathway and specifically, germ cell development in gonads, were identified using analysis of RNA-Seq transcriptomes of male and female SBT gonad cells. Expression profiles of transcripts from ovary and testis cells were compared, as well as testis germ cell-enriched fraction prepared with Percoll gradient, as used in GCT studies. Ovary cells demonstrated over-expression of genes related to stem cell maintenance, while in testis cells, transcripts encoding for reproduction-associated receptors, sex steroids and hormone synthesis and signaling genes were over-expressed. Within the testis cells, the Percoll-enriched fraction showed over-expression of genes that are related to post-meiosis germ cell populations. Gonad development and germ cell related genes were identified from SBT gonads and their expression patterns in ovary and testis cells were determined. These expression patterns correlate with the reproductive developmental stage of the sampled fish. The majority of the genes described in this study were sequenced for the first time in T. maccoyii. The wealth of SBT gonadal and germ cell-related gene sequences made publicly available by this study provides an extensive resource for further GCT and reproductive molecular biology studies of this commercially valuable fish.

Massive expression of germ cell-specific genes is a hallmark of cancer and a potential target for novel treatment development.

PubMed

Bruggeman, Jan Willem; Koster, Jan; Lodder, Paul; Repping, Sjoerd; Hamer, Geert

2018-06-15

Cancer cells have been found to frequently express genes that are normally restricted to the testis, often referred to as cancer/testis (CT) antigens or genes. Because germ cell-specific antigens are not recognized as "self" by the innate immune system, CT-genes have previously been suggested as ideal candidate targets for cancer therapy. The use of CT-genes in cancer therapy has thus far been unsuccessful, most likely because their identification has relied on gene expression in whole testis, including the testicular somatic cells, precluding the detection of true germ cell-specific genes. By comparing the transcriptomes of micro-dissected germ cell subtypes, representing the main developmental stages of human spermatogenesis, with the publicly accessible transcriptomes of 2617 samples from 49 different healthy somatic tissues and 9232 samples from 33 tumor types, we here discover hundreds of true germ cell-specific cancer expressed genes. Strikingly, we found these germ cell cancer genes (GC-genes) to be widely expressed in all analyzed tumors. Many GC-genes appeared to be involved in processes that are likely to actively promote tumor viability, proliferation and metastasis. Targeting these true GC-genes thus has the potential to inhibit tumor growth with infertility being the only possible side effect. Moreover, we identified a subset of GC-genes that are not expressed in spermatogonial stem cells. Targeting of this GC-gene subset is predicted to only lead to temporary infertility, as untargeted spermatogonial stem cells can recover spermatogenesis after treatment. Our GC-gene dataset enables improved understanding of tumor biology and provides multiple novel targets for cancer treatment.

SERCA directs cell migration and branching across species and germ layers

PubMed Central

Lansdale, Nick; Navarro, Sonia; Truong, Thai V.; Bower, Dan J.; Featherstone, Neil C.; Connell, Marilyn G.; Al Alam, Denise; Frey, Mark R.; Trinh, Le A.; Fernandez, G. Esteban; Warburton, David; Fraser, Scott E.; Bennett, Daimark; Jesudason, Edwin C.

2017-01-01

ABSTRACT Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding. PMID:28821490

High-dose ICE With Amifostine

ClinicalTrials.gov

2017-01-19

Bladder Cancer; Brain and Central Nervous System Tumors; Carcinoma of Unknown Primary; Extragonadal Germ Cell Tumor; Head and Neck Cancer; Kidney Cancer; Lung Cancer; Ovarian Cancer; Sarcoma; Testicular Germ Cell Tumor; Unspecified Adult Solid Tumor, Protocol Specific

Quality of Life and Survivorship Care in Patients Undergoing Hyperthermic Intraperitoneal Chemotherapy (HIPEC)

ClinicalTrials.gov

2017-05-25

Advanced Malignant Mesothelioma; Carcinoma of the Appendix; Ovarian Sarcoma; Ovarian Stromal Cancer; Pseudomyxoma Peritonei; Recurrent Colon Cancer; Recurrent Malignant Mesothelioma; Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Stage III Colon Cancer; Stage III Ovarian Epithelial Cancer; Stage III Ovarian Germ Cell Tumor; Stage IV Colon Cancer; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Unspecified Childhood Solid Tumor, Protocol Specific

Ultrastructural and immunocytochemical characterization of ameloblast-enamel adhesion at maturation stage in amelogenesis in Macaca fuscata tooth germ.

PubMed

Sawada, Takashi

2015-12-01

Maturation-stage ameloblasts are firmly bound to the tooth enamel by a basal lamina-like structure. The mechanism underlying this adhesion, however, remains to be fully clarified. The goal of this study was to investigate the mechanism underlying adhesion between the basal lamina-like structure and the enamel in monkey tooth germ. High-resolution immunogold labeling was performed to localize amelotin and laminin 332 at the interface between ameloblasts and tooth enamel. Minute, electron-dense strands were observed on the enamel side of the lamina densa, extending into the degrading enamel matrix to produce a well-developed fibrous layer (lamina fibroreticularis). In un-demineralized tissue sections, mineral crystals smaller than those in the bulk of the enamel were observed adhering to these strands where they protruded into the surface enamel. Immunogold particles reactive for amelotin were preferentially localized on these strands in the fibrous layer. On the other hand, those for laminin 332 were localized solely in the lamina densa; none were observed in the fibrous layer. These results suggest that the fibrous layer of the basal lamina-like structure is partly composed of amelotin molecules, and that these molecules facilitate ameloblast-enamel adhesion by promoting mineralization of the fibrous layer during the maturation stage of amelogenesis.

Characterization of the fibrillar layer at the epithelial-mesenchymal junction in tooth germs.

PubMed

Sawada, T; Inoue, S

1994-12-01

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys (Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8-9 nm in width, resembling a "basotubule", the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the "cords" forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The "fibrillar layer" therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules.

Production of maternal-zygotic mutant zebrafish by germ-line replacement.

PubMed

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F

2002-11-12

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.

Production of maternal-zygotic mutant zebrafish by germ-line replacement

PubMed Central

Ciruna, Brian; Weidinger, Gilbert; Knaut, Holger; Thisse, Bernard; Thisse, Christine; Raz, Erez; Schier, Alexander F.

2002-01-01

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies. PMID:12397179

Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor.

PubMed

Katz, D; Niederberger, C; Slaughter, G R; Cooney, A J

1997-10-01

Nuclear receptors, such as those for androgens, estrogens, and progesterones, control many reproductive processes. Proteins with structures similar to these receptors, but for which ligands have not yet been identified, have been termed orphan nuclear receptors. One of these orphans, germ cell nuclear factor (GCNF), has been shown to be germ cell specific in the adult and, therefore, may also participate in the regulation of reproductive functions. In this paper, we examine more closely the expression patterns of GCNF in germ cells to begin to define spatio-temporal domains of its activity. In situ hybridization showed that GCNF messenger RNA (mRNA) is lacking in the testis of hypogonadal mutant mice, which lack developed spermatids, but is present in the wild-type testis. Thus, GCNF is, indeed, germ cell specific in the adult male. Quantitation of the specific in situ hybridization signal in wild-type testis reveals that GCNF mRNA is most abundant in stage VII round spermatids. Similarly, Northern analysis and specific in situ hybridization show that GCNF expression first occurs in testis of 20-day-old mice, when round spermatids first emerge. Therefore, in the male, GCNF expression occurs postmeiotically and may participate in the morphological changes of the maturing spermatids. In contrast, female expression of GCNF is shown in growing oocytes that have not completed the first meiotic division. Thus, GCNF in the female is expressed before the completion of meiosis. Finally, the nature of the two different mRNAs that hybridize to the GCNF complementary DNA was studied. Although both messages contain the DNA binding domain, only the larger message is recognized by a probe from the extreme 3' untranslated region. In situ hybridization with these differential probes demonstrates that both messages are present in growing oocytes. In addition, the coding region and portions of the 3' untranslated region of the GCNF complementary DNA are conserved in the rat.

Levofloxacin to Prevent Infection Following Chemotherapy in Treating Patients With Solid Tumors or Lymphoma

ClinicalTrials.gov

2013-08-01

Brain and Central Nervous System Tumors; Breast Cancer; Extragonadal Germ Cell Tumor; Infection; Lung Cancer; Lymphoma; Ovarian Cancer; Small Intestine Cancer; Testicular Germ Cell Tumor; Unspecified Adult Solid Tumor, Protocol Specific

Ixabepilone in Treating Young Patients With Solid Tumors or Leukemia That Haven't Responded to Therapy

ClinicalTrials.gov

2012-03-14

Brain and Central Nervous System Tumors; Childhood Germ Cell Tumor; Extragonadal Germ Cell Tumor; Kidney Cancer; Leukemia; Liver Cancer; Neuroblastoma; Ovarian Cancer; Sarcoma; Unspecified Childhood Solid Tumor, Protocol Specific

Perspectives on testicular germ cell neoplasms.

PubMed

Cheng, Liang; Lyu, Bingjian; Roth, Lawrence M

2017-01-01

Our knowledge of testicular germ cell neoplasms has progressed in the last few decades due to the description of germ cell neoplasia in situ (GCNIS) and a variety of specific forms of intratubular germ cell neoplasia, the discovery of isochromosome 12p and its importance in the development of invasiveness in germ cell tumors (GCTs), the identification of specific transcription factors for GCTs, and the recognition that a teratomatous component in mixed GCT represents terminal differentiation. Isochromosome 12p and 12p overrepresentation, collectively referred to as 12p amplification, are fundamental abnormalities that account for many types of malignant GCTs of the testis. Embryonal carcinoma is common in the testis but rare in the ovary, whereas the converse is true for mature cystic teratoma. Spermatocytic tumor occurs only in the testis; it has not been described in the ovary or extragonadal sites. The origin of ovarian mature cystic teratoma is similar to that of prepubertal-type testicular teratoma and dermoid cyst at any age in that it arises from a nontransformed germ cell, whereas postpubertal-type testicular teratoma arises from a malignant germ cell, most commonly through the intermediary of GCNIS. Somatic neoplasms, often referred to as monodermal teratomas, arise not infrequently from mature cystic teratoma of the ovary, whereas such neoplasms are rare in testicular teratoma with the exception of carcinoid. Integration of classical morphologic observations and emerging novel molecular studies will result in better understanding of the pathogenesis of GCTs and will optimize patient therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Blastema cells derived from New Zealand white rabbit's pinna carry stemness properties as shown by differentiation into insulin producing, neural, and osteogenic lineages representing three embryonic germ layers.

PubMed

Saeinasab, Morvarid; Matin, Maryam M; Rassouli, Fatemeh B; Bahrami, Ahmad Reza

2016-05-01

Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit's pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins, Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.

Germ cell cluster organization and oogenesis in the tardigrade Dactylobiotus parthenogeneticus Bertolani, 1982 (Eutardigrada, Murrayidae).

PubMed

Poprawa, Izabela; Hyra, Marta; Rost-Roszkowska, Magdalena Maria

2015-07-01

Germ cell cluster organization and the process of oogenesis in Dactylobiotus parthenogeneticus have been described using transmission electron microscopy and light microscopy. The reproductive system of D. parthenogeneticus is composed of a single, sac-like, meroistic ovary and a single oviduct that opens into the cloaca. Two zones can be distinguished in the ovary: a small germarium that is filled with oogonia and a vitellarium that is filled with germ cell clusters. The germ cell cluster, which has the form of a modified rosette, consists of eight cells that are interconnected by stable cytoplasmic bridges. The cell that has the highest number of stable cytoplasmic bridges (four bridges) finally develops into the oocyte, while the remaining cells become trophocytes. Vitellogenesis of a mixed type occurs in D. parthenogeneticus. One part of the yolk material is produced inside the oocyte (autosynthesis), while the second part is synthesized in the trophocytes and transported to the oocyte through the cytoplasmic bridges. The eggs are covered with two envelopes: a thin vitelline envelope and a three-layered chorion. The surface of the chorion forms small conical processes, the shape of which is characteristic for the species that was examined. In our paper, we present the first report on the rosette type of germ cell clusters in Parachela.

Germ cell specification and ovary structure in the rotifer Brachionus plicatilis.

PubMed

Smith, James M; Cridge, Andrew G; Dearden, Peter K

2010-08-02

The segregation of the germline from somatic tissues is an essential process in the development of all animals. Specification of the primordial germ cells (PGCs) takes place via different strategies across animal phyla; either specified early in embryogenesis by the inheritance of maternal determinants in the cytoplasm of the oocyte ('preformation') or selected later in embryonic development from undifferentiated precursors by a localized inductive signal ('epigenesis'). Here we investigate the specification and development of the germ cells in the rotifer Brachionus plicatilis, a member of the poorly-characterized superphyla Lophotrochozoa, by isolating the Brachionus homologues of the conserved germ cell markers vasa and nanos, and examining their expression using in situ hybridization. Bpvasa and Bpnos RNA expression have very similar distributions in the Brachionus ovary, showing ubiquitous expression in the vitellarium, with higher levels in the putative germ cell cluster. Bpvas RNA expression is present in freshly laid eggs, remaining ubiquitous in embryos until at least the 96 cell stage after which expression narrows to a small cluster of cells at the putative posterior of the embryo, consistent with the developing ovary. Bpnos RNA expression is also present in just-laid eggs but expression is much reduced by the four-cell stage and absent by the 16-cell stage. Shortly before hatching of the juvenile rotifer from the egg, Bpnos RNA expression is re-activated, located in a subset of posterior cells similar to those expressing Bpvas at the same stage. The observed expression of vasa and nanos in the developing B. plicatilis embryo implies an epigenetic origin of primordial germ cells in Rotifer.

RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos.

PubMed

Miranda-Rodríguez, Jerónimo Roberto; Salas-Vidal, Enrique; Lomelí, Hilda; Zurita, Mario; Schnabel, Denhi

2017-01-01

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Sex determination in mammalian germ cells

PubMed Central

Spiller, Cassy M; Bowles, Josephine

2015-01-01

Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model. PMID:25791730

Localization of early germ cells in a stony coral, Euphyllia ancora: potential implications for a germline stem cell system in coral gametogenesis

NASA Astrophysics Data System (ADS)

Shikina, Shinya; Chung, Yi-Jou; Wang, Hsiang-Ming; Chiu, Yi-Ling; Shao, Zih-Fang; Lee, Yan-Horn; Chang, Ching-Fong

2015-06-01

Most corals exhibit annual or multiple gametogenic cycles. Thus far, coral gametogenesis has been studied in many species and locations during the past three decades; however, currently, only a few papers exist that describe the origin of germ cells, such as germline stem cells (GSCs), which support the continuous production of gametes in every reproductive cycle. To address this issue, in this study, we focused on and identified piwi gene, which has been used as a marker of germline cells, including GSCs, in various metazoans, in a scleractinian coral, Euphyllia ancora. Reverse-transcription PCR and Western blotting analyses revealed that E. ancora piwi-like ( Eapiwi) is expressed in mesentery tissues where the sites of gametogenesis are located for both sexes. Immunohistochemistry with a specific antibody against Eapiwi revealed strong immunoreactivity in the spermatogonia in males and in the oogonia and early oocytes in females, demonstrating that Eapiwi could be used as an early germ cell marker in E. ancora. Subsequent immunohistochemical analyses regarding the spatial and temporal distribution patterns of early germ cells in mesentery tissues revealed that early germ cells were present throughout the year in the mesentery tissue we examined, regardless of the sexual reproductive cycle. In particular, small numbers of early germ cells were observed in specific sites of mesentery tissues with fully matured gonads in both sexes. These early germ cells were not released together with mature gametes during the spawning period and remained in the mesentery tissues. These results suggested that these early germ cells most likely serve as a reservoir of germline cells and that some of these cells would produce differentiated germ cells for the upcoming sexual reproduction period; hence, these cells would function as GSCs. Our data provide new information for understanding continuous gamete production in corals.

Development of Strategies to Increase Enrollment in Clinical Trials for Children With Cancer

ClinicalTrials.gov

2014-02-12

Brain and Central Nervous System Tumors; Childhood Germ Cell Tumor; Extragonadal Germ Cell Tumor; Leukemia; Liver Cancer; Lymphoma; Neuroblastoma; Ovarian Cancer; Psychosocial Effects of Cancer and Its Treatment; Sarcoma; Unspecified Childhood Solid Tumor, Protocol Specific

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

PubMed

Sakib, Sadman; Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2016-01-01

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

Upright Imaging of Drosophila Egg Chambers

PubMed Central

Manning, Lathiena; Starz-Gaiano, Michelle

2015-01-01

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective. PMID:25867882

Etoposide damages female germ cells in the developing ovary.

PubMed

Stefansdottir, Agnes; Johnston, Zoe C; Powles-Glover, Nicola; Anderson, Richard A; Adams, Ian R; Spears, Norah

2016-08-11

As with many anti-cancer drugs, the topoisomerase II inhibitor etoposide is considered safe for administration to women in the second and third trimesters of pregnancy, but assessment of effects on the developing fetus have been limited. The purpose of this research was to examine the effect of etoposide on germ cells in the developing ovary. Mouse ovary tissue culture was used as the experimental model, thus allowing us to examine effects of etoposide on all stages of germ cell development in the same way, in vitro. Fetal ovaries from embryonic day 13.5 CD1 mice or neonatal ovaries from postnatal day 0 CD1 mice were cultured with 50-150 ng ml(-1) or 50-200 ng ml(-1) etoposide respectively, concentrations that are low relative to that in patient serum. When fetal ovaries were treated prior to follicle formation, etoposide resulted in dose-dependent damage, with 150 ng ml(-1) inducing a near-complete absence of healthy follicles. In contrast, treatment of neonatal ovaries, after follicle formation, had no effect on follicle numbers and only a minor effect on follicle health, even at 200 ng ml(-1). The sensitivity of female germ cells to etoposide coincided with topoisomerase IIα expression: in the developing ovary of both mouse and human, topoisomerase IIα was expressed in germ cells only prior to follicle formation. Exposure of pre-follicular ovaries, in which topoisomerase IIα expression was germ cell-specific, resulted in a near-complete elimination of germ cells prior to follicle formation, with the remaining germ cells going on to form unhealthy follicles by the end of culture. In contrast, exposure to follicle-enclosed oocytes, which no longer expressed topoisomerase IIα in the germ cells, had no effect on total follicle numbers or health, the only effect seen specific to transitional follicles. Results indicate the potential for adverse effects on fetal ovarian development if etoposide is administered to pregnant women when germ cells are not yet enclosed within ovarian follicles, a process that starts at approximately 17 weeks gestation and is only complete towards the end of pregnancy.

The Crystal Structure of the Drosophila Germline Inducer Oskar Identifies Two Domains with Distinct Vasa Helicase- and RNA-Binding Activities.

PubMed

Jeske, Mandy; Bordi, Matteo; Glatt, Sebastian; Müller, Sandra; Rybin, Vladimir; Müller, Christoph W; Ephrussi, Anne

2015-07-28

In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro

PubMed Central

2014-01-01

Background Recent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro. Methods Ovarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging. Results Differential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming. Conclusions Presence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine. PMID:24568237

Germ cell specification and ovary structure in the rotifer Brachionus plicatilis

PubMed Central

2010-01-01

Background The segregation of the germline from somatic tissues is an essential process in the development of all animals. Specification of the primordial germ cells (PGCs) takes place via different strategies across animal phyla; either specified early in embryogenesis by the inheritance of maternal determinants in the cytoplasm of the oocyte ('preformation') or selected later in embryonic development from undifferentiated precursors by a localized inductive signal ('epigenesis'). Here we investigate the specification and development of the germ cells in the rotifer Brachionus plicatilis, a member of the poorly-characterized superphyla Lophotrochozoa, by isolating the Brachionus homologues of the conserved germ cell markers vasa and nanos, and examining their expression using in situ hybridization. Results Bpvasa and Bpnos RNA expression have very similar distributions in the Brachionus ovary, showing ubiquitous expression in the vitellarium, with higher levels in the putative germ cell cluster. Bpvas RNA expression is present in freshly laid eggs, remaining ubiquitous in embryos until at least the 96 cell stage after which expression narrows to a small cluster of cells at the putative posterior of the embryo, consistent with the developing ovary. Bpnos RNA expression is also present in just-laid eggs but expression is much reduced by the four-cell stage and absent by the 16-cell stage. Shortly before hatching of the juvenile rotifer from the egg, Bpnos RNA expression is re-activated, located in a subset of posterior cells similar to those expressing Bpvas at the same stage. Conclusions The observed expression of vasa and nanos in the developing B. plicatilis embryo implies an epigenetic origin of primordial germ cells in Rotifer. PMID:20849649

Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells.

PubMed

Panneerdoss, Subbarayalu; Viswanadhapalli, Suryavathi; Abdelfattah, Nourhan; Onyeagucha, Benjamin C; Timilsina, Santosh; Mohammad, Tabrez A; Chen, Yidong; Drake, Michael; Vuori, Kristiina; Kumar, T Rajendra; Rao, Manjeet K

2017-09-19

Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

The importance of basonuclin 2 in adult mice and its relation to basonuclin 1.

PubMed

Vanhoutteghem, Amandine; Delhomme, Brigitte; Hervé, Françoise; Nondier, Isabelle; Petit, Jean-Maurice; Araki, Masatake; Araki, Kimi; Djian, Philippe

2016-05-01

BNC2 is an extremely conserved zinc finger protein with important functions in the development of craniofacial bones and male germ cells. Because disruption of the Bnc2 gene in mice causes neonatal lethality, the function of the protein in adult animals has not been studied. Until now BNC2 was considered to have a wider tissue distribution than its paralog, BNC1, but the precise cell types expressing Bnc2 are largely unknown. We identify here the cell types containing BNC2 in the mouse and we show the unexpected presence of BNC1 in many BNC2-containing cells. BNC1 and BNC2 are colocalized in male and female germ cells, ovarian epithelial cells, sensory neurons, hair follicle keratinocytes and connective cells of organ capsules. In many cell lineages, the two basonuclins appear and disappear synchronously. Within the male germ cell lineage, BNC1 and BNC2 are found in prospermatogonia and undifferentiated spermatogonia, and disappear abruptly from differentiating spermatogonia. During oogenesis, the two basonuclins accumulate specifically in maturing oocytes. During the development of hair follicles, BNC1 and BNC2 concentrate in the primary hair germs. As follicle morphogenesis proceeds, cells possessing BNC1 and BNC2 invade the dermis and surround the papilla. During anagen, BNC1 and BNC2 are largely restricted to the basal layer of the outer root sheath and the matrix. During catagen, the compartment of cells possessing BNC1 and BNC2 regresses, and in telogen, the two basonuclins are confined to the secondary hair germ. During the next anagen, the BNC1/BNC2-containing cell population regenerates the hair follicle. By examining Bnc2(-/-) mice that have escaped the neonatal lethality usually associated with lack of BNC2, we demonstrate that BNC2 possesses important functions in many of the cell types where it resides. Hair follicles of postnatal Bnc2(-/-) mice do not fully develop during the first cycle and thereafter remain blocked in telogen. It is concluded that the presence of BNC2 in the secondary hair germ is required to regenerate the transient segment of the follicle. Postnatal Bnc2(-/-) mice also show severe dwarfism, defects in oogenesis and alterations of palatal rugae. Although the two basonuclins possess very similar zinc fingers and are largely coexpressed, BNC1 cannot substitute for BNC2. This is shown incontrovertibly in knockin mice expressing Bnc1 instead of Bnc2 as these mice invariably die at birth with craniofacial abnormalities undistinguishable from those of Bnc2(-/-) mice. The function of the basonuclins in the secondary hair germ is of particular interest. Copyright © 2016 Elsevier B.V. All rights reserved.

Childhood Central Nervous System Germ Cell Tumors Treatment (PDQ®)—Health Professional Version

Cancer.gov

Treatment for children with central nervous system germ cell tumors (GCT) depend upon the specific tumor type. Options include radiation therapy, chemotherapy, surgery (in various combinations) and stem cell rescue. Get detailed information about GCTs in this clinician summary.

A male contraceptive targeting germ cell adhesion.

PubMed

Mruk, Dolores D; Wong, Ching-Hang; Silvestrini, Bruno; Cheng, C Yan

2006-11-01

Throughout spermatogenesis, developing germ cells remain attached to Sertoli cells via testis-specific anchoring junctions. If adhesion between these cell types is compromised, germ cells detach from the seminiferous epithelium and infertility often results. Previously, we reported that Adjudin is capable of inducing germ cell loss from the epithelium. In a small subset of animals, however, oral administration of Adjudin (50 mg per kg body weight (b.w.) for 29 d) resulted in adverse effects such as liver inflammation and muscle atrophy. Here, we report a novel approach in which Adjudin is specifically targeted to the testis by conjugating Adjudin to a recombinant follicle-stimulating hormone (FSH) mutant, which serves as its 'carrier'. Using this approach, infertility was induced in adult rats when 0.5 microg Adjudin per kg b.w. was administered intraperitoneally, which was similar to results when 50 mg per kg b.w. was given orally. This represents a substantial increase in Adjudin's selectivity and efficacy as a male contraceptive.

Sexual dimorphism in parental imprint ontogeny and contribution to embryonic development.

PubMed

Bourc'his, Déborah; Proudhon, Charlotte

2008-01-30

Genomic imprinting refers to the functional non-equivalence of parental genomes in mammals that results from the parent-of-origin allelic expression of a subset of genes. Parent-specific expression is dependent on the germ line acquisition of DNA methylation marks at imprinting control regions (ICRs), coordinated by the DNA-methyltransferase homolog DNMT3L. We discuss here how the gender-specific stages of DNMT3L expression may have influenced the various sexually dimorphic aspects of genomic imprinting: (1) the differential developmental timing of methylation establishment at paternally and maternally imprinted genes in each parental germ line, (2) the differential dependence on DNMT3L of parental methylation imprint establishment, (3) the unequal duration of paternal versus maternal methylation imprints during germ cell development, (4) the biased distribution of methylation-dependent ICRs towards the maternal genome, (5) the different genomic organization of paternal versus maternal ICRs, and finally (6) the overwhelming contribution of maternal germ line imprints to development compared to their paternal counterparts.

Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

PubMed

Kubota, Hiroshi; Wu, Xin; Goodyear, Shaun M; Avarbock, Mary R; Brinster, Ralph L

2011-08-01

Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

"Life in a Germ-Free World":

PubMed Central

Kirk, Robert G. W.

2012-01-01

Summary: This article examines a specific technology, the germ-free "isolator," tracing its development across three sites: (1) the laboratory for the production of standard laboratory animals, (2) agriculture for the efficient production of farm animals, and (3) the hospital for the control and prevention of cross-infection and the protection of individuals from infection. Germ-free technology traveled across the laboratory sciences, clinical and veterinary medicine, and industry, yet failed to become institutionalized outside the laboratory. That germ-free technology worked was not at issue. Working, however, was not enough. Examining the history of a technology that failed to find widespread application reveals the labor involved in aligning cultural, societal, and material factors necessary for successful medical innovation. PMID:23000838

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

PubMed

Suzuki, Atsushi; Niimi, Yuki; Shinmyozu, Kaori; Zhou, Zhi; Kiso, Makoto; Saga, Yumiko

2016-01-01

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development. © 2015 The Authors.

A potential germ cell-specific marker in Japanese flounder, Paralichthys olivaceus: identification and characterization of lymphocyte antigen 75 (Ly75/CD205)

NASA Astrophysics Data System (ADS)

Yang, Yang; Liu, Qinghua; Ma, Daoyuan; Song, Zongchen; Li, Jun

2018-04-01

Some germ cell marker genes, such as vasa, nanos, and dead end (dnd), have been identified in fish. Recently, lymphocyte antigen 75 (Ly75/CD205) has been identified as a mitotic germ cell-specific cell-surface marker in several fish species. In this study, the Japanese flounder ly75 homolog (ly75) was cloned and its expression pattern in gonads was analyzed. The full-length cDNA of ly75 was 7 346 bp, with an open reading frame (ORF) of 5 229 bp. The ORF encoded a protein containing 1 742 amino acids with a predicted molecular mass of 196.89 kDa. In adult tissues, ly75 transcripts were detected in all analyzed tissues but abundantly in the testis. In in-situ hybridization analyses, ly75 mRNA was predominantly localized in oocytes in the ovary and spermatogonia in the testis, but ly75 mRNA was not detected in oogonia, spermatocytes, spermatids, or spermatozoa. These results indicated that ly75 could be a potential germ cell-specific marker in P. olivaceus, as in other fishes.

Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

PubMed

Hao, Xiao-Xia; Chen, Su-Ren; Tang, Ji-Xin; Li, Jian; Cheng, Jin-Mei; Jin, Cheng; Wang, Xiu-Xia; Liu, Yi-Xun

2016-07-01

SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Influence of caecal microflora and of two dietary protein levels on the adaptation of the exocrine pancreas: comparative study in germ-free and conventional rats.

PubMed

Lhoste, E F; Catala, I; Fiszlewicz, M; Gueugneau, A M; Popot, F; Vaissade, P; Corring, T; Szylit, O

1996-03-01

Dietary proteins are degraded by both endogenous enzymes and the caecal microflora. In conventional rats the enzyme content of the pancreas depends on the amount of dietary protein. The influence of the caecal microflora on this process is unknown. We report here the effect of the caecal microflora on pancreatic enzymes (proteases, amylase (EC 3.2.1.1), lipase (EC 3.1.1.3)) and on colonic metabolites (NH3, urea, short-chain fatty acids). Germ-free and conventional male Fischer rats were fed for 3 weeks with a diet containing 220 or 450 g protein/kg provided as a mixture of fish concentrate and soyabean isolate. The excretion of NH3 and the pH were specifically increased by the high-protein diet in the germ-free rats. The higher production of isobutyrate, valerate and isovalerate in conventional rats fed on the high-protein diet reflected a high bacterial proteolytic activity since these short-chain fatty acids are specific indicators of this activity. The microflora hydrolysed urea to NH3 and maintained the pH at neutrality whatever the amount of protein in the diet since there were changes in germ-free rats but not in conventional ones. In germ-free rats, amylase, trypsin (EC 3.4.21.4), elastase (EC 3.4.21.36) and carboxypeptidase A (EC 3.4.17.1) specific activities were significantly lower than in conventional rats. The adaptation of the pancreas to the 450 g protein/kg diet was not impaired by the bacterial status except for the specific activity of chymotrypsin (EC 3.4.21.1) which was more increased by this diet in germ-free than in conventional rats. Moreover, the specific activity of lipase increased only in conventional rats fed on the 450 g protein/kg diet. In conclusion, we observed a relationship between the enzyme content of the pancreas and the presence or absence of the caecal microflora suggesting that bacterial fermentation influences pancreatic function.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

Origin and development of the germ line in sea stars

PubMed Central

Wessel, Gary M.; Fresques, Tara; Kiyomoto, Masato; Yajima, Mamiko; Zazueta, Vanesa

2014-01-01

This review summarizes and integrates our current understanding of how sea stars make gametes. Although little is known of the mechanism of germ line formation in these animals, recent results point to specific cells and to cohorts of molecules in the embryos and larvae that may lay the ground work for future research efforts. A coelomic outpocketing forms in the posterior of the gut in larvae, referred to as the posterior enterocoel (PE), that when removed, significantly reduces the number of germ cell later in larval growth. This same PE structure also selectively accumulates several germ-line associated factors – vasa, nanos, piwi – and excludes factors involved in somatic cell fate. Since its formation is relatively late in development, these germ cells may form by inductive mechanisms. When integrated into the morphological observations of germ cells and gonad development in larvae, juveniles, and adults, the field of germ line determination appears to have a good model system to study inductive germ line determination to complement the recent work on the molecular mechanisms in mice. We hope this review will also guide investigators interested in germ line determination and regulation of the germ line in how these animals can help in this research field. The review is not intended to be comprehensive – sea star reproduction has been studied over 100 years and many reviews are comprehensive in their coverage of, for example, seasonal growth of the gonads in response to light, nutrient, and temperature. Rather the intent of this review is to help the reader focus on new experimental results attached to the historical underpinnings of how the germ cell functions in sea stars with particular emphasis to clarify the important areas of priority for future research. PMID:24648114

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka.

PubMed

Yuan, Yongming; Li, Mingyou; Hong, Yunhan

2014-07-15

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development. Copyright © 2014 Elsevier B.V. All rights reserved.

[Study of enzymes of xenobiotic metabolism in the evaluation of quality of protein-containing wheat germ flakes and wallpaper flour].

PubMed

Martinchuk, A N; E En Gyn; Safronova, A M; Peskova, E V

1991-01-01

Intake of wheat upholstery meal by growing rats was attended by a sharp decrease in the content and activity of xenobiotic metabolism enzymes in the hepatic microsomes, that was caused by the low biological value of the meal proteins. Hepatic microsomes of the rats that were fed with wheat germ flakes showed increased specific content of cytochromes P-450 and b5, but the total blood protein content per 100 g of body mass was lower than during casein consumption. No significant changes were detected in hydroxylation rate of benz(a)pyrene, aniline and ethylmorphine. During consumption of wheat germ flakes induction of UDP-glucuronide-transferase was detected in hepatic microsomes. Wheat germ flakes induced a 5-fold increase of Se-dependent glutathione peroxidase activity. Wheat germ flakes produced no significant effect on glutathione-S-aryltransferase and glutathione reductase activity.

Functional tooth restoration utilising split germs through re-regionalisation of the tooth-forming field

PubMed Central

Yamamoto, Naomi; Oshima, Masamitsu; Tanaka, Chie; Ogawa, Miho; Nakajima, Kei; Ishida, Kentaro; Moriyama, Keiji; Tsuji, Takashi

2015-01-01

The tooth is an ectodermal organ that arises from a tooth germ under the regulation of reciprocal epithelial-mesenchymal interactions. Tooth morphogenesis occurs in the tooth-forming field as a result of reaction-diffusion waves of specific gene expression patterns. Here, we developed a novel mechanical ligation method for splitting tooth germs to artificially regulate the molecules that control tooth morphology. The split tooth germs successfully developed into multiple correct teeth through the re-regionalisation of the tooth-forming field, which is regulated by reaction-diffusion waves in response to mechanical force. Furthermore, split teeth erupted into the oral cavity and restored physiological tooth function, including mastication, periodontal ligament function and responsiveness to noxious stimuli. Thus, this study presents a novel tooth regenerative technology based on split tooth germs and the re-regionalisation of the tooth-forming field by artificial mechanical force. PMID:26673152

Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors.

PubMed

Galli, Uwe M; Sauter, Marlies; Lecher, Bernd; Maurer, Simone; Herbst, Hermann; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

2005-04-28

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

Functional role of EMMPRIN in the formation and mineralisation of dental matrix in mouse molars.

PubMed

Xie, Ming; Xing, Guofang; Hou, Liwen; Bao, Jing; Chen, Yuqing; Jiao, Ting; Zhang, Fuqiang

2015-02-01

Our previous research has shown that the extracellular matrix metalloproteinase inducer (EMMPRIN) is expressed during and may function in the early development of tooth germs. In the present study, we observed the specific expression of EMMPRIN in ameloblasts and odontoblasts during the middle and late stages of tooth germ development using immunohistochemistry. Furthermore, to extend our understanding of the function of EMMPRIN in odontogenesis, we used an anti-EMMPRIN function-blocking antibody to remove EMMPRIN activity in tooth germ culture in vitro. Both the formation and mineralisation of dental hard tissues were suppressed in the tooth germ culture after the abrogation of EMMPRIN. Meanwhile, significant reductions in VEGF, MMP-9, ALPL, ameloblastin, amelogenin and enamelin expression were observed in antibody-treated tooth germ explants compared to control and normal serum-treated explants. The current results illustrate that EMMPRIN may play a critical role in the processing and maturation of the dental matrix.

Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

PubMed

Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

2009-11-01

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

Balbiani body, nuage and sponge bodies--term plasm pathway players.

PubMed

Kloc, Malgorzata; Jedrzejowska, Izabela; Tworzydlo, Waclaw; Bilinski, Szczepan M

2014-07-01

In many animal species, germ cells are specified by maternally provided, often asymmetrically localized germ cell determinant, termed the germ plasm. It has been shown that in model organisms such as Xenopus laevis, Danio rerio and Drosophila melanogaster germ plasm components (various proteins, mRNAs and mitochondria) are delivered to the proper position within the egg cell by germline specific organelles, i.e. Balbiani bodies, nuage accumulations and/or sponge bodies. In the present article, we review the current knowledge on morphology, molecular composition and functioning of these organelles in main lineages of arthropods and different ovary types on the backdrop of data derived from the studies of the model vertebrate species. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Germ cell membrane lipids in spermatogenesis].

PubMed

Wang, Ting; Shi, Xiao; Quan, Song

2016-05-01

Spermatogenesis is a complex developmental process in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. During spermatogenesis, membrane remodeling takes place, and cell membrane permeability and liquidity undergo phase-specific changes, which are all associated with the alteration of membrane lipids. Lipids are important components of the germ cell membrane, whose volume and ratio fluctuate in different phases of spermatogenesis. Abnormal lipid metabolism can cause spermatogenic dysfunction and consequently male infertility. Germ cell membrane lipids are mainly composed of cholesterol, phospholipids and glycolipids, which play critical roles in cell adhesion and signal transduction during spermatogenesis. An insight into the correlation of membrane lipids with spermatogenesis helps us to better understand the mechanisms of spermatogenesis and provide new approaches to the diagnosis and treatment of male infertility.

CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline

PubMed Central

Campbell, Anne C.; Updike, Dustin L.

2015-01-01

Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. PMID:25968310

Autophagy and Apoptosis Act as Partners to Induce Germ Cell Death after Heat Stress in Mice

PubMed Central

Zhang, Mianqiu; Jiang, Min; Bi, Ye; Zhu, Hui; Zhou, Zuomin; Sha, Jiahao

2012-01-01

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis. PMID:22848486

Key apoptotic pathways for heat-induced programmed germ cell death in the testis.

PubMed

Hikim, Amiya P Sinha; Lue, Yanhe; Yamamoto, Cindy M; Vera, Yanira; Rodriguez, Susana; Yen, Pauline H; Soeng, Kevin; Wang, Christina; Swerdloff, Ronald S

2003-07-01

Short-term exposure (43 C for 15 min) of the rat testis to mild heat results within 6 h in stage- and cell-specific activation of germ cell apoptosis. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Here we show that the relocation of Bax is accompanied by cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7 and cleavage of poly(ADP) ribose polymerase. Furthermore, early in apoptosis, a significant amount of Bax also accumulates in endoplasmic reticulum, as assessed by Western blot analyses of fractionated testicular lysates. In additional studies using the FasL-defective gld mice, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also endoplasmic reticulum-dependent pathways are the key apoptotic pathways for heat-induced germ cell death in the testis.

Anti-Inflammatory Activity of Citric Acid-Treated Wheat Germ Extract in Lipopolysaccharide-Stimulated Macrophages.

PubMed

Jeong, Hee-Yeong; Choi, Yong-Seok; Lee, Jae-Kang; Lee, Beom-Joon; Kim, Woo-Ki; Kang, Hee

2017-07-10

Until recently, fermentation was the only processing used to improve the functionality of wheat germ. The release of 2,6-dimethoxy-1,4-benzoquinone (DMBQ) from hydroquinone glycosides during the fermentation process is considered a marker of quality control. Here, we treated wheat germ extract with citric acid (CWG) to release DMBQ and examined the anti-inflammatory activity of this extract using a lipopolysaccharide-activated macrophage model. Treatment of wheat germ with citric acid resulted in detectable release of DMBQ but reduced total phenolic and total flavonoid contents compared with untreated wheat germ extract (UWG). CWG inhibited secretion of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-12 and the synthesis of cyclooxygenase-2, while UWG only decreased IL-12 production. CWG and UWG induced high levels of anti-inflammatory IL-10 and heme oxygenase-1. CWG specifically inhibited phosphorylation of NF-κB p65 and p38 kinase at 15 min after LPS stimulation. Our study showed that citric acid treatment enhanced the anti-inflammatory activity of wheat germ extract.

Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation

NASA Astrophysics Data System (ADS)

Wang, Szu-Chieh; Hsu, Hao-Jen; Lin, Gee-Way; Wang, Ting-Fang; Chang, Chun-Che; Lin, Ming-Der

2015-09-01

Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Pattern formation in early embryogenesis of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mglinets, V.A.

1995-07-01

Establishment of egg polarity, separation of germ layers, and the appearance of animal-vegetal, dorsoventral, and anteroposterior axes in Xenopus laevis embryos are considered. The control of these processes by gene coding for growth factors, protooncogens, and homeobox-containing genes is also been reviewed.

A germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans.

PubMed

Mainpal, Rana; Nance, Jeremy; Yanowitz, Judith L

2015-10-15

Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation. © 2015. Published by The Company of Biologists Ltd.

[Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers].

PubMed

Wang, Yue-Chun; Zhang, Yuan

2008-06-25

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

PubMed Central

Meyer, E; Butler, A; Dubrana, K; Duharcourt, S; Caron, F

1997-01-01

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences. PMID:9199294

Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming

PubMed Central

Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

2016-01-01

The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells. PMID:27606421

Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming.

PubMed

Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

2016-09-01

The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells.

Mechanical analysis of a heat-shock induced developmental defect

NASA Astrophysics Data System (ADS)

Crews, Sarah M.; McCleery, W. Tyler; Hutson, M. Shane

2014-03-01

Embryonic development in Drosophila is a complex process involving coordinated movements of mechanically interacting tissues. Perturbing this system with a transient heat shock can result in a number of developmental defects. In particular, a heat shock applied during the earliest morphogenetic movements of gastrulation can lead to apparent recovery, but then subsequent morphogenetic failure 5-6 hours later during germ band retraction. The process of germ band retraction requires an intact amnioserosa - a single layered extra-embryonic epithelial tissue - and heat shock at gastrulation can induce the later opening of holes in the amnioserosa. These holes are highly correlated with failures of germ band retraction. These holes could be caused by a combination of mechanical weakness in the amnioserosa or local increases in mechanical stress. Here, we assess the role of mechanical stress using confocal imaging to compare cell and tissue morphology in the amnioserosa of normal and heat-shocked embryos and laser hole drilling to map the stress field around the times and locations at which heat-shock induced holes open.

Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis.

PubMed

Ye, Huan; Li, Chuang-Ju; Yue, Hua-Mei; Du, Hao; Yang, Xiao-Ge; Yoshino, Tasuku; Hayashida, Takao; Takeuchi, Yutaka; Wei, Qi-Wei

2017-05-01

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon. Copyright © 2017 Elsevier Inc. All rights reserved.

YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

PubMed

Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

2012-03-01

The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

Twenty years of embryonic stem cell research in farm animals

USDA-ARS?s Scientific Manuscript database

Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that the ESC can maintain an undifferentiated state indefinitely, self renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the...

Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

PubMed

Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S

2016-08-01

Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures obtained in monolayer differentiation had a big nucleus and a surrounding ZP4 coat, the unique attributes of a female gamete. These oocyte-like structures, in extended cultures, showed embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence. This, as per our knowledge, is first such study in higher mammals, especially farm animals, and assumes significance for its potential use in transgenic animal production, elite animal conservation and propagation, augmentation of reproductive performance in poor breeding buffalo species, and as a model for understanding human germ cell formation. Copyright © 2016 Elsevier Inc. All rights reserved.

Generation of six multiple sclerosis patient-derived induced pluripotent stem cell lines.

PubMed

Miquel-Serra, L; Duarri, A; Muñoz, Y; Kuebler, B; Aran, B; Costa, C; Martí, M; Comabella, M; Malhotra, S; Montalban, X; Veiga, A; Raya, A

2017-10-01

Multiple sclerosis (MS) is considered a chronic autoimmune disease of the central nervous system that leads to gliosis, demyelination, axonal damage and neuronal death. The MS disease aetiology is unknown, though a polymorphism of the TNFRSF1A gene, rs1800693, is known to confer an increased risk for MS. Using retroviral delivery of reprogramming transgenes, we generated six MS patient-specific iPSC lines with two distinct genotypes, CC or TT, of the polymorphism rs1800693. iPSC lines had normal karyotype, expressed pluripotency genes and differentiated into the three germ layers. These lines offer a good tool to study MS pathomechanisms and for drug testing. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Generation of induced pluripotent stem cells from a patient with Best Dystrophy carrying 11q12.3 (BEST1 (VMD2)) mutation.

PubMed

Hsu, Chih-Chien; Lu, Huai-En; Chuang, Jen-Hua; Ko, Yu-Ling; Tsai, Yi-Ching; Tai, Hsiao-Yun; Yarmishyn, Aliaksandr A; Hwang, De-Kuang; Wang, Mong-Lien; Yang, Yi-Ping; Chen, Shih-Jen; Peng, Chi-Hsien; Chiou, Shih-Hwa; Lin, Tai-Chi

2018-04-03

Best disease (BD), also termed Best vitelliform macular dystrophy (BVMD), is a juvenile-onset form of macular degeneration and central visual loss. In this report, we generated an induced pluripotent stem cell (iPSC) line, TVGH-iPSC-012-04, from the peripheral blood mononuclear cells of a female patient with BD by using the Sendai virus delivery system. The resulting iPSCs retained the disease-causing DNA mutation, expressed pluripotent markers and could differentiate into three germ layers. We believe that BD patient-specific iPSCs provide a powerful in vitro model for evaluating the pathological phenotypes of the disease. Copyright © 2018. Published by Elsevier B.V.

Identification of Germ Plasm-Associated Transcripts by Microarray Analysis of Xenopus Vegetal Cortex RNA

PubMed Central

Cuykendall, Tawny N.; Houston, Douglas W.

2011-01-01

RNA localization is a common mechanism for regulating cell structure and function. Localized RNAs in Xenopus oocytes are critical for early development, including germline specification by the germ plasm. Despite the importance of these localized RNAs, only approximately 25 have been identified and fewer are functionally characterized. Using microarrays, we identified a large set of localized RNAs from the vegetal cortex. Overall, our results indicate a minimum of 275 localized RNAs in oocytes, or 2–3% of maternal transcripts, which are in general agreement with previous findings. We further validated vegetal localization for 24 candidates and further characterized three genes expressed in the germ plasm. We identified novel germ plasm expression for reticulon 3.1, exd2 (a novel exonuclease-domain encoding gene), and a putative noncoding RNA. Further analysis of these and other localized RNAs will likely identify new functions of germ plasm and facilitate the identification of cis-acting RNA localization elements. PMID:20503379

Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

PubMed

Yoshida, Keita; Hozumi, Akiko; Treen, Nicholas; Sakuma, Tetsushi; Yamamoto, Takashi; Shirae-Kurabayashi, Maki; Sasakura, Yasunori

2017-03-15

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona. Copyright © 2017 Elsevier Inc. All rights reserved.

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance.

PubMed

Wermann, Hendrik; Stoop, Hans; Gillis, Ad J M; Honecker, Friedemann; van Gurp, Ruud J H L M; Ammerpohl, Ole; Richter, Julia; Oosterhuis, J Wolter; Bokemeyer, Carsten; Looijenga, Leendert H J

2010-08-01

Differences in the global methylation pattern, ie hyper- as well as hypo-methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5-(m)cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell-specific marker VASA, showed increased expression. Following treatment with 5-azacytidine, TCam-2 cells were analysed using a high-throughput methylation screen for changes in the methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.

New markers for tracking endoderm induction and hepatocyte differentiation from human pluripotent stem cells

PubMed Central

Holtzinger, Audrey; Streeter, Philip R.; Sarangi, Farida; Hillborn, Scott; Niapour, Maryam; Ogawa, Shinichiro; Keller, Gordon

2015-01-01

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population, broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study, we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach, we identified two endoderm-specific antibodies, HDE1 and HDE2, which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential, whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination, the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. PMID:26493401

Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES.

PubMed

Pfeiffer, Martin J; Quaranta, Roberto; Piccini, Ilaria; Fell, Jakob; Rao, Jyoti; Röpke, Albrecht; Seebohm, Guiscard; Greber, Boris

2018-01-30

Master cell fate determinants are thought to induce specific cell lineages in gastrulation by orchestrating entire gene programs. The T-box transcription factor EOMES (eomesodermin) is crucially required for the development of the heart-yet it is equally important for endoderm specification suggesting that it may act in a context-dependent manner. Here, we define an unrecognized interplay between EOMES and the WNT signaling pathway in controlling cardiac induction by using loss and gain-of-function approaches in human embryonic stem cells. Dose-dependent EOMES induction alone can fully replace a cocktail of signaling molecules otherwise essential for the specification of cardiogenic mesoderm. Highly efficient cardiomyocyte programming by EOMES mechanistically involves autocrine activation of canonical WNT signaling via the WNT3 ligand, which necessitates a shutdown of this axis at a subsequent stage. Our findings provide insights into human germ layer induction and bear biotechnological potential for the robust production of cardiomyocytes from engineered stem cells.

CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline.

PubMed

Campbell, Anne C; Updike, Dustin L

2015-05-15

Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. © 2015. Published by The Company of Biologists Ltd.

Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad

PubMed Central

Jameson, Samantha A.; Natarajan, Anirudh; Cool, Jonah; DeFalco, Tony; Maatouk, Danielle M.; Mork, Lindsey; Munger, Steven C.; Capel, Blanche

2012-01-01

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates. PMID:22438826

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche.

PubMed

Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

2014-05-13

Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Human testis and testis cancer specimens from orchidectomies were cultured in 'hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche.

Ovarian mixed germ cell tumor with yolk sac and teratomatous components in a dog.

PubMed

Robinson, Nicholas A; Manivel, J Carlos; Olson, Erik J

2013-05-01

Mixed germ cell tumors of the ovary have rarely been reported in veterinary species. A 3-year-old intact female Labrador Retriever dog was presented for lethargy, abdominal distention, and a midabdominal mass. An exploratory laparotomy revealed a large (23 cm in diameter) left ovarian tumor and multiple small (2-3 cm in diameter) pale tan masses on the peritoneum and abdominal surface of the diaphragm. Histological examination of the left ovary revealed a mixed germ cell tumor with a yolk sac component with rare Schiller-Duval bodies and a teratomatous component comprised primarily of neural differentiation. The abdominal metastases were solely comprised of the yolk sac component. The yolk sac component was diffusely immunopositive for cytokeratin with scattered cells reactive for α-fetoprotein and placental alkaline phosphatase. Within the teratomatous component, the neuropil was diffusely immunopositive for S100, neuron-specific enolase, and neurofilaments with a few glial fibrillary acidic protein immunopositive cells. Ovarian germ cell tumors may be pure and consist of only 1 germ cell element or may be mixed and include more than 1 germ cell element, such as teratoma and yolk sac tumor.

Expression of germline markers in three species of amphioxus supports a preformation mechanism of germ cell development in cephalochordates

PubMed Central

2013-01-01

Background In a previous study, we showed that the cephalochordate amphioxus Branchiostoma floridae has localized maternal transcripts of conserved germ cell markers Vasa and Nanos in its early embryos. These results provided strong evidence to support a preformation mechanism for primordial germ cell (PGC) development in B. floridae. Results In this study, we further characterize the expression of B. floridae homologs of Piwi and Tudor, which play important roles in germline development in diverse metazoan animals. We show that maternal mRNA of one of the identified Piwi-like homologs, Bf-Piwil1, also colocalizes with Vasa in the vegetal germ plasm and has zygotic expression in both the putative PGCs and the tail bud, suggesting it may function in both germline and somatic stem cells. More interestingly, one Tudor family gene, Bf-Tdrd7, is only expressed maternally and colocalizes with Vasa in germ plasm, suggesting that it may function exclusively in germ cell specification. To evaluate the conservation of the preformation mechanism among amphioxus species, we further analyze Vasa, Nanos, Piwil1, and Tdrd7 expression in two Asian amphioxus species, B. belcheri and B. japonicum. Their maternal transcripts all localize in similar patterns to those seen in B. floridae. In addition, we labeled putative PGCs with Vasa antibody to trace their dynamic distribution in developing larvae. Conclusions We identify additional germ plasm components in amphioxus and demonstrate the molecular distinction between the putative germline stem cells and somatic stem cells. Moreover, our results suggest that preformation may be a conserved mechanism for PGC specification among Branchiostoma species. Our Vasa antibody staining results suggest that after the late neurula stage, amphioxus PGCs probably proliferate with the tail bud cells during posterior elongation and are deposited near the forming myomere boundaries. Subsequently, these PGCs would concentrate at the ventral tip of the myoseptal walls to form the gonad anlagen. PMID:23777831

Multidimensional representations: The knowledge domain of germs held by students, teachers and medical professionals

NASA Astrophysics Data System (ADS)

Rua, Melissa Jo

The present study examined the understandings held by 5th, 8th, and 11th-grade students, their teachers and medical professionals about germs. Specifically, this study describes the content and structure of students' and adults' conceptions in the areas of germ contraction, transmission, and treatment of infectious and non-infectious diseases caused by microorganisms. Naturalistic and empirical research methods were used to investigate participants' conceptions. Between and within group similarities were found using data from concept maps on the topic "flu," drawings of germs, a 20 word card sort related to germs and illness, and a semi-structured interview. Concept maps were coded according to techniques by Novak and Gowan (1984). Drawings of germs were coded into four main categories (bacteria, viruses, animal cell, other) and five subcategories (disease, caricature, insect, protozoa, unclassified). Cluster patterns for the card sorts of each group were found using multidimensional scaling techniques. Six coding categories emerged from the interview transcripts: (a) transmission, (b) treatment, (c) effect of weather on illness, (d) immune response, (e) location of germs, and (f) similarities and differences between bacteria and viruses. The findings showed students, teachers and medical professionals have different understandings about bacteria and viruses and the structures of those understandings vary. Gaps or holes in the participants knowledge were found in areas such as: (a) how germs are transmitted, (b) where germs are found, (c) how the body transports and uses medicine, (d) how the immune system functions, (e) the difference between vaccines and non-prescription medicines, (f) differences that exist between bacteria and viruses, and (g) bacterial resistance to medication. The youngest students relied heavily upon personal experiences with germs rather than formal instruction when explaining their conceptions. As a result, the influence of media was evident in the students' understandings and images of microbes. Students also viewed germs as a human problem rather than seeing microorganisms as an independent member of the ecosystem. Teachers' explanations about germs varied in explicitness based on the grade level they taught while medical professionals based their understandings on formal knowledge and tended to use explicit technical language in their explanations of the phenomena.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

PubMed

Tao, Yu; Zheng, Weisheng; Jiang, Yonghua; Ding, Guitao; Hou, Xinfeng; Tang, Yitao; Li, Yueying; Gao, Shuai; Chang, Gang; Zhang, Xiaobai; Liu, Wenqiang; Kou, Xiaochen; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

2014-12-21

Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

Suppression of Radiation-Induced Testicular Germ Cell Apoptosis by 2,5-Hexanedione Pretreatment. III. Candidate Gene Analysis Identifies a Role for Fas in the Attenuation of X-ray–Induced Apoptosis

PubMed Central

Campion, Sarah N.; Sandrof, Moses A.; Yamasaki, Hideki; Boekelheide, Kim

2010-01-01

Germ cell apoptosis directly induced by x-radiation (x-ray) exposure is stage specific, with a higher incidence in stage II/III seminiferous tubules. A priming exposure to the Sertoli cell toxicant 2,5-hexanedione (HD) results in a marked reduction in x-ray–induced germ cell apoptosis in these affected stages. Because of the stage specificity of these responses, examination of associated gene expression in whole testis tissue has clear limitations. Laser capture microdissection (LCM) of specific cell populations in the testis is a valuable technique for investigating the responses of different cell types following toxicant exposure. LCM coupled with quantitative real-time PCR was performed to examine the expression of apoptosis-related genes at both early (3 h) and later (12 h) time points after x-ray exposure, with or without the priming exposure to HD. The mRNAs examined include Fas, FasL, caspase 3, bcl-2, p53, PUMA, and AEN, which were identified either by literature searches or microarray analysis. Group 1 seminiferous tubules (stages I–VI) exhibited the greatest changes in gene expression. Further analysis of this stage group (SG) revealed that Fas induction by x-ray is significantly attenuated by HD co-exposure. Selecting only for germ cells from seminiferous tubules of the most sensitive SG has provided further insight into the mechanisms involved in the co-exposure response. It is hypothesized that following co-exposure, germ cells adapt to the lack of Sertoli cell support by reducing the Fas response to normal FasL signals. These findings provide a better understanding and appreciation of the tissue complexity and technical difficulties associated with examining gene expression in the testis. PMID:20616204

Effects of the Transforming Growth Factor Beta Signaling Pathway on the Differentiation of Chicken Embryonic Stem Cells into Male Germ Cells

PubMed Central

Zhang, Yani; Wang, Yingjie; Zuo, Qisheng; Li, Dong; Zhang, Wenhui; Lian, Chao; Tang, Beibei; Xiao, Tianrong; Wang, Man; Wang, Kehua

2016-01-01

Abstract The objectives of the present study were to screen for key gene and signaling pathways involved in the production of male germ cells in poultry and to investigate the effects of the transforming growth factor beta (TGF-β) signaling pathway on the differentiation of chicken embryonic stem cells (ESCs) into male germ cells. The ESCs, primordial germ cells, and spermatogonial stem cells (SSCs) were sorted using flow cytometry for RNA sequencing (RNA-seq) technology. Male chicken ESCs were induced using 40 ng/mL of bone morphogenetic protein 4 (BMP4). The effects of the TGF-β signaling pathway on the production of chicken SSCs were confirmed by morphology, quantitative real-time polymerase chain reaction, and immunocytochemistry. One hundred seventy-three key genes relevant to development, differentiation, and metabolism and 20 signaling pathways involved in cell reproduction, differentiation, and signal transduction were identified by RNA-seq. The germ cells formed agglomerates and increased in number 14 days after induction by BMP4. During the induction process, the ESCs, Nanog, and Sox2 marker gene expression levels decreased, whereas expression of the germ cell-specific genes Stra8, Dazl, integrin-α6, and c-kit increased. The results indicated that the TGF-β signaling pathway participated in the differentiation of chicken ESCs into male germ cells. PMID:27906584

Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares.

PubMed

Kobayashi, Toru; Honryo, Tomoki; Agawa, Yasuo; Sawada, Yoshifumi; Tapia, Ileana; Macìas, Karla A; Cano, Amado; Scholey, Vernon P; Margulies, Daniel; Yagishita, Naoki

2015-06-01

To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 ± 3.2 cells), compared to that at 1 dph (11 ± 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Use of guar by-products in high-production laying hen diets.

PubMed

Gutierrez, O; Zhang, C; Cartwright, A L; Carey, J B; Bailey, C A

2007-06-01

A 5x5 Latin square experiment was conducted to evaluate the effect of feeding low concentrations of guar germ or a combination of guar germ and hull (guar meal) in high-production laying hen diets. A total of 125 Lohmann laying hens (21 wk old) of similar BW were randomly assigned to 5 blocks. Each block was divided into 5 experimental units, consisting of 5 hens per unit. Hens were fed either a nonguar control diet, or 1 of 4 diets containing either 2.5 or 5% guar germ, or 2.5 or 5% guar meal over a 20-wk trial period (five 4-wk periods). No significant differences were observed when feeding either 2.5 or 5% guar germ or meal (P>0.05) on hen-day egg production or feed consumption. Significant differences in egg weight, total egg mass per hen, and feed conversion ratio were detected in hens fed 2.5% guar meal, whereas they remained unchanged for diets containing either level of guar germ or 5% guar meal. Feeding either level of guar germ or guar meal did not affect shell quality (shell thickness, egg breaking force, and specific gravity), Haugh units, or egg yolk color (L*, a*, b*). The results showed that both guar germ and guar meal can be fed to high-production laying hens at up to 5% without adverse effects on laying hen performance.

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

PubMed

Xue, Fei; Ma, Yinghong; Chen, Y Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang; Xu, Jie

2012-08-01

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama.

PubMed

Klisch, K; Contreras, D A; Sun, X; Brehm, R; Bergmann, M; Alberio, R

2011-11-01

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and β-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcβ1-4)Galβ1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

Evaluation of in vitro spermatogenesis system effectiveness to study genes behavior: monitoring the expression of the testis specific 10 (Tsga10) gene as a model.

PubMed

Miryounesi, Mohammad; Nayernia, Karim; Mobasheri, Maryam Beigom; Dianatpour, Mahdi; Oko, Richard; Savad, Shahram; Modarressi, Mohammad Hossein

2014-10-01

In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESC) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th, and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes. Expression of Tsga10 in RNA and protein levels was then analyzed. Transition from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed forms of the related protein was similar to those in vivo as well. Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.

Embryological evidence for a possible polyphyletic origin of the recent amphibians.

PubMed

Nieuwkoop, P D; Sutasurya, L A

1976-02-01

The markedly different mode of mesoderm formation in anuran and urodelan amphibians (which is related to the early double-layered nature of the anuran blastula wall in contrast to its single-layered nature in the urodeles), but particularly the fundamentally different place and mode of origin of the primordial germ cells in the two groups of amphibians, strongly pleads in favour of a very ancient bifurcation in the phylogenetic history of the two groups, even suggesting a polyphyletic origin from different ancestral fishes.

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

PubMed

Ewulonu, U K; Snyder, L; Silver, L M; Schimenti, J C

1996-03-01

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.

Normal embryonic and germ cell development in mice lacking alpha 1,3-fucosyltransferase IX (Fut9) which show disappearance of stage-specific embryonic antigen 1.

PubMed

Kudo, Takashi; Kaneko, Mika; Iwasaki, Hiroko; Togayachi, Akira; Nishihara, Shoko; Abe, Kuniya; Narimatsu, Hisashi

2004-05-01

Stage-specific embryonic antigen 1 (SSEA-1), an antigenic epitope defined as a Lewis x carbohydrate structure, is expressed during the 8-cell to blastocyst stages in mouse embryos and in primordial germ cells, undifferentiated embryonic stem cells, and embryonic carcinoma cells. For many years, SSEA-1 has been implicated in the development of mouse embryos as a functional carbohydrate epitope in cell-to-cell interaction during morula compaction. In a previous study, alpha 1,3-fucosyltransferase IX (Fut9) exhibited very strong activity for the synthesis of Lewis x compared to other alpha 1,3-fucosyltransferases in an in vitro substrate specificity assay. Fut4 and Fut9 transcripts were expressed in mouse embryos. The Fut9 transcript was detected in embryonic-day-13.5 gonads containing primordial germ cells, but the Fut4 transcript was not. In order to identify the role of SSEA-1 and determine the key enzyme for SSEA-1 synthesis in vivo, we have generated Fut9-deficient (Fut9(-/-)) mice. Fut9(-/-) mice develop normally, with no gross phenotypic abnormalities, and are fertile. Immunohistochemical analysis revealed an absence of SSEA-1 expression in early embryos and primordial germ cells of Fut9(-/-) mice. Therefore, we conclude that expression of the SSEA-1 epitope in the developing mouse embryo is not essential for embryogenesis in vivo.

Combination Chemotherapy in Treating Young Patients With Advanced Solid Tumors

ClinicalTrials.gov

2013-05-01

Childhood Central Nervous System Choriocarcinoma; Childhood Central Nervous System Embryonal Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Central Nervous System Germinoma; Childhood Central Nervous System Mixed Germ Cell Tumor; Childhood Central Nervous System Teratoma; Childhood Central Nervous System Yolk Sac Tumor; Recurrent Childhood Brain Stem Glioma; Recurrent Childhood Central Nervous System Embryonal Tumor; Unspecified Childhood Solid Tumor, Protocol Specific

Sunitinib in Treating Young Patients With Refractory Solid Tumors

ClinicalTrials.gov

2014-01-27

Central Nervous System Metastases; Childhood Central Nervous System Choriocarcinoma; Childhood Central Nervous System Embryonal Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Central Nervous System Germinoma; Childhood Central Nervous System Mixed Germ Cell Tumor; Childhood Central Nervous System Teratoma; Childhood Central Nervous System Yolk Sac Tumor; Recurrent Childhood Central Nervous System Embryonal Tumor; Unspecified Childhood Solid Tumor, Protocol Specific

Treatment-related Cardiovascular Late-effects and Exercise Training Countermeasures in Testicular Germ Cell Cancer Survivorship

PubMed Central

Christensen, Jesper F; Bandak, Mikkel; Campbell, Anna; Jones, Lee W.; Højman, Pernille

2016-01-01

Background Treatment of testicular germ cell cancer constitutes a major success story in modern oncology. Today, the vast majority of patients are cured by a therapeutic strategy using one or more highly effective components including surgery (orchiectomy), radiotherapy and/or chemotherapy. However, the excellent cancer specific survival comes at considerable costs, as individuals with a history of germ cell cancer experience serious long-term complications, including markedly increased risk of cardiovascular morbidities and premature cardiovascular death. The factors responsible, as well as their mode of action, are not fully understood and there is a lack of knowledge concerning optimal evidence-based long-term follow-up strategies. Results Here, we present the growing body of evidence suggesting that germ cell cancer patients as a consequence of the different treatment components, are subjected to toxicities, which individually, and synergistically, can cause physiological impairments leading to sub-clinical or clinical cardiovascular disorders the ‘multiple-hit hypothesis’). Furthermore, we discuss the efficacy and utility of structured exercise training to ameliorate treatment-induced cardiovascular dysfunction to prevent premature onset of clinical cardiovascular disease in germ cell cancer survivors, with a view towards highlighting future directions of exercise-based survivorship research in the germ cell cancer setting. Conclusion Since exercise training may have the potential to ameliorate and/or reverse long-term cardiovascular disease sequelae in germ cell cancer survivors, a strong rationale exists for the promotion of exercise-oncology research in this setting, in order to provide exercise-recommendations for optimal germ cell cancer survivorship. PMID:25751759

Treatment-related cardiovascular late effects and exercise training countermeasures in testicular germ cell cancer survivorship.

PubMed

Christensen, Jesper F; Bandak, Mikkel; Campbell, Anna; Jones, Lee W; Højman, Pernille

2015-05-01

Treatment of testicular germ cell cancer constitutes a major success story in modern oncology. Today, the vast majority of patients are cured by a therapeutic strategy using one or more highly effective components including surgery (orchiectomy), radiotherapy and/or chemotherapy. However, the excellent cancer-specific survival comes at considerable costs, as individuals with a history of germ cell cancer experience serious long-term complications, including markedly increased risk of cardiovascular morbidities and premature cardiovascular death. The factors responsible, as well as their mode of action, are not fully understood and there is a lack of knowledge concerning optimal evidence-based long-term follow-up strategies. Here, we present the growing body of evidence suggesting that germ cell cancer patients as a consequence of the different treatment components, are subjected to toxicities, which individually, and synergistically, can cause physiological impairments leading to sub-clinical or clinical cardiovascular disorders (i.e. the 'multiple-hit hypothesis'). Furthermore, we discuss the efficacy and utility of structured exercise training to ameliorate treatment-induced cardiovascular dysfunction to prevent premature onset of clinical cardiovascular disease in germ cell cancer survivors, with a view towards highlighting future directions of exercise-based survivorship research in the germ cell cancer setting. As exercise training may have the potential to ameliorate and/or reverse long-term cardiovascular disease sequelae in germ cell cancer survivors, a strong rationale exists for the promotion of exercise oncology research in this setting, in order to provide exercise recommendations for optimal germ cell cancer survivorship.

Genetic changes associated with testicular cancer susceptibility.

PubMed

Pyle, Louise C; Nathanson, Katherine L

2016-10-01

Testicular germ cell tumor (TGCT) is a highly heritable cancer primarily affecting young white men. Genome-wide association studies (GWAS) have been particularly effective in identifying multiple common variants with strong contribution to TGCT risk. These loci identified through association studies have implicated multiple genes as associated with TGCT predisposition, many of which are unique among cancer types, and regulate processes such as pluripotency, sex specification, and microtubule assembly. Together these biologically plausible genes converge on pathways involved in male germ cell development and maturation, and suggest that perturbation of them confers susceptibility to TGCT, as a developmental defect of germ cell differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs*

PubMed Central

Ortogero, Nicole; Schuster, Andrew S.; Oliver, Daniel K.; Riordan, Connor R.; Hong, Annie S.; Hennig, Grant W.; Luong, Dickson; Bao, Jianqiang; Bhetwal, Bhupal P.; Ro, Seungil; McCarrey, John R.; Yan, Wei

2014-01-01

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3′-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs. PMID:25320077

LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

PubMed

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-04-08

Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

PubMed Central

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-01-01

Summary Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis. PMID:24749077

Highly water pressurized brown rice improves cognitive dysfunction in senescence-accelerated mouse prone 8 and reduces amyloid beta in the brain.

PubMed

Okuda, Michiaki; Fujita, Yuki; Katsube, Takuya; Tabata, Hiromasa; Yoshino, Katsumi; Hashimoto, Michio; Sugimoto, Hachiro

2018-03-27

Alzheimer's disease (AD) is the most common form of dementia and the number of AD patients continues to increase worldwide. Components of the germ layer and bran of Brown rice (BR) help maintain good health and prevent AD. Because the germ layer and bran absorb little water and are very hard and difficult to cook, they are often removed during processing. To solve these problems, in this study, we tried to use a high-pressure (HP) technique. We produced the highly water pressurized brown rice (HPBR) by pressurizing BR at 600 MPa, and then we fed it to an AD mouse model, senescence-accelerated mouse prone 8, to investigate the therapeutic effects of HPBR on cognitive dysfunction by Y-maze spatial memory test. HP treatment increased the water absorbency of BR without nutrient loss. HPBR ameliorated cognitive dysfunction and reduced the levels of amyloid-β, which is a major protein responsible for AD, in the brain. These results suggest that HPBR is effective for preventing AD.

ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells.

PubMed

Pettinato, Giuseppe; Vanden Berg-Foels, Wendy S; Zhang, Ning; Wen, Xuejun

2014-01-01

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.

Cell tracking supports secondary gastrulation in the moon jellyfish Aurelia.

PubMed

Gold, David A; Nakanishi, Nagayasu; Hensley, Nicholai M; Hartenstein, Volker; Jacobs, David K

2016-11-01

The moon jellyfish Aurelia exhibits a dramatic reorganization of tissue during its metamorphosis from planula larva to polyp. There are currently two competing hypotheses regarding the fate of embryonic germ layers during this metamorphosis. In one scenario, the original endoderm undergoes apoptosis and is replaced by a secondary endoderm derived from ectodermal cells. In the second scenario, both ectoderm and endoderm remain intact through development. In this study, we performed a pulse-chase experiment to trace the fate of larval ectodermal cells. We observed that prior to metamorphosis, ectodermal cells that proliferated early in larval development concentrate at the future oral end of the polyp. During metamorphosis, these cells migrate into the endoderm, extending all the way to the aboral portion of the gut. We therefore reject the hypothesis that larval endoderm remains intact during metamorphosis and provide additional support for the "secondary gastrulation" hypothesis. Aurelia appears to offer the first and only described case where a cnidarian derives its endoderm twice during normal development, adding to a growing body of evidence that germ layers can be dramatically reorganized in cnidarian life cycles.

Sexual dimorphic expression of dnd in germ cells during sex reversal and its requirement for primordial germ cell survival in protogynous hermaphroditic grouper.

PubMed

Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Liu, Xiao-Chun; Li, Shui-Sheng; Wang, Yang; Gui, Jian-Fang

2017-06-01

Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper. Copyright © 2017 Elsevier Inc. All rights reserved.

Hanging drop cultures of human testis and testis cancer samples: a model used to investigate activin treatment effects in a preserved niche

PubMed Central

Jørgensen, A; Young, J; Nielsen, J E; Joensen, U N; Toft, B G; Rajpert-De Meyts, E; Loveland, K L

2014-01-01

Background: Testicular germ cell tumours of young adults, seminoma or non-seminomas, are preceded by a pre-invasive precursor, carcinoma in situ (CIS), understood to arise through differentiation arrest of embryonic germ cells. Knowledge about the malignant transformation of germ cells is currently limited by the lack of experimental models. The aim of this study was to establish an experimental tissue culture model to maintain normal and malignant germ cells within their niche and allow investigation of treatment effects. Methods: Human testis and testis cancer specimens from orchidectomies were cultured in ‘hanging drops' and effects of activin A and follistatin treatment were investigated in seminoma cultures. Results: Testis fragments with normal spermatogenesis or CIS cells were cultured for 14 days with sustained proliferation of germ cells and CIS cells and without increased apoptosis. Seminoma cultures survived 7 days, with proliferating cells detectable during the first 5 days. Activin A treatment significantly reduced KIT transcript and protein levels in seminoma cultures, thereby demonstrating a specific treatment response. Conclusions: Hanging drop cultures of human testis and testis cancer samples can be employed to delineate mechanisms governing growth of normal, CIS and tumorigenic germ cells retained within their niche. PMID:24781282

DND protein functions as a translation repressor during zebrafish embryogenesis.

PubMed

Kobayashi, Manami; Tani-Matsuhana, Saori; Ohkawa, Yasuka; Sakamoto, Hiroshi; Inoue, Kunio

2017-03-04

Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

Dissecting Germ Cell Metabolism through Network Modeling.

PubMed

Whitmore, Leanne S; Ye, Ping

2015-01-01

Metabolic pathways are increasingly postulated to be vital in programming cell fate, including stemness, differentiation, proliferation, and apoptosis. The commitment to meiosis is a critical fate decision for mammalian germ cells, and requires a metabolic derivative of vitamin A, retinoic acid (RA). Recent evidence showed that a pulse of RA is generated in the testis of male mice thereby triggering meiotic commitment. However, enzymes and reactions that regulate this RA pulse have yet to be identified. We developed a mouse germ cell-specific metabolic network with a curated vitamin A pathway. Using this network, we implemented flux balance analysis throughout the initial wave of spermatogenesis to elucidate important reactions and enzymes for the generation and degradation of RA. Our results indicate that primary RA sources in the germ cell include RA import from the extracellular region, release of RA from binding proteins, and metabolism of retinal to RA. Further, in silico knockouts of genes and reactions in the vitamin A pathway predict that deletion of Lipe, hormone-sensitive lipase, disrupts the RA pulse thereby causing spermatogenic defects. Examination of other metabolic pathways reveals that the citric acid cycle is the most active pathway. In addition, we discover that fatty acid synthesis/oxidation are the primary energy sources in the germ cell. In summary, this study predicts enzymes, reactions, and pathways important for germ cell commitment to meiosis. These findings enhance our understanding of the metabolic control of germ cell differentiation and will help guide future experiments to improve reproductive health.

A Genomewide RNAi Screen for Genes That Affect the Stability, Distribution and Function of P Granules in Caenorhabditis elegans

PubMed Central

Updike, Dustin L.; Strome, Susan

2009-01-01

P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their “germ granule” counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells. PMID:19805813

HMGA2 expression distinguishes between different types of postpubertal testicular germ cell tumour.

PubMed

Kloth, Lars; Gottlieb, Andrea; Helmke, Burkhard; Wosniok, Werner; Löning, Thomas; Burchardt, Käte; Belge, Gazanfer; Günther, Kathrin; Bullerdiek, Jörn

2015-10-01

The group of postpubertal testicular germ cell tumours encompasses lesions with highly diverse differentiation - seminomas, embryonal carcinomas, yolk sac tumours, teratomas and choriocarcinomas. Heterogeneous differentiation is often present within individual tumours and the correct identification of the components is of clinical relevance. HMGA2 re-expression has been reported in many tumours, including testicular germ cell tumours. This is the first study investigating HMGA2 expression in a representative group of testicular germ cell tumours with the highly sensitive method of quantitative real-time PCR as well as with immunohistochemistry. The expression of HMGA2 and HPRT was measured using quantitative real-time PCR in 59 postpubertal testicular germ cell tumours. Thirty specimens contained only one type of tumour and 29 were mixed neoplasms. With the exception of choriocarcinomas, at least two pure specimens from each subgroup of testicular germ cell tumour were included. In order to validate the quantitative real-time PCR data and gather information about the localisation of the protein, additional immunohistochemical analysis with an antibody specific for HMGA2 was performed in 23 cases. Expression of HMGA2 in testicular germ cell tumours depended on the histological differentiation. Seminomas and embryonal carcinomas showed no or very little expression, whereas yolk sac tumours strongly expressed HMGA2 at the transcriptome as well as the protein level. In teratomas, the expression varied and in choriocarcinomas the expression was moderate. In part, these results contradict data from previous studies but HMGA2 seems to represent a novel marker to assist pathological subtyping of testicular germ cell tumours. The results indicate a critical role in yolk sac tumours and some forms of teratoma.

X-ray micro-analysis of the mineralization patterns in developing enamel in hamster tooth germs exposed to fluoride in vitro during the secretory phase of amelogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyaruu, D.M.; Blijleven, N.; Hoeben-Schornagel, K.

1989-09-01

The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray microanalysis technique. The aim of this study was to obtain semi-quantitative data on the F(-)-induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F(-)-treated enamel is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence ofmore » 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used as a measure of the mineral content in the enamel. The aprismatic pre-exposure enamel, deposited in vivo before the onset of culture, was the most hypermineralized region in the fluorotic enamel, i.e., it contained the highest amount of calcium measured. The degree of the F(-)-induced hypermineralization gradually decreased (but was not abolished) in the more mature regions of the enamel. The unmineralized enamel matrix secreted during the initial F- treatment in vitro mineralized during the subsequent culture without F-. The calcium content in this enamel layer was in the same order of magnitude as that recorded for the newly deposited enamel in control tooth germs cultured without F-.« less

Developmental origins of epigenetic transgenerational inheritance

PubMed Central

Hanson, Mark A.; Skinner, Michael K.

2016-01-01

Abstract Environmental factors can induce epigenetic alterations in the germ cells that can potentially be transmitted transgenerationally. This non-genetic form of inheritance is termed epigenetic transgenerational inheritance and has been shown in a variety of species including plants, flies, worms, fish, rodents, pigs, and humans. This phenomenon operates during specific critical windows of exposure, linked to the developmental biology of the germ cells (sperm and eggs). Therefore, concepts of the developmental origins of transgenerational inheritance of phenotypic variation and subsequent disease risk need to include epigenetic processes affecting the developmental biology of the germ cell. These developmental impacts on epigenetic transgenerational inheritance, in contrast to multigenerational exposures, are the focus of this Perspective. PMID:27390622

New insights into human primordial germ cells and early embryonic development from single-cell analysis.

PubMed

Otte, Jörg; Wruck, Wasco; Adjaye, James

2017-08-01

Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications. © 2017 Federation of European Biochemical Societies.

Drosophila as a model system to unravel the layers of innate immunity to infection

PubMed Central

Kounatidis, Ilias; Ligoxygakis, Petros

2012-01-01

Summary Innate immunity relies entirely upon germ-line encoded receptors, signalling components and effector molecules for the recognition and elimination of invading pathogens. The fruit fly Drosophila melanogaster with its powerful collection of genetic and genomic tools has been the model of choice to develop ideas about innate immunity and host–pathogen interactions. Here, we review current research in the field, encompassing all layers of defence from the role of the microbiota to systemic immune activation, and attempt to speculate on future directions and open questions. PMID:22724070

Drosophila as a model system to unravel the layers of innate immunity to infection.

PubMed

Kounatidis, Ilias; Ligoxygakis, Petros

2012-05-01

Innate immunity relies entirely upon germ-line encoded receptors, signalling components and effector molecules for the recognition and elimination of invading pathogens. The fruit fly Drosophila melanogaster with its powerful collection of genetic and genomic tools has been the model of choice to develop ideas about innate immunity and host-pathogen interactions. Here, we review current research in the field, encompassing all layers of defence from the role of the microbiota to systemic immune activation, and attempt to speculate on future directions and open questions.

Sequence and expression pattern of the germ line marker vasa in honey bees and stingless bees

PubMed Central

2009-01-01

Queens and workers of social insects differ in the rates of egg laying. Using genomic information we determined the sequence of vasa, a highly conserved gene specific to the germ line of metazoans, for the honey bee and four stingless bees. The vasa sequence of social bees differed from that of other insects in two motifs. By RT-PCR we confirmed the germ line specificity of Amvasa expression in honey bees. In situ hybridization on ovarioles showed that Amvasa is expressed throughout the germarium, except for the transition zone beneath the terminal filament. A diffuse vasa signal was also seen in terminal filaments suggesting the presence of germ line cells. Oocytes showed elevated levels of Amvasa transcripts in the lower germarium and after follicles became segregated. In previtellogenic follicles, Amvasa transcription was detected in the trophocytes, which appear to supply its mRNA to the growing oocyte. A similar picture was obtained for ovarioles of the stingless bee Melipona quadrifasciata, except that Amvasa expression was higher in the oocytes of previtellogenic follicles. The social bees differ in this respect from Drosophila, the model system for insect oogenesis, suggesting that changes in the sequence and expression pattern of vasa may have occurred during social evolution. PMID:21637523

Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults

USDA-ARS?s Scientific Manuscript database

Bird eggs and are in contact with intestinal microbiota prior to oviposition, but are protected from bacterial translocation by a glycoprotein cuticle layer, the shell and internal membranes. In a preliminary study, turkey eggs were hatched in a germ-free environment. Firmicutes 16S rRNA gene was de...

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

Spermatogenesis, a study of germ cell development, is a long, orderly, and well-defined process occurring in seminiferous tubules of the testis. It is a temporal event whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa over a period of several weeks. Spermatogenesis is characterized by three specific functional phases: proliferation, meiosis, and differentiation, and it involves spermatogonia, spermatocytes, and spermatids. Germ cells at steps of development form various cellular associations or stages, with 6, 12, and 14 specific stages being identified in human, mouse, and rat, respectively. The stages evolve over time in a given area of the seminiferous tubule forming a cycle of the seminiferous epithelium that has a well-defined duration for a given species. In this part, we discuss the proliferation and meiotic phase whereby spermatogonia undergo several mitotic divisions to form spermatocytes that undergo two meiotic divisions to form haploid spermatids. In the rat, spermatogonia can be subdivided into several classes: stem cells (A(s)), proliferating cells (A(pr), A(al)), and differentiating cells (A(1)-A(4), In, B). They are dependent on a specific microenvironment (niche) contributed by Sertoli, myoid, and Leydig cells for proper development. Spermatogonia possess several surface markers whereby they can be identified from each other. During meiosis, spermatocytes undergo chromosomal pairing, synapsis, and genetic exchange as well as transforming into haploid cells following meiosis. The meiotic cells form specific structural entities such as the synaptonemal complex and sex body. Many genes involved in spermatogonial renewal and the meiotic process have been identified and shown to be essential for this event. Copyright 2009 Wiley-Liss, Inc.

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Modulation of WNT signaling activity is key to the formation of the embryonic head.

PubMed

Fossat, Nicolas; Jones, Vanessa; Garcia-Garcia, Maria J; Tam, Patrick P L

2012-01-01

The formation of the embryonic head begins with the assembly of the progenitor tissues of the brain, the head and face primordia and the foregut that are derived from the primary germ layers during gastrulation. Specification of the anterior-posterior polarity of major body parts and the morphogenesis of the head and brain specifically is driven by inductive signals including those mediated by BMP, Nodal, FGF and WNT. A critical role of β-catenin dependent WNT signalling activity for head morphogenesis has been revealed through the analysis of the phenotypic impact of loss of function mutation of an antagonist: DKK1, a transcriptional repressor: GSC; and the outcome of interaction of Dkk1 with genes coding three components of the canonical signalling pathway: the ligand WNT3, the co-receptor LRP6 and the transcriptional co-factor, β-catenin. The findings highlight the requirement of a stringent control of the timing, domain and level of canonical WNT signalling activity for the formation of the embryonic head.

Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning

PubMed Central

Morgani, Sophie M; Metzger, Jakob J; Nichols, Jennifer

2018-01-01

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species. PMID:29412136

Circulating tumor cells in patients with testicular germ cell tumors.

PubMed

Nastały, Paulina; Ruf, Christian; Becker, Pascal; Bednarz-Knoll, Natalia; Stoupiec, Małgorzata; Kavsur, Refik; Isbarn, Hendrik; Matthies, Cord; Wagner, Walter; Höppner, Dirk; Fisch, Margit; Bokemeyer, Carsten; Ahyai, Sascha; Honecker, Friedemann; Riethdorf, Sabine; Pantel, Klaus

2014-07-15

Germ cell tumors (GCTs) represent the most frequent malignancies among young men, but little is known about circulating tumor cells (CTCs) in these tumors. Considering their heterogeneity, CTCs were investigated using two independent assays targeting germ cell tumor and epithelial cell-specific markers, and results were correlated with disease stage, histology, and serum tumor markers. CTCs were enriched from peripheral blood (n = 143 patients) and testicular vein blood (TVB, n = 19 patients) using Ficoll density gradient centrifugation. For CTC detection, a combination of germ cell tumor (anti-SALL4, anti-OCT3/4) and epithelial cell-specific (anti-keratin, anti-EpCAM) antibodies was used. In parallel, 122 corresponding peripheral blood samples were analyzed using the CellSearch system. In total, CTCs were detected in 25 of 143 (17.5%) peripheral blood samples, whereas only 11.5% of patients were CTC-positive when considering exclusively the CellSearch assay. The presence of CTCs in peripheral blood correlated with clinical stage (P < 0.001) with 41% of CTC positivity in patients with metastasized tumors and 100% in patients with relapsed and chemotherapy-refractory disease. Histologically, CTC-positive patients suffered more frequently from nonseminomatous primary tumors (P < 0.001), with higher percentage of yolk sac (P < 0.001) and teratoma (P = 0.004) components. Furthermore, CTC detection was associated with elevated serum levels of α-fetoprotein (AFP; P = 0.025), β-human chorionic gonadotropin (βHCG; P = 0.002), and lactate dehydrogenase (LDH; P = 0.002). Incidence and numbers of CTCs in TVB were much higher than in peripheral blood. The inclusion of germ cell tumor-specific markers improves CTC detection in GCTs. CTCs occur frequently in patients with more aggressive disease, and there is a gradient of CTCs with decreasing numbers from the tumor-draining vein to the periphery. ©2014 American Association for Cancer Research.

Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells.

PubMed

Kurimoto, Kazuki; Yabuta, Yukihiro; Hayashi, Katsuhiko; Ohta, Hiroshi; Kiyonari, Hiroshi; Mitani, Tadahiro; Moritoki, Yoshinobu; Kohri, Kenjiro; Kimura, Hiroshi; Yamamoto, Takuya; Katou, Yuki; Shirahige, Katsuhiko; Saitou, Mitinori

2015-05-07

Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression pattern of G protein-coupled receptor 30 in human seminiferous tubular cells.

PubMed

Oliveira, Pedro F; Alves, Marco G; Martins, Ana D; Correia, Sara; Bernardino, Raquel L; Silva, Joaquina; Barros, Alberto; Sousa, Mário; Cavaco, José E; Socorro, Sílvia

2014-05-15

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERβ) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

Germ plasm anchoring is a dynamic state that requires persistent trafficking.

PubMed

Sinsimer, Kristina S; Lee, Jack J; Thiberge, Stephan Y; Gavis, Elizabeth R

2013-12-12

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, we show that RNP particle motility is required for long-term germ plasm retention. We propose that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Epoxides Derived from Dietary Dihomo-Gamma-Linolenic Acid Induce Germ Cell Death in C. elegans.

PubMed

Deline, Marshall; Keller, Julia; Rothe, Michael; Schunck, Wolf-Hagen; Menzel, Ralph; Watts, Jennifer L

2015-10-21

Dietary fats are not created equally, slight differences in structure lead to crucial differences in function. Muticellular organisms use polyunsaturated fatty acid as substrates to produce potent signaling molecules crucial for many physiological processes, including reproduction. Here we explored the mechanism responsible for germ cell loss induced by dietary supplementation of dihomo-gamma-linolenic acid (DGLA, 20:3n-6) in the roundworm Caenorhabditis elegans. In this study we found that C. elegans CYP-33E2 activity produces a range of epoxy and hydroxy metabolites from dietary DGLA. Knockdown of cyp-33E2 suppressed the DGLA-induced sterility phenotype. Additionally, direct exposure of two specific DGLA-derived epoxy products, 8,9- and 14,15-epoxyeicosadienoic acids, produced germ cell abnormalities in the C. elegans gonad. We propose that sterility is mediated by the production of toxic DGLA-derived epoxides that trigger germ cell destruction. These studies are the first to establish a biological activity for a CYP-produced metabolite of DGLA.

Chemotherapy-Induced Depletion of OCT4-Positive Cancer Stem Cells in a Mouse Model of Malignant Testicular Cancer.

PubMed

Pierpont, Timothy M; Lyndaker, Amy M; Anderson, Claire M; Jin, Qiming; Moore, Elizabeth S; Roden, Jamie L; Braxton, Alicia; Bagepalli, Lina; Kataria, Nandita; Hu, Hilary Zhaoxu; Garness, Jason; Cook, Matthew S; Capel, Blanche; Schlafer, Donald H; Southard, Teresa; Weiss, Robert S

2017-11-14

Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Essential role of citron kinase in cytokinesis of spermatogenic precursors.

PubMed

Cunto, Ferdinando Di; Imarisio, Sara; Camera, Paola; Boitani, Carla; Altruda, Fiorella; Silengo, Lorenzo

2002-12-15

During spermatogenesis, the first morphological indication of spermatogonia differentiation is incomplete cytokinesis, followed by the assembly of stable intercellular cytoplasmic communications. This distinctive feature of differentiating male germ cells has been highly conserved during evolution, suggesting that regulation of the cytokinesis endgame is a crucial aspect of spermatogenesis. However, the molecular mechanisms underlying testis-specific regulation of cytokinesis are still largely unknown. Citron kinase is a myotonin-related protein acting downstream of the GTPase Rho in cytokinesis control. We previously reported that Citron kinase knockout mice are affected by a complex neurological syndrome caused by cytokinesis block and apoptosis of specific neuronal precursors. In this report we show that, in addition, these mice display a dramatic testicular impairment, with embryonic and postnatal loss of undifferentiated germ cells and complete absence of mature spermatocytes. By contrast, the ovaries of mutant females appear essentially normal. Developmental analysis revealed that the cellular depletion observed in mutant testes is caused by increased apoptosis of undifferentiated and differentiating precursors. The same cells display a severe cytokinesis defect, resulting in the production of multinucleated cells and apoptosis. Our data indicate that Citron kinase is specifically required for cytokinesis of the male germ line.

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

Permissive and instructive anterior patterning rely on mRNA localization in the wasp embryo.

PubMed

Brent, Ava E; Yucel, Gozde; Small, Stephen; Desplan, Claude

2007-03-30

The long-germ mode of embryogenesis, in which segments arise simultaneously along the anteriorposterior axis, has evolved several times in different lineages of the holometabolous, or fully metamorphosing, insects. Drosophila's long-germ fate map is established largely by the activity of the dipteran-specific Bicoid (Bcd) morphogen gradient, which operates both instructively and permissively to accomplish anterior patterning. By contrast, all nondipteran long-germ insects must achieve anterior patterning independently of bcd. We show that bcd's permissive function is mimicked in the wasp by a maternal repression system in which anterior localization of the wasp ortholog of giant represses anterior expression of the trunk gap genes so that head and thorax can properly form.

Generation of induced pluripotent stem cells derived from an autosomal dominant polycystic kidney disease patient with a p.Ser1457fs mutation in PKD1.

PubMed

Huang, Ching-Ying; Ho, Ming-Ching; Lee, Jia-Jung; Hwang, Daw-Yang; Ko, Hui-Wen; Cheng, Yu-Che; Hsu, Yu-Hung; Lu, Huai-En; Chen, Hung-Chun; Hsieh, Patrick C H

2017-10-01

Autosomal dominant polycystic kidney disease is one of the most prevalent forms of inherited cystic kidney disease, and can be characterized by kidney cyst formation and enlargement. Here we report the generation of a Type 1 ADPKD disease iPS cell line, IBMS-iPSC-012-12, which retains the conserved deletion of PKD1, normal karyotype and exhibits the properties of pluripotent stem cells such as ES-like morphology, expression of pluripotent markers and capacity to differentiate into all three germ layers. Our results show that we have successfully generated a patient-specific iPS cell line with a mutation in PKD1 for study of renal disease pathophysiology. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Molecular cloning and characterization of oocyte-specific Pat1a in Rana rugosa frogs.

PubMed

Nakamura, Yoriko; Iwasaki, Takehiro; Umei, Yosuke; Saotome, Kazuhiro; Nakajima, Yukiko; Kitahara, Shoichi; Uno, Yoshinobu; Matsuda, Yoichi; Oike, Akira; Kodama, Maho; Nakamura, Masahisa

2015-10-01

The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a germ cell-specific protein-and Pat1a, Vasa-immunopositive signals were observed in all of the germ cells, whereas Pat1a signals were confined to the growing oocytes (50-200 μm in diameter), and absent from small germ cells (<50 μm in diameter). Forty days after testosterone injection into tadpoles to induce female-to-male sex-reversal, Pat1a-immunoreactive oocytes had disappeared completely from the sex-reversed gonad, but Vasa-positive small germ cells persisted. Thus, Pat1a would be a good marker for identifying the sexual status of the sex-reversing gonad in amphibians. In addition, fluorescence in situ hybridization analysis showed Pat1a to have an autosomal locus, suggesting that Pat1a transcription is probably regulated by a tissue-specific transcription factor in R. rugosa. © 2015 Wiley Periodicals, Inc.

Altered Lipid Homeostasis in Sertoli Cells Stressed by Mild Hyperthermia

PubMed Central

Vallés, Ana S.; Aveldaño, Marta I.; Furland, Natalia E.

2014-01-01

Spermatogenesis is known to be vulnerable to temperature. Exposures of rat testis to moderate hyperthermia result in loss of germ cells with survival of Sertoli cells (SC). Because SC provide structural and metabolic support to germ cells, our aim was to test the hypothesis that these exposures affect SC functions, thus contributing to germ cell damage. In vivo, regularly repeated exposures (one of 15 min per day, once a day during 5 days) of rat testes to 43°C led to accumulation of neutral lipids. This SC-specific lipid function took 1–2 weeks after the last of these exposures to be maximal. In cultured SC, similar daily exposures for 15 min to 43°C resulted in significant increase in triacylglycerol levels and accumulation of lipid droplets. After incubations with [3H]arachidonate, the labeling of cardiolipin decreased more than that of other lipid classes. Another specifically mitochondrial lipid metabolic function, fatty acid oxidation, also declined. These lipid changes suggested that temperature affects SC mitochondrial physiology, which was confirmed by significantly increased degrees of membrane depolarization and ROS production. This concurred with reduced expression of two SC-specific proteins, transferrin, and Wilms' Tumor 1 protein, markers of SC secretion and differentiation functions, respectively, and with an intense SC cytoskeletal perturbation, evident by loss of microtubule network (α-tubulin) and microfilament (f-actin) organization. Albeit temporary and potentially reversible, hyperthermia-induced SC structural and metabolic alterations may be long-lasting and/or extensive enough to respond for the decreased survival of the germ cells they normally foster. PMID:24690895

The origin of mesoderm in phoronids

NASA Technical Reports Server (NTRS)

Freeman, Gary; Martindale, Mark Q.

2002-01-01

Descriptive studies of phoronid development have concluded that the mesoderm of these animals originates from the endoderm during gastrulation. This interpretation has been tested by labeling one blastomere of 4- through 16-cell embryos and examining the position and germ layers occupied by the labeled clones of cells in the larva. No 2 injections gave rise to identical clones of cells, suggesting that the cleavage program does not generate cells of unique identity and that cell fates are established at later developmental time points. In many cases, a relatively large sector composed of ectodermal cells was labeled. When these labeled cells were adjacent to the mouth or anus of the larva, muscle and mesenchyme cells originated from the labeled clones. Under these circumstances, nerve cells also originated from these labeled sectors. These labeling studies also showed that endodermal cells can give rise to mesodermal and neural cells. These results suggest that nerve and muscle cells are induced to form at ectodermal-endodermal boundaries from both germ layers. These marking experiments also confirmed the observation that nerve cells originate both from the apical organ and the trunk region and show for the first time that the intestine originates by ingression of posterior ectoderm.

A Theory of L 1-Dissipative Solvers for Scalar Conservation Laws with Discontinuous Flux

NASA Astrophysics Data System (ADS)

Andreianov, Boris; Karlsen, Kenneth Hvistendahl; Risebro, Nils Henrik

2011-07-01

We propose a general framework for the study of L 1 contractive semigroups of solutions to conservation laws with discontinuous flux: u_t + mathfrak{f}(x,u)_x=0, qquad mathfrak{f}(x,u)= left\\{begin{array}{ll} f^l(u),& x < 0,\\ f^r(u), & x > 0, right.quadquadquad (CL) where the fluxes f l , f r are mainly assumed to be continuous. Developing the ideas of a number of preceding works ( Baiti and Jenssen in J Differ Equ 140(1):161-185, 1997; Towers in SIAM J Numer Anal 38(2):681-698, 2000; Towers in SIAM J Numer Anal 39(4):1197-1218, 2001; Towers et al. in Skr K Nor Vidensk Selsk 3:1-49, 2003; Adimurthi et al. in J Math Kyoto University 43(1):27-70, 2003; Adimurthi et al. in J Hyperbolic Differ Equ 2(4):783-837, 2005; Audusse and Perthame in Proc Roy Soc Edinburgh A 135(2):253-265, 2005; Garavello et al. in Netw Heterog Media 2:159-179, 2007; Bürger et al. in SIAM J Numer Anal 47:1684-1712, 2009), we claim that the whole admissibility issue is reduced to the selection of a family of "elementary solutions", which are piecewise constant weak solutions of the form c(x)=c^l11_{left\\{{x < 0}right\\}}+c^r11_{left\\{{x > 0}right\\}}. We refer to such a family as a "germ". It is well known that (CL) admits many different L 1 contractive semigroups, some of which reflect different physical applications. We revisit a number of the existing admissibility (or entropy) conditions and identify the germs that underly these conditions. We devote specific attention to the "vanishing viscosity" germ, which is a way of expressing the "Γ-condition" of D iehl (J Hyperbolic Differ Equ 6(1):127-159, 2009). For any given germ, we formulate "germ-based" admissibility conditions in the form of a trace condition on the flux discontinuity line { x = 0} [in the spirit of V ol'pert (Math USSR Sbornik 2(2):225-267, 1967)] and in the form of a family of global entropy inequalities [following K ruzhkov (Math USSR Sbornik 10(2):217-243, 1970) and C arrillo (Arch Ration Mech Anal 147(4):269-361, 1999)]. We characterize those germs that lead to the L 1-contraction property for the associated admissible solutions. Our approach offers a streamlined and unifying perspective on many of the known entropy conditions, making it possible to recover earlier uniqueness results under weaker conditions than before, and to provide new results for other less studied problems. Several strategies for proving the existence of admissible solutions are discussed, and existence results are given for fluxes satisfying some additional conditions. These are based on convergence results either for the vanishing viscosity method (with standard viscosity or with specific viscosities "adapted" to the choice of a germ), or for specific germ-adapted finite volume schemes.

A caprine chimera produced by injection of embryonic germ cells into a blastocyst.

PubMed

Jia, W; Yang, W; Lei, A; Gao, Z; Yang, C; Hua, J; Huang, W; Ma, X; Wang, H; Dou, Z

2008-02-01

This report details a chimeric goat derived by injecting caprine embryonic germ (EG) cells into a host blastocyst. The EG cells, isolated from the primordial genital ridge of white Guanzhong goat fetuses (28-42 days of pregnancy), had alkaline phosphatase activity and several stem cell markers, including SSEA-1, c-kit, and Nanog. Ten to 20EG cells were microinjected into the blastocoelic cavity of a host blastocyst collected from a black goat following natural service. Twenty-nine injected blastocysts were transferred into nine white surrogate goats. One of the recipients maintained pregnancy to term and gave birth to three kids: one male, one female, and a dead, malformed fetus of undetermined gender; all three fetuses were black, but the female and the malformed fetus each had a large white spot on their head. Based on PCR and microsatellite DNA assay, the female and the malformed fetus were monozygotic twins and chimeras. Microsatellite assay on various tissues from the dead fetus (including skin, blood, liver, placenta, lung, heart, spleen, muscle, and brain), revealed that these tissues and organs were chimeric and contained cells derived from EG cells. In conclusion, caprine EG cells differentiated into all three germ layers in vivo.

Activation of the mouse Oct4 promoter in medaka embryonic stem cells and its use for ablation of spontaneous differentiation.

PubMed

Hong, Yunhan; Winkler, Christoph; Liu, Tongming; Chai, Guixuan; Schartl, Manfred

2004-07-01

The determination and maintenance of the cell fate is ultimately due to differential gene activity. In the mouse, expression of the transcription factor Oct4 is high in totipotent inner cell mass, germ cells and undifferentiated embryonic stem (ES) cells, but dramatically reduced or extinct upon differentiation. Here, we show that medaka blastula embryos and cells of the ES cell line MES1 are able to activate the Oct4 promoter. Ectopic expression of a fusion gene for beta-galactosidase and neomycin resistance from the Oct4 promoter conferred resistance to G418. G418 selection led to a homogeneous population of undifferentiated ES cells which were able to undergo induced or directed differentiation into various cell types including neuron-like cells and melanocytes. Furthermore, GFP-labeled GOF18geo-MES1 cells after differentiation ablation were able to contribute to a wide variety of organ systems derived from all the three germ layers. Most importantly, we show that drug ablation of differentiation on the basis of Oct4 promoter is a useful tool to improve ES cell cultivation and chimera formation: MES1 cells after differentiation ablation appeared to be better donors than the parental MES1 line, as the permissive number of input donor cells increases from 100 to 200, resulting in an enhanced degree of chimerism. Taken together, some transcription factors and cis-acting regulatory sequences controlling totipotency-specific gene expression appear to be conserved between mammals and fish, and medaka ES cells offer an in vitro system for characterizing the expression of totipotency-specific genes such as putative Oct4 homologs from fish.

Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

PubMed Central

Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

2012-01-01

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

Surgery and Combination Chemotherapy in Treating Children With Extracranial Germ Cell Tumors

ClinicalTrials.gov

2017-12-07

Childhood Embryonal Tumor; Childhood Extracranial Germ Cell Tumor; Childhood Extragonadal Germ Cell Tumor; Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Childhood Teratoma; Ovarian Embryonal Carcinoma; Ovarian Yolk Sac Tumor; Stage II Malignant Testicular Germ Cell Tumor; Stage IIA Ovarian Germ Cell Tumor; Stage IIB Ovarian Germ Cell Tumor; Stage IIC Ovarian Germ Cell Tumor; Stage III Malignant Testicular Germ Cell Tumor; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIC Ovarian Germ Cell Tumor; Testicular Choriocarcinoma and Yolk Sac Tumor; Testicular Embryonal Carcinoma

Instruction at the Hopkins Marine Station

DTIC Science & Technology

1992-07-29

foI homtadcodnaio.. caronavirus nucleocapsid protein. wheat germ bial ~ ~ %~H2A (5), mussel sperm nuclear protein 03 [6), and man chromofsome...wvpi,~Tninev PM"p Johne HiWA~aa Unuw~rsaty &Dio of Medicine, Balw,,.vv, Manh land 21205 The two germ -line- specific Sp histione classes Treatment of...composit conical morphology of the male pronucleus- Mal, pro- serine-proline adjacent to two basic amino acids (lyo hucl*I inhibite’d with I nsMGDMAP

High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors.

PubMed

Shekhani, Mohammed Talha; Barber, John R; Bezerra, Stephania M; Heaphy, Christopher M; Gonzalez Roibon, Nilda Diana; Taheri, Diana; Reis, Leonardo O; Guner, Gunes; Joshu, Corinne E; Netto, George J; Meeker, Alan K

2016-08-01

Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well. Copyright © 2016. Published by Elsevier Inc.

Prognostic value of CD66b positive tumor-infiltrating neutrophils in testicular germ cell tumor.

PubMed

Yamada, Yuta; Nakagawa, Tohru; Sugihara, Toru; Horiuchi, Takamasa; Yoshizaki, Uran; Fujimura, Tetsuya; Fukuhara, Hiroshi; Urano, Tomohiko; Takayama, Kenichi; Inoue, Satoshi; Kume, Haruki; Homma, Yukio

2016-11-18

Prognostic value of immune cells is not clear in testicular germ cell tumors (TGCTs). We aimed to investigate the prognostic value of tumor-infiltrating neutrophils in TGCTs. A total of 102 patients who underwent orchiectomy for TGCT were investigated for CD66b positive tumor-infiltrating neutrophils (CD66b + TINs). Immmunostaining for CD66b was performed in 102 sections as described. Clinicopathological parameters as well as cancer specific survival and overall survival were assessed for correlation with CD66b + TIN density. High density group was significantly correlated with tumor diameter ≥ 10 cm, presence of nodal/distant metastasis, S stage, diagnosis of nonseminomatous germ cell tumor (NGCT), and presence of venous invasion (p = 0.0198, p < 0.0001, p = 0.0275, p = 0.0004, and p = 0.0287, respectively). It was also significantly associated with cancer-specific and overall survival (logrank p = 0.0036, and p = 0.0002, respectively). Multivariate analysis showed that increased CD66b + TIN was an independent prognostic factor for overall survival (p = 0.0095). Increased CD66b + TIN was significantly associated with presence of metastasis, S stage, and nonseminomatous germ cell tumor diagnosis. It was also an independent prognostic factor of overall survival in patients with TGCT.

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa).

PubMed

Barfield, Sarah; Aglyamova, Galina V; Matz, Mikhail V

2016-01-13

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. © 2016 The Author(s).

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa)

PubMed Central

Barfield, Sarah; Aglyamova, Galina V.; Matz, Mikhail V.

2016-01-01

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. PMID:26763699

GPR30, the non-classical membrane G protein related estrogen receptor, is overexpressed in human seminoma and promotes seminoma cell proliferation.

PubMed

Chevalier, Nicolas; Vega, Aurélie; Bouskine, Adil; Siddeek, Bénazir; Michiels, Jean-François; Chevallier, Daniel; Fénichel, Patrick

2012-01-01

Testicular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs) with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A) are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30) mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium). The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation. We report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells) and germ cells (spermatogonia and spermatocytes). GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist) and by siRNA invalidation. These results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for seminomas.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

PubMed

Dores, C; Rancourt, D; Dobrinski, I

2015-05-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling. © 2015 American Society of Andrology and European Academy of Andrology.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis

PubMed Central

Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2015-01-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here we report the use of stirred suspension bioreactors to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: stirred suspension bioreactor followed by differential plating. After 66 hours of culture, germ cell enrichment in stirred suspension bioreactors provided 7.3±1.0 fold (n=9), differential plating 9.8±2.4 fold (n=6) and combination of both methods resulted in 9.1±0.3 fold enrichment of germ cells from the initial germ cell population (n=3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the stirred suspension bioreactor allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability due to handling. PMID:25877677

Germ stem cells are active in postnatal mouse ovary under physiological conditions

PubMed Central

Guo, Kun; Li, Chao-hui; Wang, Xin-yi; He, Da-jian; Zheng, Ping

2016-01-01

STUDY HYPOTHESIS Are active ovarian germ stem cells present in postnatal mouse ovaries under physiological conditions? STUDY FINDING Active ovarian germ stem cells exist and function in adult mouse ovaries under physiological conditions. WHAT IS KNOWN ALREADY In vitro studies suggested the existence of germ stem cells in postnatal ovaries of mouse, pig and human. However, in vivo studies provided evidence against the existence of active germ stem cells in postnatal mouse ovaries. Thus, it remains controversial whether such germ stem cells really exist and function in vivo in postnatal mammalian ovaries. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Octamer-binding transcription factor 4 (Oct4)-MerCreMer transgenic mice were crossed with R26R-enhanced yellow fluorescent protein (EYFP) mice to establish a tamoxifen-inducible tracing system so that Oct4-expressing potential ovarian germ stem cells in young adult mice (5–6 weeks old) can be labeled with EYFP. The germ cell activities of DNA replication, mitotic division, entry into meiosis and progression to primordial follicle stage were investigated by means of immunofluorescent staining of ovarian tissues collected at different time points post-tamoxifen injection (1 day, 3 days, 2 months and 4 months). Meiosis entry and primordial follicle formation were also measured by EYFP-labeled single-cell RT–PCR. Germ cell proliferation and mitotic division were examined through 5-bromodeoxyuridine triphosphate incorporation assay. At each time point, ovaries from two to three animals were used for each set of experiment. MAIN RESULTS AND THE ROLE OF CHANCE By labeling the Oct4-expressing small germ cells and tracing their fates for up to 4 months, we observed persistent meiosis entry and primordial follicle replenishment. Furthermore, we captured the transient processes of mitotic DNA replication as well as mitotic division of the marked germ cells at various time periods after tracing. These lines of evidence unambiguously support the presence of active germ stem cells in postnatal ovaries and their function in replenishing primordial follicle pool under physiological conditions. Moreover, we pointed out that Oct4+ deleted in azoospermia-like (Dazl)− but not Oct4+Dazl+ or Oct4+ DEAD (Asp–Glu–Ala–Asp) Box Polypeptide 4 (Ddx4)+ cells contain a population of germ stem cells in mouse ovary. LIMITATIONS, REASONS FOR CAUTION This study was conducted in mice. Whether or not the results are applicable to human remain unclear. The future work should aim at identifying the specific ovarian germ stem cell marker and evaluating the significance of these stem cells to normal ovarian function. WIDER IMPLICATIONS OF THE FINDINGS Clarifying the existence of active germ stem cells and their functional significance in postnatal mammalian ovaries could provide new insights in understanding the mechanism of ovarian aging and failure. LARGE SCALE DATA Not applicable. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Key Basic Research Program of China (grant number 2012CBA01300) and the National Natural Science Foundation of China to P.Z. (31571484). No competing interests are reported. PMID:26916381

Testicular seminomatous mixed germ cell tumor with choriocarcinoma and teratoma with secondary somatic malignancy: a case report.

PubMed

Aneja, Amandeep; Bhattacharyya, Siddharth; Mydlo, Jack; Inniss, Susan

2014-01-01

Testicular tumors are a heterogeneous group of neoplasms exhibiting diverse histopathology and can be classified as seminomatous and non-seminomatous germ cell tumor types. Mixed germ cell tumors contain more than one germ cell component and various combinations have been reported. Here, we present a rare case of a mixed germ cell tumor composed of seminoma, choriocarcinoma and teratoma with a secondary somatic malignancy. A 31-year-old Caucasian man presented with splenic rupture to our hospital. A right-sided testicular swelling had been present for 6 months and his alpha-fetoprotein, beta-human chorionic gonadotropin, and lactose dehydrogenase were increased. An ultrasound of his scrotum revealed an enlarged right testis with heterogeneous echogenicity. Multiple hypervascular lesions were noted in his liver and spleen. He underwent transcatheter embolization therapy of his splenic artery followed by splenectomy and right-sided orchiectomy. A computed tomography scan also showed metastasis to both lungs. During his last follow up after four cycles of cisplatin-based chemotherapy, the level of tumor markers had decreased, decreases in the size of his liver and pulmonary lesions were noted but new sclerotic lesions were evident in his thoracolumbar region raising concern for bony metastasis. Prognosis of testicular tumor depends mainly on the clinical stage, but emergence of a sarcomatous component presents a challenge in the treatment of germ cell tumors and the histological subtype of this component can be used as a guide to specific chemotherapy in these patients.

Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization

PubMed Central

García-López, Jesús; Alonso, Lola; Cárdenas, David B.; Artaza-Alvarez, Haydeé; Hourcade, Juan de Dios; Martínez, Sergio; Brieño-Enríquez, Miguel A.; del Mazo, Jesús

2015-01-01

The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes. PMID:25805854

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wyrobek, Andrew J.; Mulvihill, John J.; Wassom, John S.

Although numerous germ-cell mutagens have been identified inanimal model systems, to date, no human germ-cell mutagens have beenconfirmed. Because the genomic integrity of our germ cells is essentialfor the continuation of the human species, a resolution of this enduringconundrum is needed. To facilitate such a resolution, we organized aworkshop at The Jackson Laboratory in Bar Harbor, Maine on September28-30, 2004. This interactive workshop brought together scientists from awide range of disciplines to assess the applicability of emergingmolecular methods for genomic analysis to the field of human germ-cellmutagenesis. Participants recommended that focused, coordinated humangerm-cell mutation studies be conducted in relation tomore » important societalexposures. Because cancer survivors represent a unique cohort withwell-defined exposures, there was a consensus that studies should bedesigned to assess the mutational impact on children born to parents whohad received certain types of mutagenic cancer chemotherapy prior toconceiving their children. Within this high-risk cohort, parents andchildren could be evaluated for inherited changes in (a) gene sequencesand chromosomal structure, (b) repeat sequences and minisatelliteregions, and (c) global gene expression and chromatin. Participants alsorecommended studies to examine trans-generational effects in humansinvolving mechanisms such as changes in imprinting and methylationpatterns, expansion of nucleotide repeats, or induction of mitochondrialDNA mutations. Workshop participants advocated establishment of abio-bank of human tissue samples that could be used to conduct amultiple-endpoint, comprehensive, and collaborative effort to detectexposure-induced heritable alterations in the human genome. Appropriateanimal models of human germ-cell mutagenes is should be used in parallelwith human studies to provide insights into the mechanisms of mammaliangerm-cell mutagenesis. Finally, participants recommended that scientificspecialty groups be convened to address specific questions regarding thepotential germ-cell mutagenicity of environmental, occupational, andlifestyle exposures. Strong support from relevant funding agencies andengagement of scientists outside the fields of genomics and germ-cellmutagenesis will be required to launch a full-scale assault on some ofthe most pressing and enduring questions in environmental mutagenesis: Dohuman germ-cell mutagens exist, what risk do they pose to futuregenerations, and are some parents at higher risk than others foracquiring and transmitting germ-cell mutations?« less

Immortalization of canine adipose-derived mesenchymal stem cells and their seminiferous tubule transplantation.

PubMed

Fang, Jia; Wei, Yudong; Teng, Xin; Zhao, Shanting; Hua, Jinlian

2018-04-01

Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs. © 2017 Wiley Periodicals, Inc.

Exposure to Endocrine Disruptor Induces Transgenerational Epigenetic Deregulation of MicroRNAs in Primordial Germ Cells

PubMed Central

Brieño-Enríquez, Miguel A.; García-López, Jesús; Cárdenas, David B.; Guibert, Sylvain; Cleroux, Elouan; Děd, Lukas; Hourcade, Juan de Dios; Pěknicová, Jana; Weber, Michael; del Mazo, Jesús

2015-01-01

In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation. PMID:25897752

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

PubMed Central

Spike, Caroline A.; Bader, Jason; Reinke, Valerie; Strome, Susan

2008-01-01

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs). PMID:18234720

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells.

PubMed

Spike, Caroline A; Bader, Jason; Reinke, Valerie; Strome, Susan

2008-03-01

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs).

Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ.

PubMed

Smailov, S K; Lee, A V; Iskakov, B K

1993-04-26

Phosphorylation of elongation factor 2 (EF-2) by specific Ca2+/calmodulin-dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF-2 can be intensively phosphorylated by the rabbit reticulocyte EF-2 kinase. Phosphorylation results in inhibition of the activity of plant EF-2 in poly(U)-dependent cell-free translation system. Thus, the activity of EF-2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF-2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF-2 kinase activity is not displayed in different organs of wheat and other higher plants.

Role of Axumin PET Scan in Germ Cell Tumor

ClinicalTrials.gov

2018-05-01

Testis Cancer; Germ Cell Tumor; Testicular Cancer; Germ Cell Tumor of Testis; Germ Cell Tumor, Testicular, Childhood; Testicular Neoplasms; Testicular Germ Cell Tumor; Testicular Yolk Sac Tumor; Testicular Choriocarcinoma; Testicular Diseases; Germ Cell Cancer Metastatic; Germ Cell Neoplasm of Retroperitoneum; Germ Cell Cancer, Nos

Intestinal Microbiota Modulates Gluten-Induced Immunopathology in Humanized Mice

PubMed Central

Galipeau, Heather J.; McCarville, Justin L.; Huebener, Sina; Litwin, Owen; Meisel, Marlies; Jabri, Bana; Sanz, Yolanda; Murray, Joseph A.; Jordana, Manel; Alaedini, Armin; Chirdo, Fernando G.; Verdu, Elena F.

2016-01-01

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk. PMID:26456581

Pazopanib Hydrochloride in Treating Young Patients With Solid Tumors That Have Relapsed or Not Responded to Treatment

ClinicalTrials.gov

2013-09-27

Childhood Central Nervous System Choriocarcinoma; Childhood Central Nervous System Embryonal Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Central Nervous System Germinoma; Childhood Central Nervous System Mixed Germ Cell Tumor; Childhood Central Nervous System Teratoma; Childhood Central Nervous System Yolk Sac Tumor; Metastatic Childhood Soft Tissue Sarcoma; Recurrent Childhood Brain Stem Glioma; Recurrent Childhood Central Nervous System Embryonal Tumor; Recurrent Childhood Soft Tissue Sarcoma; Recurrent Childhood Visual Pathway Glioma; Unspecified Childhood Solid Tumor, Protocol Specific

A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection

NASA Astrophysics Data System (ADS)

Elliott, David J.; Bourgeois, Cyril F.; Klink, Albrecht; Stévenin, James; Cooke, Howard J.

2000-05-01

RNA-binding motif (RBM) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein-protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.

Dough performance, quality and shelf life of flat bread supplemented with fractions of germinated date seed.

PubMed

Hejri-Zarifi, Sudiyeh; Ahmadian-Kouchaksaraei, Zahra; Pourfarzad, Amir; Khodaparast, Mohammad Hossein Haddad

2014-12-01

Germinated palm date seeds were milled into two fractions: germ and residue. Dough rheological characteristics, baking (specific volume and sensory evaluation), and textural properties (at first day and during storage for 5 days) were determined in Barbari flat bread. Germ and residue fractions were incorporated at various levels ranged in 0.5-3 g/100 g of wheat flour. Water absorption, arrival time and gelatination temperature were decreased by germ fraction but accompanied by an increasing effect on the mixing tolerance index and degree of softening in most levels. Although improvement in dough stability was monitored but specific volume of bread was not affected by both fractions. Texture analysis of bread samples during 5 days of storage indicated that both fractions of germinated date seeds were able to diminish bread staling. Avrami non-linear regression equation was chosen as useful mathematical model to properly study bread hardening kinetics. In addition, principal component analysis (PCA) allowed discriminating among dough and bread specialties. Partial least squares regression (PLSR) models were applied to determine the relationships between sensory and instrumental data.

Autoantibodies against Leydig cells in patients after spermatic cord torsion.

PubMed Central

Zanchetta, R; Mastrogiacomo, I; Graziotti, P; Foresta, C; Betterle, C

1984-01-01

This study is aimed at searching for the presence of circulating antibodies against frozen sections of human testis, ovary and trophoblast in patients that had spermatic cord torsion. Sixty-eight sera samples were studied. Nine patients (13.2%) were positive for organ specific anti-testis autoantibodies. Six patients were positive for antibodies against Leydig cells: five were positive only with the indirect immunofluorescence technique of complement fixing (ITT/CF), the sixth patient was positive only with the indirect immunofluorescence technique (ITT). The other three patients were positive for antibodies against germ line cells: two patients were positive with both techniques, the third was positive only with indirect immunofluorescence technique. Eight of these patients were negative for antibodies against adrenal cortex while only one case was positive with indirect immunofluorescence technique both on adrenal cortex and Leydig cells. Human lyophilized testis absorbed the reactive antibodies against Leydig cells and germ line cells, while adrenal cortex and lyophilized testosterone were ineffective. This study shows the identification of a specific antibody against Leydig cells and germ line cells in patients after spermatic cord torsion. PMID:6362937

Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

PubMed

Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

2015-09-01

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.

Specialized rules of gene transcription in male germ cells: the CREM paradigm.

PubMed

Monaco, Lucia; Kotaja, Noora; Fienga, Giulia; Hogeveen, Kevin; Kolthur, Ullas S; Kimmins, Sarah; Brancorsini, Stefano; Macho, Betina; Sassone-Corsi, Paolo

2004-12-01

Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding protein family and its associated cofactors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator cAMP response element modulator (CREM) represents an established paradigm. Somatic cell activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CREB-binding protein. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, activator of CREM in the testis (ACT), which confers a powerful, phosphorylation-independent activation capacity. The function of ACT was found to be regulated by the testis-specific kinesin KIF17b. Here we discuss some aspects of the testis-specific transcription machinery, whose function is essential for the process of spermatogenesis.

Palifosfamide in Treating Patients With Recurrent Germ Cell Tumors

ClinicalTrials.gov

2015-06-11

Adult Central Nervous System Germ Cell Tumor; Adult Teratoma; Malignant Extragonadal Germ Cell Tumor; Malignant Extragonadal Non-Seminomatous Germ Cell Tumor; Extragonadal Seminoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Stage IV Extragonadal Non-Seminomatous Germ Cell Tumor; Stage IV Extragonadal Seminoma; Stage IV Ovarian Germ Cell Tumor

The Ectopic Expression of the Wheat Puroindoline Genes Increase Germ Size and Seed Oil Content in Transgenic Corn

PubMed Central

Zhang, Jinrui; Martin, John M.; Beecher, Brian; Lu, Chaofu; Hannah, L. Curtis; Wall, Michael L.; Altosaar, Illimar; Giroux, Michael J.

2014-01-01

Plant oil content and composition improvement is a major goal of plant breeding and biotechnology. The Puroindoline a and b (PINA and PINB) proteins together control whether wheat seeds are soft or hard textured and share a similar structure to that of plant non-specific lipid-transfer proteins. Here we transformed corn (Zea mays L.) with the wheat (Triticum aestivum L.) puroindoline genes (Pina and Pinb) to assess their effects upon seed oil content and quality. Pina and Pinb coding sequences were introduced into corn under the control of a corn Ubiquitin promoter. Three Pina/Pinb expression positive transgenic events were evaluated over two growing seasons. The results showed that Pin expression increased germ size significantly without negatively impacting seed size. Germ yield increased 33.8% while total seed oil content was increased by 25.23%. Seed oil content increases were primarily the result of increased germ size. This work indicates that higher oil content corn hybrids having increased food or feed value could be produced via puroindoline expression. PMID:20725765

Human primordial germ cell formation is diminished by exposure to environmental toxicants acting through the AHR signaling pathway.

PubMed

Kee, Kehkooi; Flores, Martha; Cedars, Marcelle I; Reijo Pera, Renee A

2010-09-01

Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

A Human Anti-Insulin IgG Autoantibody Apparently Arises Through Clonal Selection from an Insulin-Specific “Germ-Line” Natural Antibody Template

PubMed Central

Ichiyoshi, Yuji; Zhou, Min; Casali, Paolo

2015-01-01

We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 κ mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and Vκ segments and used them to generate the full (unmutated) “germ-line revertant” of the “wild-type” (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human Cγ1 and Cκ expression system. The full “germ-line revertant” bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (Avrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 × 10−8 vs 4.91 × 10−9 g/μl). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and Vκ segments, that the increased Avrel for insulin of mAb13 when compared with its full “germ-line revertant” counterpart was entirely dependent on the mutations in the VH not those in the Vκ chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser→Gly, Ser→Thr, and Ser→Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 × 10−8 to 5.14 × 10−9 g/μl) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 × 10−3 to 1.54 × 10−3 s−1)but not an increase in association rate constant (Kon, from 2.60 × 104 to 2.61 × 104 M−1 s−1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor expressing a specific surface receptor for Ag in unmutated configuration, and drive it through affinity maturation. They also show that binding of insulin by such a receptor can be enhanced or completely abrogated by a single amino acid change. PMID:7995943

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

PubMed

Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

2011-01-01

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barton, M.K.; Schedl, T.B.; Kimble, J.

1987-01-01

The authors have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3 (gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3 (gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All ninemore » fem-3 (gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3 (gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.« less

Formation of the Embryonic Head in the Mouse: Attributes of a Gene Regulatory Network.

PubMed

Tam, Patrick P L; Fossat, Nicolas; Wilkie, Emilie; Loebel, David A F; Ip, Chi Kin; Ramialison, Mirana

2016-01-01

The embryonic head is the first major body part to be constructed during embryogenesis. The allocation and the assembly of the progenitor tissues, which start at gastrulation, are accompanied by the spatiotemporal activity of transcription factors and signaling pathways that drives lineage specification, germ layer formation, and cell/tissue movement. The morphogenesis, regionalization, and patterning of the brain and craniofacial structures rely on the function of LIM-domain, homeodomain, and basic helix-loop-helix transcription factors. These factors constitute the central nodes of a gene regulatory network (GRN) which encompasses and intersects with signaling pathways involved with head formation. It is predicted that the functional output of this "head GRN" impacts on cellular function and cell-cell interactions that are essential for lineage differentiation and tissue modeling, which are key processes underpinning the formation of the head. © 2016 Elsevier Inc. All rights reserved.

Mosaicism of a TCOF1 mutation in an individual clinically unaffected with Treacher Collins syndrome.

PubMed

Shoo, Brenda A; McPherson, Elizabeth; Jabs, Ethylin Wang

2004-04-01

Treacher Collins syndrome (TCS) or mandibulofacial dysostosis is an autosomal dominant disorder of craniofacial development with 60% of its cases arising de novo. Other modes of inheritance such as autosomal recessive, gonadal mosaicism, and chromosomal rearrangement have also been proposed. This syndrome can result from TCOF1 gene mutations. In this study we identified a TCOF1 1408delAG heterozygous mutation in a patient with the clinical diagnosis of TCS. This same mutation was found in the clinically unaffected mother's leukocytes, hair root bulbs, buccal mucosa, urine, and stool. The mother has a clinically unaffected child and the maternal grandparents do not have the mutation. Because the mother has the mutation in cells derived from all three germ layers, we suspected the mutation was nonpenetrant. However, we could not detect the mutation in her skin fibroblasts, suggesting she is mosaic secondary to cell type specific selection. Copyright 2003 Wiley-Liss, Inc.

Integration-free induced pluripotent stem cells derived from a patient with autosomal recessive Alport syndrome (ARAS).

PubMed

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS).

PubMed

Kuebler, Bernd; Aran, Begoña; Miquel-Serra, Laia; Muñoz, Yolanda; Ars, Elisabet; Bullich, Gemma; Furlano, Monica; Torra, Roser; Marti, Merce; Veiga, Anna; Raya, Angel

2017-12-01

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Derivation of an induced pluripotent stem cell line (MUSIi004-A) from dermal fibroblasts of a 48-year-old spinocerebellar ataxia type 3 patient.

PubMed

Ritthaphai, Alisa; Wattanapanitch, Methichit; Pithukpakorn, Manop; Heepchantree, Worapa; Soi-Ampornkul, Rungtip; Mahaisavariya, Panchalee; Triwongwaranat, Daranporn; Pattanapanyasat, Kovit; Vatanashevanopakorn, Chinnavuth

2018-05-21

Dermal fibroblasts were obtained from a 48-year-old female patient with spinocerebellar ataxia type 3 (SCA3). Fibroblasts were reprogrammed by nucleofection with episomal plasmids, carrying L-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1 and shRNA against p53. The SCA3 patient-specific iPSC line, MUSIi004-A, was characterized by immunofluorescence staining to verify the expression of pluripotent markers. The iPSC line exhibited an ability to differentiate into three germ layers by embryoid body (EB) formation. Karyotypic analysis of the MUSIi004-A line was normal. The mutant allele was still present in the iPSC line. This iPSC line represents a useful tool for studying neurodegeneration in SCA3. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Intratubular transplantation as a strategy for establishing animal models of testicular germ cell tumours

PubMed Central

Li, Yunmin; Kido, Tatsuo; Luo, Jinping; Fukuda, Michiko; Dobrinski, Ina; Lau, Yun-Fai Chris

2008-01-01

Testicular germ cell tumours (TGCTs) are prevalent cancers among young men. Currently, there is no reliable animal model for TGCTs. To establish such animal models, we have explored the possibility of intratubular testicular transplantation as means to deliver tumour cells into the seminiferous tubules of host animals. Our results demonstrated that transplanted cells could effectively populate the testis of a recipient mouse and develop into TGCTs. In addition, the donor cells could be transfected with a specific transgene before transplantation, thereby providing an approach to evaluate the specific effects of gene functions in the oncogenic processes. Hence, depending on selection of specific donor cells or mixtures of donor cells, transplantation models of TGCTs could be significant for studies on the pathogenesis, diagnosis and therapies of such a prevalent and important cancer in men. PMID:18808526

Differential proliferation and metabolic activity of Sertoli cells in the testes of broiler and layer breeder chickens.

PubMed

Faure, Mélanie; Guibert, Edith; Crochet, Sabine; Chartrin, Pascal; Brillard, Jean-Pierre; Collin, Anne; Froment, Pascal

2017-07-01

Decades of genetic selection have generated 2 different, highly specialized types of chickens in which 1 type, known as the layer-type chicken, expresses high laying performance while the other type, known as the broiler-type chicken, is dedicated to the production of fast-growing birds. Selected lines for the latter type often express disorders in their reproductive performance including early sexual maturation and accelerated, non-reversible seasonal decline of their semen production and mating behavior. The aim of the present study was to characterize some metabolic markers of the Sertoli cell populations. Sertoli cells are somatic cells known to support, coordinate, nourish, and protect the germ cell populations from onset to the end of their meiotic process. Comparisons of gonadal development between males of the 2 genetic types taken at their pre-pubertal period indicated that the testes of layer-type chickens are significantly less developed than in broiler-type males taken at the same age. In addition, cultures of purified Sertoli cells from the 2 types revealed in vitro a higher proliferative capacity when issued from layer compared to broiler-type chickens. This was associated with a higher expression of the genes involved in the beta-oxidation of fatty acids (CPT1; PPARβ) as well as a 4-fold increase in the Lactate Dehydrogenase-A expression and activity. In contrast, Sertoli cells from broiler-type chickens presented an elevated activity of citrate synthase and mitochondria, suggesting a better efficacy of aerobic metabolism in Sertoli cells from broiler compared to layer-type chickens. Moreover, the testis from broiler-type chickens seems to be more sensitive to oxidative stress due to the lower global antioxidant capacity compared to layer-type chickens.In conclusion, these results suggest that the metabolic activity of testicular tissues is different in the layer and broiler breeder chickens. The aerobic metabolism more prevalent in broiler-type chickens could be a factor to reduce the male fertility such as germ cell quality. © 2017 Poultry Science Association Inc.

Nanoscale biophysical properties of the cell surface galactosaminogalactan from the fungal pathogen Aspergillus fumigatus

NASA Astrophysics Data System (ADS)

Beaussart, Audrey; El-Kirat-Chatel, Sofiane; Fontaine, Thierry; Latgé, Jean-Paul; Dufrêne, Yves F.

2015-09-01

Many fungal pathogens produce cell surface polysaccharides that play essential roles in host-pathogen interactions. In Aspergillus fumigatus, the newly discovered polysaccharide galactosaminogalactan (GAG) mediates adherence to a variety of substrates through molecular mechanisms that are poorly understood. Here we use atomic force microscopy to unravel the localization and adhesion of GAG on living fungal cells. Using single-molecule imaging with tips bearing anti-GAG antibodies, we found that GAG is massively exposed on wild-type (WT) germ tubes, consistent with the notion that this glycopolymer is secreted by the mycelium of A. fumigatus, while it is lacking on WT resting conidia and on germ tubes from a mutant (Δuge3) deficient in GAG. Imaging germ tubes with tips bearing anti-β-glucan antibodies shows that exposure of β-glucan is strongly increased in the Δuge3 mutant, indicating that this polysaccharide is masked by GAG during hyphal growth. Single-cell force measurements show that expression of GAG on germ tubes promotes specific adhesion to pneumocytes and non-specific adhesion to hydrophobic substrates. These results provide a molecular foundation for the multifunctional adhesion properties of GAG, thus suggesting it could be used as a potential target in anti-adhesion therapy and immunotherapy. Our methodology represents a powerful approach for characterizing the nanoscale organization and adhesion of cell wall polysaccharides during fungal morphogenesis, thereby contributing to increase our understanding of their role in biofilm formation and immune responses.

A practical guide for evaluating gonadal germ cell tumor predisposition in differences of sex development.

PubMed

Pyle, Louise C; Nathanson, Katherine L

2017-06-01

Differences of Sex Development (DSD) includes a wide spectrum of etiologies and phenotypes. A subset of individuals with DSDs are predisposed to gonadal germ cell tumor (GCT). In this setting, GCT risk varies widely, depending on the DSD molecular etiology and penetrance. Prognostication based on molecular diagnosis remains challenging, as natural history data specific to recently identified molecular causes of DSD is lacking. In this review, we provide a framework for the clinical geneticist to consider GCT tumor risk in the patient with DSD. We discuss germ cell development and etiology of GCT growth, along with parameters to consider when recommending prophylactic gonadectomy including fertility, hormonal output, and malignant GTC treatment outcomes. Shortly after the 2006 reorganization of DSD nomenclature, literature reviews of natural history publications stratified GCT risk by a chromosomal, pathological, and hormonal taxonomy. Our 2017 literature review reveals a larger body of publications. However, the broad DSD GCT risk stratification within the 2006 taxonomy remains stable. We discuss precise GCT risk assessment for specific diagnoses, including androgen insensitivity, Smith-Lemli-Opitz, and 46,XY with MAP3K1 mutations and gonadal dysgenesis, as examples. We also examine the GCT risk in non-DSD syndromes, in addition to the cancer risks in DSD patients with dimorphic gonads and genitalia. This review is intended to provide a nuanced assessment of relative germ cell tumor risk in the DSD patient, including modern precise molecular diagnosis, for use by the clinical geneticist. © 2017 Wiley Periodicals, Inc.

Why do primordial germ cells migrate through an embryo and what does it mean for biological evolution?

PubMed

Olovnikov, A M

2013-10-01

An explanation of the role of primordial germ cell (PGC) migration during embryogenesis is proposed. According to the hypothesis, various PGCs during their migrations through an early embryo are contacting with anlagen of organs and acquiring nonidentical organ specificities. An individual PGC gets such an organ specificity, which corresponds to specificity of the first anlage with which this PGC has the first contact. As a result, the cellular descendants of PGCs (oocytes or spermatocytes) will express nonidentical organ-specific receptors, hence becoming functionally heterogeneous. Therefore, each clone of germ cells becomes capable of recognizing specifically the molecular signals that correspond only to "its" organ of the body. Such signals are produced by the body's organ when it functions in an extreme mode. Signals from the "exercising" organ of the body are delivered to the gonad only via the brain retransmitter, which is composed of neurons grouped as virtual organs of a homunculus. Homunculi are so-called somatotopic maps of the skeletomotor and other parts of the body represented in the brain. Signals, as complexes of regulatory RNAs and proteins, are transported from the "exercising" organ of the body to the corresponding virtual organ of the homunculus where they are processed and then forwarded to the gonad. The organ-specific signal will be selectively recognized by certain gametocytes according to their organ specificity, and then it will initiate the directed epimutation in the gametocyte genome. The nonrandomness of the gene order in chromosomes, that is the synteny and genetic map, is controlled by the so-called creatron that consolidates the soma and germline into a united system, providing the possibility of evolutionary responses of an organism to environmental influences.

Growth advantage of Streptococcus thermophilus over Lactobacillus bulgaricus in vitro and in the gastrointestinal tract of gnotobiotic rats.

PubMed

Ben-Yahia, L; Mayeur, C; Rul, F; Thomas, M

2012-09-01

The yoghurt bacteria, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, are alleged to have beneficial effects on human health. The objective of this study was to characterise growth, biochemical activity and competitive behaviour of these two bacteria in vitro and in vivo. S. thermophilus LMD-9 and L. bulgaricus ATCC 11842 growth and lactate production were monitored in different media and in the gastrointestinal tract (GIT) of germ-free rats. In vitro, particularly in milk, S. thermophilus had a selective growth advantage over L. bulgaricus. The GIT of germ-free rats not supplemented with lactose was colonised by S. thermophilus but not by L. bulgaricus. Both bacteria were able to colonise the GIT of germ-free rats supplemented with 45 g/l lactose in their drinking water. However, if germ-free rats were inoculated with a mixture of the two bacteria and were supplemented with lactose, S. thermophilus rapidly and extensively colonised the GIT (1010 cfu/g faeces) at the expense of L. bulgaricus, which remained in most cases at levels <102 cfu/g faeces. S. thermophilus specifically produced L-lactate, while L. bulgaricus produced only D-lactate, both in vitro and in vivo. S. thermophilus showed competitive and growth advantage over L. bulgaricus in vitro as well as in vivo in the GIT of germ-free rats and, accordingly, L-lactate was the main lactate isomer produced.

Organization of cytokeratin cytoskeleton and germ plasm in the vegetal cortex of Xenopus laevis oocytes depends on coding and non-coding RNAs: Three-dimensional and ultrastructural analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kloc, Malgorzata; Bilinski, Szczepan; Dougherty, Matthew T.

2007-05-01

Recent studies discovered a novel structural role of RNA in maintaining the integrity of the mitotic spindle and cellular cytoskeleton. In Xenopus laevis, non-coding Xlsirts and coding VegT RNAs play a structural role in anchoring localized RNAs, maintaining the organization of the cytokeratin cytoskeleton and germinal granules in the oocyte vegetal cortex and in subsequent development of the germline in the embryo. We studied the ultrastructural effects of antisense oligonucleotide driven ablation of Xlsirts and VegT RNAs on the organization of the cytokeratin, germ plasm and other components of the vegetal cortex. We developed a novel method to immunolabel andmore » visualize cytokeratin at the electron microscopy level, which allowed us to reconstruct the ultrastructural organization of the cytokeratin network relative to the components of the vegetal cortex in Xenopus oocytes. The removal of Xlsirts and VegT RNAs not only disrupts the cytokeratin cytoskeleton but also has a profound transcript-specific effect on the anchoring and distribution of germ plasm islands and their germinal granules and the arrangement of yolk platelets within the vegetal cortex. We suggest that the cytokeratin cytoskeleton plays a role in anchoring of germ plasm islands within the vegetal cortex and germinal granules within the germ plasm islands.« less

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin

PubMed Central

Oulhen, Nathalie; Wessel, Gary M.

2016-01-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3′UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. PMID:27424271

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

PubMed

Oulhen, Nathalie; Wessel, Gary M

2016-10-01

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of mitosis-meiosis transition by the ubiquitin ligase β-TrCP in male germ cells.

PubMed

Nakagawa, Tadashi; Zhang, Teng; Kushi, Ryo; Nakano, Seiji; Endo, Takahiro; Nakagawa, Makiko; Yanagihara, Noriko; Zarkower, David; Nakayama, Keiko

2017-11-15

The mitosis-meiosis transition is essential for spermatogenesis. Specific and timely downregulation of the transcription factor DMRT1, and consequent induction of Stra8 expression, is required for this process in mammals, but the molecular mechanism has remained unclear. Here, we show that β-TrCP, the substrate recognition component of an E3 ubiquitin ligase complex, targets DMRT1 for degradation and thereby controls the mitosis-meiosis transition in mouse male germ cells. Conditional inactivation of β-TrCP2 in male germ cells of β-TrCP1 knockout mice resulted in sterility due to a lack of mature sperm. The β-TrCP-deficient male germ cells did not enter meiosis, but instead underwent apoptosis. The induction of Stra8 expression was also attenuated in association with the accumulation of DMRT1 at the Stra8 promoter in β-TrCP-deficient testes. DMRT1 contains a consensus β-TrCP degron sequence that was found to bind β-TrCP. Overexpression of β-TrCP induced the ubiquitylation and degradation of DMRT1. Heterozygous deletion of Dmrt1 in β-TrCP-deficient spermatogonia increased meiotic cells with a concomitant reduction of apoptosis. Collectively, our data indicate that β-TrCP regulates the transition from mitosis to meiosis in male germ cells by targeting DMRT1 for degradation. © 2017. Published by The Company of Biologists Ltd.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

PubMed Central

Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

2015-01-01

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

Smad2 and Smad3 have differential sensitivity in relaying TGFβ signaling and inversely regulate early lineage specification

PubMed Central

Liu, Ling; Liu, Xu; Ren, Xudong; Tian, Yue; Chen, Zhenyu; Xu, Xiangjie; Du, Yanhua; Jiang, Cizhong; Fang, Yujiang; Liu, Zhongliang; Fan, Beibei; Zhang, Quanbin; Jin, Guohua; Yang, Xiao; Zhang, Xiaoqing

2016-01-01

The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity and differential transcriptional activity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events. PMID:26905010

Smad2 and Smad3 have differential sensitivity in relaying TGFβ signaling and inversely regulate early lineage specification.

PubMed

Liu, Ling; Liu, Xu; Ren, Xudong; Tian, Yue; Chen, Zhenyu; Xu, Xiangjie; Du, Yanhua; Jiang, Cizhong; Fang, Yujiang; Liu, Zhongliang; Fan, Beibei; Zhang, Quanbin; Jin, Guohua; Yang, Xiao; Zhang, Xiaoqing

2016-02-24

The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity and differential transcriptional activity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

PubMed

Sułkowski, Maciej; Konieczny, Paweł; Chlebanowska, Paula; Majka, Marcin

2018-01-09

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase ( HSV-TK ). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Medaka embryonic stem cells are capable of generating entire organs and embryo-like miniatures.

PubMed

Hong, Ni; He, Bei Ping; Schartl, Manfred; Hong, Yunhan

2013-03-01

Embryonic stem (ES) cells have the potency to produce many cell types of the embryo and adult body. Upon transplantation into early host embryos, ES cells are able to differentiate into various specialized cells and contribute to host tissues and organs of all germ layers. Here we present data in the fish medaka (Oryzias latipes) that ES cells have a novel ability to form extra organs and even embryo-like miniatures. Upon transplantation as individual cells according to the standard procedure, ES cells distributed widely to various organ systems of 3 germ layers. Upon transplantation as aggregates, ES cells were able to form extra organs, including the hematopoietic organ and contracting heart. We show that localized ES cell transplantation often led to the formation of extra axes that comprised essentially of either host cells or donor ES cells. These extra axes were associated with the head region of the embryo proper or formed at ectopic sites on the yolk sac. Surprisingly, certain ectopic axes were even capable of forming embryo-like miniatures. We conclude that ES cells have the ability to form entire organs and even embryo-like miniatures under proper environmental conditions. This finding points to a new possibility to generate ES cell-derived axes and organs.

Retinoic Acid Signalling and the Control of Meiotic Entry in the Human Fetal Gonad

PubMed Central

Kinnell, Hazel L.; Anderson, Richard A.; Saunders, Philippa T. K.

2011-01-01

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8–9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis. PMID:21674038

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

PubMed Central

Lee, Janet; Baek, Jeong-Hwa; Choi, Kyu-Sil; Kim, Hyun-Soo; Park, Hye-Young; Ha, Geun-Hyoung; Park, Ho; Lee, Kyo-Won; Lee, Chang Geun; Yang, Dong-Yun; Moon, Hyo Eun; Paek, Sun Ha; Lee, Chang-Woo

2013-01-01

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs. PMID:23324348

Modeling to Optimize Terminal Stem Cell Differentiation

PubMed Central

Gallicano, G. Ian

2013-01-01

Embryonic stem cell (ESC), iPCs, and adult stem cells (ASCs) all are among the most promising potential treatments for heart failure, spinal cord injury, neurodegenerative diseases, and diabetes. However, considerable uncertainty in the production of ESC-derived terminally differentiated cell types has limited the efficiency of their development. To address this uncertainty, we and other investigators have begun to employ a comprehensive statistical model of ESC differentiation for determining the role of intracellular pathways (e.g., STAT3) in ESC differentiation and determination of germ layer fate. The approach discussed here applies the Baysian statistical model to cell/developmental biology combining traditional flow cytometry methodology and specific morphological observations with advanced statistical and probabilistic modeling and experimental design. The final result of this study is a unique tool and model that enhances the understanding of how and when specific cell fates are determined during differentiation. This model provides a guideline for increasing the production efficiency of therapeutically viable ESCs/iPSCs/ASC derived neurons or any other cell type and will eventually lead to advances in stem cell therapy. PMID:24278782

GPR30, the Non-Classical Membrane G Protein Related Estrogen Receptor, Is Overexpressed in Human Seminoma and Promotes Seminoma Cell Proliferation

PubMed Central

Chevalier, Nicolas; Vega, Aurélie; Bouskine, Adil; Siddeek, Bénazir; Michiels, Jean-François; Chevallier, Daniel; Fénichel, Patrick

2012-01-01

Background Testicular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs) with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A) are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30) mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium). The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation. Results We report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells) and germ cells (spermatogonia and spermatocytes). GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist) and by siRNA invalidation. Conclusion These results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for seminomas. PMID:22496838

Identification of rat serum alkaline phosphatase isoenzyme by means of wheat germ agglutinin.

PubMed

Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

1997-01-01

Wheat germ agglutinin (WGA) precipitates bone type serum alkaline phosphatase (sALP) isoenzyme specifically. The precipitates are composed of the macromolecules of WGA and "bone type sALP" (WGA-ALP complex). In order to use bone type sALP as a marker in polyacrylamide gel electrophoresis (PAGE), a method to separate "bone type sALP" from the "WGA-ALP complex" was established by using N-acetyl-D-glucosamine (GlcNAc)-Sepharose 6E column chromatography. It was concluded that this method is useful for clinical examination in the rat.

A demonstration of the H3 trimethylation ChIP-seq analysis of galline follicular mesenchymal cells and male germ cells.

PubMed

Chokeshaiusaha, Kaj; Puthier, Denis; Nguyen, Catherine; Sananmuang, Thanida

2018-06-01

Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions (Fold-change≥2, FDR≤0.01) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 ( CRABP1 ), growth differentiation factor 10 ( GDF10 ), and gremlin 1 ( GREM1 ) gene explorations. The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes- CRABP1 , GDF10 , and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

Intestinal microbiota modulates gluten-induced immunopathology in humanized mice.

PubMed

Galipeau, Heather J; McCarville, Justin L; Huebener, Sina; Litwin, Owen; Meisel, Marlies; Jabri, Bana; Sanz, Yolanda; Murray, Joseph A; Jordana, Manel; Alaedini, Armin; Chirdo, Fernando G; Verdu, Elena F

2015-11-01

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

GPCRs Direct Germline Development and Somatic Gonad Function in Planarians

PubMed Central

Saberi, Amir; Beets, Isabel; Schoofs, Liliane; Newmark, Phillip A.

2016-01-01

Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development. PMID:27163480

GPCRs Direct Germline Development and Somatic Gonad Function in Planarians.

PubMed

Saberi, Amir; Jamal, Ayana; Beets, Isabel; Schoofs, Liliane; Newmark, Phillip A

2016-05-01

Planarians display remarkable plasticity in maintenance of their germline, with the ability to develop or dismantle reproductive tissues in response to systemic and environmental cues. Here, we investigated the role of G protein-coupled receptors (GPCRs) in this dynamic germline regulation. By genome-enabled receptor mining, we identified 566 putative planarian GPCRs and classified them into conserved and phylum-specific subfamilies. We performed a functional screen to identify NPYR-1 as the cognate receptor for NPY-8, a neuropeptide required for sexual maturation and germ cell differentiation. Similar to NPY-8, knockdown of this receptor results in loss of differentiated germ cells and sexual maturity. NPYR-1 is expressed in neuroendocrine cells of the central nervous system and can be activated specifically by NPY-8 in cell-based assays. Additionally, we screened the complement of GPCRs with expression enriched in sexually reproducing planarians, and identified an orphan chemoreceptor family member, ophis, that controls differentiation of germline stem cells (GSCs). ophis is expressed in somatic cells of male and female gonads, as well as in accessory reproductive tissues. We have previously shown that somatic gonadal cells are required for male GSC specification and maintenance in planarians. However, ophis is not essential for GSC specification or maintenance and, therefore, defines a secondary role for planarian gonadal niche cells in promoting GSC differentiation. Our studies uncover the complement of planarian GPCRs and reveal previously unappreciated roles for these receptors in systemic and local (i.e., niche) regulation of germ cell development.

High-Dose Chemotherapy, Total-Body Irradiation, and Autologous Stem Cell Transplantation or Bone Marrow Transplantation in Treating Patients With Hematologic Cancer or Solid Tumors

ClinicalTrials.gov

2013-05-07

Breast Cancer; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Testicular Germ Cell Tumor; Unspecified Adult Solid Tumor, Protocol Specific; Unspecified Childhood Solid Tumor, Protocol Specific

Disrupting the male germ line to find infertility and contraception targets.

PubMed

Archambeault, Denise R; Matzuk, Martin M

2014-05-01

Genetically-manipulated mouse models have become indispensible for broadening our understanding of genes and pathways related to male germ cell development. Until suitable in vitro systems for studying spermatogenesis are perfected, in vivo models will remain the gold standard for inquiry into testicular function. Here, we discuss exciting advances that are allowing researchers faster, easier, and more customizable access to their mouse models of interest. Specifically, the trans-NIH Knockout Mouse Project (KOMP) is working to generate knockout mouse models of every gene in the mouse genome. The related Knockout Mouse Phenotyping Program (KOMP2) is performing systematic phenotypic analysis of this genome-wide collection of knockout mice, including fertility screening. Together, these programs will not only uncover new genes involved in male germ cell development but also provide the research community with the mouse models necessary for further investigations. In addition to KOMP/KOMP2, another promising development in the field of mouse models is the advent of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas technology. Utilizing 20 nucleotide guide sequences, CRISPR/Cas has the potential to introduce sequence-specific insertions, deletions, and point mutations to produce null, conditional, activated, or reporter-tagged alleles. CRISPR/Cas can also successfully target multiple genes in a single experimental step, forgoing the multiple generations of breeding traditionally required to produce mouse models with deletions, insertions, or mutations in multiple genes. In addition, CRISPR/Cas can be used to create mouse models carrying variants identical to those identified in infertile human patients, providing the opportunity to explore the effects of such mutations in an in vivo system. Both the KOMP/KOMP2 projects and the CRISPR/Cas system provide powerful, accessible genetic approaches to the study of male germ cell development in the mouse. A more complete understanding of male germ cell biology is critical for the identification of novel targets for potential non-hormonal contraceptive intervention. Copyright © 2014. Published by Elsevier Masson SAS.

Alvocidib and Oxaliplatin With or Without Fluorouracil and Leucovorin Calcium in Treating Patients With Relapsed or Refractory Germ Cell Tumors

ClinicalTrials.gov

2017-01-20

Recurrent Extragonadal Seminoma; Recurrent Malignant Extragonadal Germ Cell Tumor; Recurrent Malignant Extragonadal Non-Seminomatous Germ Cell Tumor; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Stage III Testicular Cancer; Stage IV Extragonadal Non-Seminomatous Germ Cell Tumor; Stage IV Extragonadal Seminoma; Stage IV Ovarian Germ Cell Tumor

Combination Chemotherapy in Treating Young Patients With Recurrent or Resistant Malignant Germ Cell Tumors

ClinicalTrials.gov

2017-11-14

Childhood Extracranial Germ Cell Tumor; Childhood Extragonadal Germ Cell Tumor; Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Ovarian Choriocarcinoma; Ovarian Embryonal Carcinoma; Ovarian Yolk Sac Tumor; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Testicular Choriocarcinoma; Testicular Choriocarcinoma and Embryonal Carcinoma; Testicular Choriocarcinoma and Yolk Sac Tumor; Testicular Embryonal Carcinoma; Testicular Embryonal Carcinoma and Yolk Sac Tumor; Testicular Yolk Sac Tumor

A novel germ cell-specific protein, SHIP1, forms a complex with chromatin remodeling activity during spermatogenesis.

PubMed

Choi, Eunyoung; Han, Cecil; Park, Inju; Lee, Boyeon; Jin, Sora; Choi, Heejin; Kim, Do Han; Park, Zee Yong; Eddy, Edward M; Cho, Chunghee

2008-12-12

To determine the mechanisms of spermatogenesis, it is essential to identify and characterize germ cell-specific genes. Here we describe a protein encoded by a novel germ cell-specific gene, Mm.290718/ZFP541, identified from the mouse spermatocyte UniGene library. The protein contains specific motifs and domains potentially involved in DNA binding and chromatin reorganization. An antibody against Mm.290718/ZFP541 revealed the existence of the protein in testicular spermatogenic cells (159 kDa) but not testicular and mature sperm. Immunostaining analysis of cells at various stages of spermatogenesis consistently showed that the protein is present in spermatocytes and round spermatids only. Transfection assays and immunofluorescence studies indicate that the protein is localized specifically in the nucleus. Proteomic analyses performed to explore the functional characteristics of Mm.290718/ZFP541 showed that the protein forms a unique complex. Other major components of the complex included histone deacetylase 1 (HDAC1) and heat-shock protein A2. Disappearance of Mm.290718/ZFP541 was highly correlated with hyperacetylation in spermatids during spermatogenesis, and specific domains of the protein were involved in the regulation of interactions and nuclear localization of HDAC1. Furthermore, we found that premature hyperacetylation, induced by an HDAC inhibitor, is associated with an alteration in the integrity of Mm.290718/ZFP541 in spermatogenic cells. Our results collectively suggest that the Mm.290718/ZFP541 complex is implicated in chromatin remodeling during spermatogenesis, and we provide further information on the previously unknown molecular mechanism. Consequently, we re-designate Mm.290718/ZFP541 as "SHIP1" representing spermatogenic cell HDAC-interacting protein 1.

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.

PubMed

Ren, Xingjie; Sun, Jin; Housden, Benjamin E; Hu, Yanhui; Roesel, Charles; Lin, Shuailiang; Liu, Lu-Ping; Yang, Zhihao; Mao, Decai; Sun, Lingzhu; Wu, Qujie; Ji, Jun-Yuan; Xi, Jianzhong; Mohr, Stephanie E; Xu, Jiang; Perrimon, Norbert; Ni, Jian-Quan

2013-11-19

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes.

PubMed

Young, Nathan P; Kamireddy, Anwesh; Van Nostrand, Jeanine L; Eichner, Lillian J; Shokhirev, Maxim Nikolaievich; Dayn, Yelena; Shaw, Reuben J

2016-03-01

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. © 2016 Young et al.; Published by Cold Spring Harbor Laboratory Press.

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

NASA Technical Reports Server (NTRS)

Gruppi, C. M.; Wolgemuth, D. J.

1993-01-01

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Use of pluripotent stem cells for reproductive medicine: are we there yet?

PubMed

Duggal, Galbha; Heindryckx, Björn; Deroo, Tom; De Sutter, Petra

2014-01-01

In recent years, pluripotent stem cells have demonstrated to be exciting tools to understand embryonic development, cell lineage specification, tissue generation and repair, and various other biological processes. In addition, the identification and isolation of germ line stem cells has given more insight into germ cell biology at the molecular level and into the underlying causes of infertility which was not possible earlier. The recent derivation of in vitro derived sperm and oocytes from pluripotent stem cells in the mouse model represents a major breakthrough in the field and substantiates the critical relevance of stem cells as a potential alternative resource for treating infertility. Although the past years have yielded compelling information in understanding germ cell development via in vitro stem cell assays, extended investigative research is necessary in order to derive fully functional 'artificial gametes' in a safe way for future therapeutic applications.

[Rhythmic beating cardiomyocytes derived from human embryonic germ (EG) cells in vitro].

PubMed

Hua, Jinlian; Xu, Xiaoming; Dou, Zhongying

2006-10-01

Embryonic germ (EG) cells are pluripotent cells derived from primordial germ cells (PGCs) of gonads, gonadal ridges and mesenteries, analogies of fetuses,with the ability to undergo both highly self-renewal and multiple differentiation. These cells in vitro can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cells of the body. The aim of this study is to investigate the potentiality of human EG cells differentiation into cardiomyocytes. Inducing human EG cells with the method of murine ES cells differentiation into cardiomyocytes, supplemented with 0.75%-1% DMSO, 20% NBS, 10(-7) mM RA and 20% cardiomyocytes conditioned medium. 20 heart-like (rhythmic beating cell masses were observed in vitro culture and delayed human EG cells, which beat spontaneously from 20-120 times per minute and maintained beating for 2-15 days, periodic acid's staining (PAS), Myoglobin and a-actin immunological histology positive were all positive and reacted with K+, Ca2+ and adrenalin. Relatively unorganized myofibrillar bundles or more organized sarcomeres, z-bands or a gap junction, the presence of desmosomes in a few cells of the cell masses was observed with transmision electron microscope, which initially demonstrated that these cells were cardiomyocytes. We could not get rhythmly beating cardiomyocytes with 0.75%-1% DMSO, 10-7 mM RA and 20% cardiomyocytes conditioned medium,but in which the percentage of cardiac alpha-actin immunostaining positive cells were increased. The results first demonstrated that human EG cells can differentiate into rhythmic beating cardiomyocytes in vitro and suggests that human EG cells may represent a new potent resource for cardiomyocytes transplantation therapy for myocardium infarction.

Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

PubMed Central

Renault, Andrew D.; Kunwar, Prabhat S.; Lehmann, Ruth

2010-01-01

In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells. PMID:20431117

Meioc maintains an extended meiotic prophase I in mice

PubMed Central

Soh, Y. Q. Shirleen; Godfrey, Alexander K.; de Rooij, Dirk G.; Page, David C.

2017-01-01

The meiosis-specific chromosomal events of homolog pairing, synapsis, and recombination occur over an extended meiotic prophase I that is many times longer than prophase of mitosis. Here we show that, in mice, maintenance of an extended meiotic prophase I requires the gene Meioc, a germ-cell specific factor conserved in most metazoans. In mice, Meioc is expressed in male and female germ cells upon initiation of and throughout meiotic prophase I. Mouse germ cells lacking Meioc initiate meiosis: they undergo pre-meiotic DNA replication, they express proteins involved in synapsis and recombination, and a subset of cells progress as far as the zygotene stage of prophase I. However, cells in early meiotic prophase—as early as the preleptotene stage—proceed to condense their chromosomes and assemble a spindle, as if having progressed to metaphase. Meioc-deficient spermatocytes that have initiated synapsis mis-express CYCLIN A2, which is normally expressed in mitotic spermatogonia, suggesting a failure to properly transition to a meiotic cell cycle program. MEIOC interacts with YTHDC2, and the two proteins pull-down an overlapping set of mitosis-associated transcripts. We conclude that when the meiotic chromosomal program is initiated, Meioc is simultaneously induced so as to extend meiotic prophase. Specifically, MEIOC, together with YTHDC2, promotes a meiotic (as opposed to mitotic) cell cycle program via post-transcriptional control of their target transcripts. PMID:28380054

Dynamic patterning by the Drosophila pair-rule network reconciles long-germ and short-germ segmentation

PubMed Central

2017-01-01

Drosophila segmentation is a well-established paradigm for developmental pattern formation. However, the later stages of segment patterning, regulated by the “pair-rule” genes, are still not well understood at the system level. Building on established genetic interactions, I construct a logical model of the Drosophila pair-rule system that takes into account the demonstrated stage-specific architecture of the pair-rule gene network. Simulation of this model can accurately recapitulate the observed spatiotemporal expression of the pair-rule genes, but only when the system is provided with dynamic “gap” inputs. This result suggests that dynamic shifts of pair-rule stripes are essential for segment patterning in the trunk and provides a functional role for observed posterior-to-anterior gap domain shifts that occur during cellularisation. The model also suggests revised patterning mechanisms for the parasegment boundaries and explains the aetiology of the even-skipped null mutant phenotype. Strikingly, a slightly modified version of the model is able to pattern segments in either simultaneous or sequential modes, depending only on initial conditions. This suggests that fundamentally similar mechanisms may underlie segmentation in short-germ and long-germ arthropods. PMID:28953896

Sperm 1: a POU-domain gene transiently expressed immediately before meiosis I in the male germ cell.

PubMed Central

Andersen, B; Pearse, R V; Schlegel, P N; Cichon, Z; Schonemann, M D; Bardin, C W; Rosenfeld, M G

1993-01-01

Members of the POU-domain gene family encode for transcriptional regulatory molecules that are important for terminal differentiation of several organ systems, including anterior pituitary, sensory neurons, and B lymphocytes. We have identified a POU-domain factor, referred to as sperm 1 (Sprm-1). This factor is most related to the transactivator Oct-3/4, which is expressed in the early embryo, primordial germ cells, and the egg. However, in contrast with Oct-3/4, rat Sprm-1 is selectively expressed during a 36- to 48-hr period immediately preceding meiosis I in male germ cells. Although the POU-domain of Sprm-1 is divergent from the POU-domains of Oct-1 and Oct-2, random-site-selection assay reveals that Sprm-1 preferentially binds to a specific variant of the classic octamer DNA-response element in which the optimal sequence differs from that preferred by Oct-1 and Pit-1. These data suggest that the Sprm-1 gene encodes a DNA-binding protein that may exert a regulatory function in meiotic events that are required for terminal differentiation of the male germ cell. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7902581

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

PubMed

Wu, J Y; Ribar, T J; Cummings, D E; Burton, K A; McKnight, G S; Means, A R

2000-08-01

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Germ-line epigenetic modification of the murine Avy allele by nutritional supplementation

PubMed Central

Cropley, Jennifer E.; Suter, Catherine M.; Beckman, Kenneth B.; Martin, David I. K.

2006-01-01

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine Avy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the Avy allele. We find that the somatic epigenetic state of Avy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts Avy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the Avy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mother's diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change. PMID:17101998

Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation.

PubMed

Cropley, Jennifer E; Suter, Catherine M; Beckman, Kenneth B; Martin, David I K

2006-11-14

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine A vy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the A vy allele. We find that the somatic epigenetic state of A vy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts A vy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the A vy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mother's diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change.

CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule.

PubMed Central

Latza, U.; Foss, H. D.; Dürkop, H.; Eitelbach, F.; Dieckmann, K. P.; Loy, V.; Unger, M.; Pizzolo, G.; Stein, H.

1995-01-01

The expression, serological detection, and possible functional role of the CD30 antigen in Hodgkin's disease and anaplastic large cell lymphoma is well documented. In embryonal carcinoma (EC), the expression of this cytokine receptor has been demonstrated only by immunohistology. Because the CD30 monoclonal antibody Ki-1 was found to cross-react with an unrelated molecule, we examined by in situ hybridization testicular germ cell neoplasms for the presence of CD30-specific transcripts. CD30 mRNA was detectable in the tumor cells of 9 of 9 cases of EC or mixed germ cell tumors with an EC component but in no other nonlymphoid tumors. Thus, the CD30 transcript expression pattern proved to be identical to the immunostaining pattern seen with the CD30-specific monoclonal antibody Ber-H2. By Northern blot analysis, CD30 transcripts could be demonstrated in the EC cell line Tera-2. Employing a highly sensitive second generation sandwich enzyme-linked immunosorbent assay, we could detect the soluble CD30 molecule in 8 of 8 sera from patients with a diagnosis of EC but not in 8 of 10 sera from patients with other testicular germ cell tumors. In fetal tissue, no CD30-expressing germ cells or epithelial cells could be observed. Thus, the cellularly expressed CD30 marker for testicular neoplasms of EC type. Moreover, the serum levels of soluble CD30 antigen seem to be a promising parameter for monitoring patients with EC. Images Figure 1 Figure 2 PMID:7856755

Expression of Apg-1, a member of the Hsp110 family, in the human testis and sperm.

PubMed

Nonoguchi, K; Tokuchi, H; Okuno, H; Watanabe, H; Egawa, H; Saito, K; Ogawa, O; Fujita, J

2001-06-01

Apg-1 encodes a heat shock protein belonging to the Hsp110 family and is inducible by a 32 degrees C to 39 degrees C heat shock in somatic cells. In mouse testicular germ cells Apg-1 mRNA is constitutively expressed depending on the developmental stage. As human Apg-1 has recently been identified, the expression of Apg-1 in the human testis and sperm was investigated. Expression and heat-inducibility of Apg-1 in the human testicular germ cell tumor cell line, NEC8, was analyzed. Using an antimouse Apg-1 antibody, expression of Apg-1 in the human testis and sperm was examined by western blotting after confirmation of the specificity of the antibody. The cells expressing Apg-1 in the testis were further determined by immunohistochemistry. Slight induction of Apg-1 mRNA was detected in NEC8 cells after 32 degrees C to 39 degrees C temperature shift. In the human testis, the antibody specifically recognized Apg-1, which was absent in the testis without germ cells (Sertoli-cell-only syndrome) or arrested at spermatogonia. Spermatocytes and spermatids, but not testicular somatic cells, were positively stained with the anti-Apg-1 antibody. By western blot analysis, Apg-1 was detected in the preparation enriched for sperm from normal volunteers and infertile patients, but not from azoospermia patients. Apg-1 is developmentally expressed in human testicular germ cells and sperm, suggesting its role in spermatogenesis and fertilization. Identification of substrates for Apg-1 chaperone activity will help elucidate its function.

Zinc is an essential trace element for spermatogenesis.

PubMed

Yamaguchi, Sonoko; Miura, Chiemi; Kikuchi, Kazuya; Celino, Fritzie T; Agusa, Tetsuro; Tanabe, Shinsuke; Miura, Takeshi

2009-06-30

Zinc (Zn) plays important roles in various biological activities but there is little available information regarding its functions in spermatogenesis. In our current study, we further examined the role of Zn during spermatogenesis in the Japanese eel (Anguilla japonica). Human CG (hCG) was injected into the animals to induce spermatogenesis, after which the concentration of Zn in the testis increased in tandem with the progression of spermatogenesis. Staining of testicular cells with a Zn-specific fluorescent probe revealed that Zn accumulates in germ cells, particularly in the mitochondria of spermatogonia and spermatozoa. Using an in vitro testicular organ culture system for the Japanese eel, production of a Zn deficiency by chelation with N,N,N',N'-tetrakis (2-pyridylemethyl)ethylenediamine (TPEN) caused apoptosis of the germ cells. However, this cell death was rescued by the addition of Zn to the cultures. Furthermore, an induced deficiency of Zn by TPEN chelation was found to inhibit the germ cell proliferation induced by 11-ketotestosterone (KT), a fish specific androgen, 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), the initiator of meiosis in fish, and estradiol-17beta (E2), an inducer of spermatogonial stem-cell renewal. We also investigated the effects of Zn deficiency on sperm motility and observed that TPEN treatment of eel sperm suppressed the rate and duration of their motility but that co-treatment with Zn blocked the effects of TPEN. Our present results thus suggest that Zn is an essential trace element for the maintenance of germ cells, the progression spermatogenesis, and the regulation of sperm motility.

Interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line

PubMed Central

Krentz, Anthony D.; Murphy, Mark W.; Zhang, Teng; Sarver, Aaron L.; Jain, Sanjay; Griswold, Michael D.; Bardwell, Vivian J.; Zarkower, David

2013-01-01

Dmrt1(doublesex and mab-3 related transcription factor 1) is a regulator of testis development in vertebrates that has been implicated in testicular germ cell tumors of mouse and human. In the fetal mouse testis Dmrt1 regulates germ cell pluripotency in a strain-dependent manner. Loss of Dmrt1 in 129Sv strain mice results in a >90% incidence of testicular teratomas, tumors consisting cells of multiple germ layers; by contrast, these tumors have never been observed in Dmrt1 mutants of C57BL/6J (B6) or mixed genetic backgrounds. To further investigate the interaction between Dmrt1 and genetic background we compared mRNA expression in wild type and Dmrt1 mutant fetal testes of 129Sv and B6 mice at embryonic day 15.5 (E15.5), prior to overt tumorigenesis. Loss of Dmrt1 caused misexpression of overlapping but distinct sets of mRNAs in the two strains. The mRNAs that were selectively affected included some that changed expression only in one strain or the other and some that changed in both strains but to a greater degree in one versus the other. In particular, loss of Dmrt1 in 129Sv testes caused a more severe failure to silence regulators of pluripotency than in B6 testes. A number of genes misregulated in 129Sv mutant testes also are misregulated in human testicular germ cell tumors (TGCTs), suggesting similar etiology between germ cell tumors in mouse and man. Expression profiling showed that DMRT1 also regulates pluripotency genes in the fetal ovary, although Dmrt1 mutant females do not develop teratomas. Pathway analysis indicated disruption of several signaling pathways in Dmrt1 mutant fetal testes, including Nodal, Notch, and GDNF. We used a Nanos3-cre knock-in allele to perform conditional gene targeting, testing the GDNF coreceptors Gfra1 and Ret for effects on teratoma susceptibility. Conditional deletion of Gfra1 but not Ret in fetal germ cells of animals outcrossed to 129Sv caused a modest but significant elevation in tumor incidence. Despite some variability in genetic background in these crosses, this result is consistent with previous genetic mapping of teratoma susceptibility loci to the region containing Gfra1. Using Nanos3-cre we also uncovered a strong genetic interaction between Dmrt1 and Nanos3, suggesting parallel functions for these two genes in fetal germ cells. Finally, we used chromatin immunoprecipitation (ChIP-seq) analysis to identify a number of potentially direct DMRT1 targets. This analysis suggested that DMRT1 controls pluripotency via transcriptional repression of Esrrb, Nr5a2/Lrh1, and Sox2. Given the strong evidence for involvement of DMRT1 in human TGCT, the downstream genes and pathways identified in this study provide potentially useful candidates for roles in the human disease. PMID:23473982

Imatinib Mesylate in Treating Patients With Progressive, Refractory, or Recurrent Stage II or Stage III Testicular or Ovarian Cancer

ClinicalTrials.gov

2013-01-15

Ovarian Dysgerminoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Ovarian Germ Cell Tumor; Stage II Malignant Testicular Germ Cell Tumor; Stage II Ovarian Germ Cell Tumor; Stage III Malignant Testicular Germ Cell Tumor; Stage III Ovarian Germ Cell Tumor; Testicular Seminoma

Growing up in a Bubble: Using Germ-Free Animals to Assess the Influence of the Gut Microbiota on Brain and Behavior

PubMed Central

Luczynski, Pauline; McVey Neufeld, Karen-Anne; Oriach, Clara Seira; Clarke, Gerard; Dinan, Timothy G.

2016-01-01

There is a growing recognition of the importance of the commensal intestinal microbiota in the development and later function of the central nervous system. Research using germ-free mice (mice raised without any exposure to microorganisms) has provided some of the most persuasive evidence for a role of these bacteria in gut-brain signalling. Key findings show that the microbiota is necessary for normal stress responsivity, anxiety-like behaviors, sociability, and cognition. Furthermore, the microbiota maintains central nervous system homeostasis by regulating immune function and blood brain barrier integrity. Studies have also found that the gut microbiota influences neurotransmitter, synaptic, and neurotrophic signalling systems and neurogenesis. The principle advantage of the germ-free mouse model is in proof-of-principle studies and that a complete microbiota or defined consortiums of bacteria can be introduced at various developmental time points. However, a germ-free upbringing can induce permanent neurodevelopmental deficits that may deem the model unsuitable for specific scientific queries that do not involve early-life microbial deficiency. As such, alternatives and complementary strategies to the germ-free model are warranted and include antibiotic treatment to create microbiota-deficient animals at distinct time points across the lifespan. Increasing our understanding of the impact of the gut microbiota on brain and behavior has the potential to inform novel management strategies for stress-related gastrointestinal and neuropsychiatric disorders. PMID:26912607

Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference

PubMed Central

Bujna, Ágnes; Vilmos, Péter; Spirohn, Kerstin; Boutros, Michael; Erdélyi, Miklós

2014-01-01

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general. PMID:24896584

Heterogeneity across the dorso-ventral axis in zebrafish EVL is regulated by a novel module consisting of sox, snail1a and max genes.

PubMed

Chen, Yi-Yen; Harris, Matthew P; Levesque, Mitchell P; Nüsslein-Volhard, Christiane; Sonawane, Mahendra

2012-01-01

In vertebrates, the dorso-ventral (DV) axis is defined by the combinatorial action of localised Wnt, FGF and Nodal signalling along with the antagonizing activities of Chordin and BMP pathways. Our knowledge of the factors that may act in concert with these core pathways to regulate early embryonic patterning is far from complete. Furthermore, while all three germ layers respond to these patterning cues, it is not clear whether in zebrafish the outermost protective epithelium, the enveloping layer (EVL), is also patterned along the DV axis. Here, we have identified a transgenic line driving GFP under a crestin promoter, which specifically labels the dorsal domain of the EVL suggesting heterogeneity in the EVL across the DV axis. Our attempts to understand how the expression from this promoter fragment is regulated specifically in the dorsal domain, have unravelled potential novel players involved in early EVL and embryonic patterning. We show that along with Nodal signalling components, four proteins Sox11b, Sox19b, Snail1a and Max are involved in regulating the size of this EVL domain. However, Chordin-BMP signalling might be dispensable for the dorso-ventral patterning of the EVL. For the first time, this transgenic line unravels the heterogeneity in the EVL and will serve as an important tool in understanding the molecular basis of the DV patterning of the EVL. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Ghrelin modulates testicular germ cells apoptosis and proliferation in adult normal rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kheradmand, Arash, E-mail: arashkheradmand@yahoo.com; Dezfoulian, Omid; Alirezaei, Masoud

Highlights: Black-Right-Pointing-Pointer Spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. Black-Right-Pointing-Pointer Numerous studies have documented the direct action of ghrelin in the modulation of apoptosis in different cell types. Black-Right-Pointing-Pointer Ghrelin may be considered as a modulator of spermatogenesis in normal adult rats. Black-Right-Pointing-Pointer Ghrelin may be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors. -- Abstract: Under normal condition in the most mammals, spermatogenesis is closely associated with the balance between germ cells proliferation and apoptosis. The present study was designed to determine the effects of ghrelin treatment on in vivomore » quality and quantity expression of apoptosis and proliferation specific indices in rat testicular germ cells. Twenty eight adult normal rats were subdivided into equal control and treatment groups. Treatment group received 3 nmol of ghrelin as subcutaneous injection for 30 consecutive days or vehicle to the control animals. The rats from each group (n = 7) were killed on days 10 and 30 and their testes were taken for immunocytochemical evaluation and caspase-3 assay. Immunohistochemical analysis indicated that the accumulations of Bax and PCNA peptides are generally more prominent in spermatocytes and spermatogonia of both groups. Likewise, the mean percentage of immunoreactive spermatocytes against Bax increased (P < 0.01) in the ghrelin-treated group on day 10, while despite of 30% increment in the Bax level of spermatocytes in the treated rats on day 30, however, it was not statistically significant. During the experimental period, only a few spermatogonia represented Bax expression and the changes of Bax immunolabling cells were negligible upon ghrelin treatment. Likewise, there were immunostaining cells against Bcl-2 in each germ cell neither in the control nor in the treated animals. In fact, ghrelin balanced Bax/Bcl-2 ratio toward at increase of Bax level in the spermatocytes and therefore may stimulate apoptosis in these germ cells. In contrast, ghrelin administration significantly suppressed proliferation-associated peptide PCNA in the spermatocytes as well as spermatogonia (P < 0.05). Whereas, caspase-3 activity did not show any marked alteration during the experiment in both groups (P > 0.05). Upstream of Bax substance parallel to down-regulation of PCNA demonstrate that ghrelin may prevent massive accumulation of germ cells during normal spermatogenesis. These observations also indicate that ghrelin may be considered as a modulator of spermatogenesis in normal adult rats and could be potentially implicated for abnormal spermatogenesis in some testicular germ cell tumors.« less

Methanol exposure interferes with morphological cell movements in the Drosophila embryo and causes increased apoptosis in the CNS.

PubMed

Mellerick, Dervla M; Liu, Heather

2004-09-05

Despite the significant contributions of tissue culture and bacterial models to toxicology, whole animal models for developmental neurotoxins are limited in availability and ease of experimentation. Because Drosophila is a well understood model for embryonic development that is highly accessible, we asked whether it could be used to study methanol developmental neurotoxicity. In the presence of 4% methanol, approximately 35% of embryos die and methanol exposure leads to severe CNS defects in about half those embryos, where the longitudinal connectives are dorsally displaced and commissure formation is severely reduced. In addition, a range of morphological defects in other germ layers is seen, and cell movement is adversely affected by methanol exposure. Although we did not find any evidence to suggest that methanol exposure affects the capacity of neuroblasts to divide or induces inappropriate apoptosis in these cells, in the CNS of germ band retracted embryos, the number of apoptotic nuclei is significantly increased in methanol-exposed embryos in comparison to controls, particularly in and adjacent to the ventral midline. Apoptosis contributes significantly to methanol neurotoxicity because embryos lacking the cell death genes grim, hid, and reaper have milder CNS defects resulting from methanol exposure than wild-type embryos. Our data suggest that when neurons and glia are severely adversely affected by methanol exposure, the damaged cells are cleared by apoptosis, leading to embryonic death. Thus, the Drosophila embryo may prove useful in identifying and unraveling mechanistic aspects of developmental neurotoxicity, specifically in relation to methanol toxicity.

AiGERM: A logic programming front end for GERM

NASA Technical Reports Server (NTRS)

Hashim, Safaa H.

1990-01-01

AiGerm (Artificially Intelligent Graphical Entity Relation Modeler) is a relational data base query and programming language front end for MCC (Mission Control Center)/STP's (Space Test Program) Germ (Graphical Entity Relational Modeling) system. It is intended as an add-on component of the Germ system to be used for navigating very large networks of information. It can also function as an expert system shell for prototyping knowledge-based systems. AiGerm provides an interface between the programming language and Germ.

Loss of the Endothelial Glycocalyx Links Albuminuria and Vascular Dysfunction

PubMed Central

Ferguson, Joanne K.; Burford, James L.; Gevorgyan, Haykanush; Nakano, Daisuke; Harper, Steven J.; Bates, David O.; Peti-Peterdi, Janos

2012-01-01

Patients with albuminuria and CKD frequently have vascular dysfunction but the underlying mechanisms remain unclear. Because the endothelial surface layer, a meshwork of surface-bound and loosely adherent glycosaminoglycans and proteoglycans, modulates vascular function, its loss could contribute to both renal and systemic vascular dysfunction in proteinuric CKD. Using Munich-Wistar-Fromter (MWF) rats as a model of spontaneous albuminuric CKD, multiphoton fluorescence imaging and single-vessel physiology measurements revealed that old MWF rats exhibited widespread loss of the endothelial surface layer in parallel with defects in microvascular permeability to both water and albumin, in both continuous mesenteric microvessels and fenestrated glomerular microvessels. In contrast to young MWF rats, enzymatic disruption of the endothelial surface layer in old MWF rats resulted in neither additional loss of the layer nor additional changes in permeability. Intravenous injection of wheat germ agglutinin lectin and its adsorption onto the endothelial surface layer significantly improved glomerular albumin permeability. Taken together, these results suggest that widespread loss of the endothelial surface layer links albuminuric kidney disease with systemic vascular dysfunction, providing a potential therapeutic target for proteinuric kidney disease. PMID:22797190

[Effect on the microbicidal efficacy of formaldehyde, glutardialdehyde, peracetic acid, chloramine T (N-chloro-4-toluenesulfonamide), m-cresol, ethanol and benzyldimethyldodecacylammonium bromide by blood (model experiments for chemical disinfection of instruments)].

PubMed

Spicher, G; Peters, J

1998-02-01

In a preceding paper (Zbl. Hyg. 191 [1991] 457-477) we reported on the dependence of the microbicidal efficacy of active agents of the disinfection of instruments on the amount of coagulated blood adhering to the instruments. In the present investigation, we were interested in the dependence of the microbicidal effects on the amount of blood in the solutions of the active agents. Test areas of 2 cm2 were contaminated with 50 and 100 microliters coagulating blood, respectively, containing cells of Staphylococcus aureus as test germ. The solutions of the microbicidal agents were contaminated with heparinized blood up to a concentration of 4% immediately before starting the disinfection and 24 hours before, respectively. After a period of action lasting 1 hour at 20 degrees C, the relative number of test germs capable of multiplying (N/N0) was determined. The concentration of the microbicidal substances reducing the relative number of test germs capable to multiply to 10(-4) served for estimating the dependence of the microbicidal efficacy of the agents on the blood content of the solutions. The experimental results depended on the thickness of the layer of coagulated blood. The dependence of the efficacy of the microbicidal substances on the blood content of the solutions was the higher the thinner the blood layer was. At a thickness of the layer of the coagulated blood of 0.25 mm, a blood content of the solution of 4%, and applying it immediately after adding the blood, the concentration of glutardialdehyde had to be 1.6 times that without blood to reach the same microbicidal efficacy. When applying the solution 24 hours after adding the blood, the concentration of glutardialdehyde had to be 4.2 times that without blood. The quaternary ammonium compound reacted faster with the blood than did glutardialdehyde; the respective factors were 2.6 and 4.5. The concentration factors of chloramine T were 3.3 and 3.8. Under the conditions of the test, peracetic acid exhibited small concentration factors: 1.3 and 1.6. The microbicidal efficacy of ethanol, formaldehyde and m-cresol soap solution was not or only slightly altered by the amount of blood in the solution of the microbicidal agent.

Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice.

PubMed

Welborn, Joshua P; Davis, Matthew G; Ebers, Steven D; Stodden, Genna R; Hayashi, Kanako; Cheatwood, Joseph L; Rao, Manjeet K; MacLean, James A

2015-07-01

The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. © 2015 by the Society for the Study of Reproduction, Inc.

Rhox8 Ablation in the Sertoli Cells Using a Tissue-Specific RNAi Approach Results in Impaired Male Fertility in Mice1

PubMed Central

Welborn, Joshua P.; Davis, Matthew G.; Ebers, Steven D.; Stodden, Genna R.; Hayashi, Kanako; Cheatwood, Joseph L.; Rao, Manjeet K.; MacLean, James A.

2015-01-01

The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility. PMID:25972016

SOX2 regulates common and specific stem cell features in the CNS and endoderm derived organs.

PubMed

Hagey, Daniel W; Klum, Susanne; Kurtsdotter, Idha; Zaouter, Cecile; Topcic, Danijal; Andersson, Olov; Bergsland, Maria; Muhr, Jonas

2018-02-01

Stem cells are defined by their capacities to self-renew and generate progeny of multiple lineages. The transcription factor SOX2 has key roles in the regulation of stem cell characteristics, but whether SOX2 achieves these functions through similar mechanisms in distinct stem cell populations is not known. To address this question, we performed RNA-seq and SOX2 ChIP-seq on embryonic mouse cortex, spinal cord, stomach and lung/esophagus. We demonstrate that, although SOX2 binds a similar motif in the different cell types, its target regions are primarily cell-type-specific and enriched for the distinct binding motifs of appropriately expressed interacting co-factors. Furthermore, cell-type-specific SOX2 binding in endodermal and neural cells is most often found around genes specifically expressed in the corresponding tissue. Consistent with this, we demonstrate that SOX2 target regions can act as cis-regulatory modules capable of directing reporter expression to appropriate tissues in a zebrafish reporter assay. In contrast, SOX2 binding sites found in both endodermal and neural tissues are associated with genes regulating general stem cell features, such as proliferation. Notably, we provide evidence that SOX2 regulates proliferation through conserved mechanisms and target genes in both germ layers examined. Together, these findings demonstrate how SOX2 simultaneously regulates cell-type-specific, as well as core transcriptional programs in neural and endodermal stem cells.

Vaccine Therapy Plus Biological Therapy in Treating Adults With Metastatic Solid Tumors

ClinicalTrials.gov

2013-06-19

Colorectal Cancer; Endometrial Cancer; Head and Neck Cancer; Liver Cancer; Lung Cancer; Melanoma (Skin); Pancreatic Cancer; Testicular Germ Cell Tumor; Unspecified Adult Solid Tumor, Protocol Specific

PHASE II TRIAL OF THE CYCLIN-DEPEDENT KINASE INHIBITOR PD 0332991 IN PATIENTS WITH CANCER

ClinicalTrials.gov

2016-08-24

Adult Solid Tumor; Adenocarcinoma of the Colon; Adenocarcinoma of the Rectum; Adult Central Nervous System Germ Cell Tumor; Adult Teratoma; Benign Teratoma; Estrogen Receptor-negative Breast Cancer; Estrogen Receptor-positive Breast Cancer; Familial Testicular Germ Cell Tumor; HER2-negative Breast Cancer; HER2-positive Breast Cancer; Male Breast Cancer; Ovarian Immature Teratoma; Ovarian Mature Teratoma; Ovarian Monodermal and Highly Specialized Teratoma; Progesterone Receptor-negative Breast Cancer; Progesterone Receptor-positive Breast Cancer; Recurrent Breast Cancer; Recurrent Colon Cancer; Recurrent Extragonadal Germ Cell Tumor; Recurrent Extragonadal Non-seminomatous Germ Cell Tumor; Recurrent Extragonadal Seminoma; Recurrent Malignant Testicular Germ Cell Tumor; Recurrent Melanoma; Recurrent Ovarian Germ Cell Tumor; Recurrent Rectal Cancer; Stage III Extragonadal Non-seminomatous Germ Cell Tumor; Stage III Extragonadal Seminoma; Stage III Malignant Testicular Germ Cell Tumor; Stage III Ovarian Germ Cell Tumor; Stage IV Breast Cancer; Stage IV Colon Cancer; Stage IV Extragonadal Non-seminomatous Germ Cell Tumor; Stage IV Extragonadal Seminoma; Stage IV Melanoma; Stage IV Ovarian Germ Cell Tumor; Stage IV Rectal Cancer; Testicular Immature Teratoma; Testicular Mature Teratoma

Effect of 3D Cultivation Conditions on the Differentiation of Endodermal Cells

PubMed Central

Petrakova, O. S.; Ashapkin, V. V.; Voroteliak, E. A.; Bragin, E. Y.; Shtratnikova, V. Y.; Chernioglo, E. S.; Sukhanov, Y. V.; Terskikh, V. V.; Vasiliev, A. V.

2012-01-01

Cellular therapy of endodermal organs is one of the most important issues in modern cellular biology and biotechnology. One of the most promising directions in this field is the study of the transdifferentiation abilities of cells within the same germ layer. A method for anin vitroinvestigation of the cell differentiation potential (the cell culture in a three-dimensional matrix) is described in this article. Cell cultures of postnatal salivary gland cells and postnatal liver progenitor cells were obtained; their comparative analysis under 2D and 3D cultivation conditions was carried out. Both cell types have high proliferative abilities and can be cultivated for more than 20 passages. Under 2D cultivation conditions, the cells remain in an undifferentiated state. Under 3D conditions, they undergo differentiation, which was confirmed by a lower cell proliferation and by an increase in the differentiation marker expression. Salivary gland cells can undergo hepatic and pancreatic differentiation under 3D cultivation conditions. Liver progenitor cells also acquire a pancreatic differentiation capability under conditions of 3D cultivation. Thus, postnatal salivary gland cells exhibit a considerable differentiation potential within the endodermal germ layer and can be used as a promising source of endodermal cells for the cellular therapy of liver pathologies. Cultivation of cells under 3D conditions is a useful model for thein vitroanalysis of the cell differentiation potential. PMID:23346379

Standard-Dose Combination Chemotherapy or High-Dose Combination Chemotherapy and Stem Cell Transplant in Treating Patients With Relapsed or Refractory Germ Cell Tumors

ClinicalTrials.gov

2018-06-08

Germ Cell Tumor; Teratoma; Choriocarcinoma; Germinoma; Mixed Germ Cell Tumor; Yolk Sac Tumor; Childhood Teratoma; Malignant Germ Cell Neoplasm; Extragonadal Seminoma; Non-seminomatous Germ Cell Tumor; Seminoma

Teratoma of the nervous system: A case series.

PubMed

Algahtani, Hussein; Shirah, Bader; Abdullah, Ahad; Bazaid, Abdulrahman

Teratoma is a common form of germ cell tumors composed of multiple tissues foreign to the site in which arise with a histological representation of all three germ cell layers. Intracranial teratomas are very rare. In this study, we report three cases of intracranial teratomas with an interesting clinical course, neuroradiology, and outcome. In addition, we review the literature and convey important messages to the neuroscience community regarding issues related to the management of these rare tumors. The present cases are interesting examples of intracranial teratoma in terms of location of the tumor and neuroimaging findings. Delay in surgical intervention may complicate the course of the disease with progressive enlargement of tumors and development of complication including hydrocephalus. Using endoscopic surgical techniques may emerge as the preferred intervention option as compared to other traditional methods. We recommend the establishment of a national and international registry for intracranial tumors. Copyright © 2017 Sociedad Española de Neurocirugía. Publicado por Elsevier España, S.L.U. All rights reserved.

Ovarian teratoma displaying a wide variety of tissue components in a broiler chicken (Gallus Domesticus): morphological heterogeneity of pluripotential germ cell during tumorigenesis.

PubMed

Ohfuji, S

2016-01-01

Spontaneous ovarian teratoma was found in a seven-week-old female Chunky broiler chicken that was slaughtered for food. On post-mortem inspection, a spherical tumor mass attaching to a juvenile ovary was found in the abdominal cavity. Histopathologically, the tumor was comprised of immature mesenchymal stroma and a variety of mature tissue elements of mesodermal and ectodermal origin. In addition, there were multiple indistinguishable tissue elements, which showed no malignant cytological features but were unidentifiable as to corresponding embryological layer of origin. These heterogeneous teratoma tissues consisted of a variety of glandular, cystic, duct-like, and tubular structures, some of which exhibited a lining by a mixture of both keratinizing/non-keratinizing stratified squamous epithelial cells and cuboidal/columnar epithelial cells. The ovarian tetatoma was considered a benign and congenital one. The highly diverse differentiation of the teratoma might have manifested a morphological aspect of intrinsic character of the pluripotential germ cells during tumorigenesis.

Phenotypic and Molecular Analysis of Mes-3, a Maternal-Effect Gene Required for Proliferation and Viability of the Germ Line in C. Elegans

PubMed Central

Paulsen, J. E.; Capowski, E. E.; Strome, S.

1995-01-01

mes-3 is one of four maternal-effect sterile genes that encode maternal components required for normal postembryonic development of the germ line in Caenorhabditis elegans. mes-3 mutant mothers produce sterile progeny, which contain few germ cells and no gametes. This terminal phenotype reflects two problems: reduced proliferation of the germ line and germ cell death. Both the appearance of the dying germ cells and the results of genetic tests indicate that germ cells in mes-3 animals undergo a necrotic-like death, not programmed cell death. The few germ cells that appear healthy in mes-3 worms do not differentiate into gametes, even after elimination of the signaling pathway that normally maintains the undifferentiated population of germ cells. Thus, mes-3 encodes a maternally supplied product that is required both for proliferation of the germ line and for maintenance of viable germ cells that are competent to differentiate into gametes. Cloning and molecular characterization of mes-3 revealed that it is the upstream gene in an operon. The genes in the operon display parallel expression patterns; transcripts are present throughout development and are not restricted to germ-line tissue. Both mes-3 and the downstream gene in the operon encode novel proteins. PMID:8601481

Characterization of migratory primordial germ cells in the aorta-gonad-mesonephros of a 4.5-week-old human embryo: a toolbox to evaluate in vitro early gametogenesis.

PubMed

Gomes Fernandes, Maria; Bialecka, Monika; Salvatori, Daniela C F; Chuva de Sousa Lopes, Susana M

2018-05-01

Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. Non-applicable. Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro. M.G.F. was funded by Fundação para a Ciência e Tecnologia (FCT) [SFRH/BD/78689/2011] and S.M.C.S.L. was funded by the Interuniversity Attraction Poles (IAP, P7/07) and the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). The authors declare no conflict of interest.

Physician-Initiated Stop-Smoking Program for Patients Receiving Treatment for Early-Stage Cancer

ClinicalTrials.gov

2015-10-06

Bladder Cancer; Breast Cancer; Colorectal Cancer; Head and Neck Cancer; Lung Cancer; Lymphoma; Prostate Cancer; Testicular Germ Cell Tumor; Tobacco Use Disorder; Unspecified Adult Solid Tumor, Protocol Specific

The HOX genes are expressed, in vivo, in human tooth germs: in vitro cAMP exposure of dental pulp cells results in parallel HOX network activation and neuronal differentiation.

PubMed

D'Antò, Vincenzo; Cantile, Monica; D'Armiento, Maria; Schiavo, Giulia; Spagnuolo, Gianrico; Terracciano, Luigi; Vecchione, Raffaela; Cillo, Clemente

2006-03-01

Homeobox-containing genes play a crucial role in odontogenesis. After the detection of Dlx and Msx genes in overlapping domains along maxillary and mandibular processes, a homeobox odontogenic code has been proposed to explain the interaction between different homeobox genes during dental lamina patterning. No role has so far been assigned to the Hox gene network in the homeobox odontogenic code due to studies on specific Hox genes and evolutionary considerations. Despite its involvement in early patterning during embryonal development, the HOX gene network, the most repeat-poor regions of the human genome, controls the phenotype identity of adult eukaryotic cells. Here, according to our results, the HOX gene network appears to be active in human tooth germs between 18 and 24 weeks of development. The immunohistochemical localization of specific HOX proteins mostly concerns the epithelial tooth germ compartment. Furthermore, only a few genes of the network are active in embryonal retromolar tissues, as well as in ectomesenchymal dental pulp cells (DPC) grown in vitro from adult human molar. Exposure of DPCs to cAMP induces the expression of from three to nine total HOX genes of the network in parallel with phenotype modifications with traits of neuronal differentiation. Our observations suggest that: (i) by combining its component genes, the HOX gene network determines the phenotype identity of epithelial and ectomesenchymal cells interacting in the generation of human tooth germ; (ii) cAMP treatment activates the HOX network and induces, in parallel, a neuronal-like phenotype in human primary ectomesenchymal dental pulp cells. 2005 Wiley-Liss, Inc.

Stage-dependency of apoptosis and the blood-testis barrier in the dogfish shark (Squalus acanthias): cadmium-induced changes as assessed by vital fluorescence techniques.

PubMed

McClusky, Leon M

2006-09-01

Naturally occurring heavy metals and synthetic compounds are potentially harmful for testicular function but evidence linking heavy metal exposure to reduced semen parameters is inconclusive. Elucidation of the exact stage at which the toxicant interferes with spermatogenesis is difficult because the various germ cell stages may have different sensitivities to any given toxicant, germ cell development is influenced by supporting testicular somatic cells and the presence of inter-Sertoli cell tight junctions create a blood-testis barrier, sequestering meiotic and postmeiotic germ cells in a special microenvironment. Sharks such as Squalus acanthias provide a suitable model for studying aspects of vertebrate spermatogenosis because of their unique features: spermatogenesis takes place within spermatocysts and relies mainly on Sertoli cells for somatic cell support; spermatocysts are linearly arranged in a maturational order across the diameter of the elongated testis; spermatocysts containing germ cells at different stages of development are topographically separated, resulting in visible zonation in testicular cross sections. We have used the vital dye acridine orange and a novel fluorescence staining technique to study this model to determine (1) the efficacy of these methods in assays of apoptosis and blood-testis barrier function, (2) the sensitivity of the various spermatogonial generations in Squalus to cadmium (as an illustrative spermatotoxicant) and (3) the way that cadmium might affect more mature spermatogenic stages and other physiological processes in the testis. Our results show that cadmium targets early spermatogenic stages, where it specifically activates a cell death program in susceptible (mature) spermatogonial clones, and negatively affects blood-testis barrier function. Since other parameters are relatively unaffected by cadmium, the effects of this toxicant on apoptosis are presumably process-specific and not attributable to general toxicity.

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis.

PubMed

Lin, Ying-Hung; Ke, Chih-Chun; Wang, Ya-Yun; Chen, Mei-Feng; Chen, Tsung-Ming; Ku, Wei-Chi; Chiang, Han-Sun; Yeh, Chung-Hsin

2017-01-05

According to recent estimates, 2%-15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins ( MGCRABGAPs ) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP-RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taru Sharma, G., E-mail: gts553@gmail.com; Dubey, Pawan K.; Verma, Om Prakash

Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBsmore » from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications.« less

Comparison of duplex PCR and phenotypic analysis in differentiating Candida dubliniensis from Candida albicans from oral samples.

PubMed

Sampath, Asanga; Weerasekera, Manjula; Dilhari, Ayomi; Gunasekara, Chinthika; Bulugahapitiya, Uditha; Fernando, Neluka; Samaranayake, Lakshman

2017-12-01

Candida dubliniensis shares a wide range of phenotypic characteristics with Candida albicans including a common trait called germ tube positivity. Hence, laboratory differentiation of these two species is cumbersome. Duplex PCR analyses for C. albicans and C. dubliniensis was performed directly on DNA extracted from a total of 122 germ tube positive isolates derived from 100 concentrated oral rinse samples from a random cohort of diabetics attending a clinic in Sri Lanka. These results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA of the yeasts. Performance efficacy of duplex PCR was then compared with phenotypic identification using a standard battery of phenotypic tests. Of the 122 germ tube positive isolates three were identified by duplex PCR as C. dubliniensis and the remainder as C. albicans. On the contrary, when the standard phenotypic tests, sugar assimilation and chlamydospore formation, were used to differentiate the two species 13 germ tube positive isolates were erroneously identified as C. dubliniensis. Duplex PCR was found to be rapid, sensitive and more specific than phenotypic identification methods in discriminating C. dubliniensis from C. albicans. This is also the first report on the oral carriage of C. dubliniensis in a Sri Lankan population.

DAZ Family Proteins, Key Players for Germ Cell Development

PubMed Central

Fu, Xia-Fei; Cheng, Shun-Feng; Wang, Lin-Qing; Yin, Shen; De Felici, Massimo; Shen, Wei

2015-01-01

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution. PMID:26327816

Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming.

PubMed

Eguizabal, C; Herrera, L; De Oñate, L; Montserrat, N; Hajkova, P; Izpisua Belmonte, J C

2016-09-01

Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428. © 2016 AlphaMed Press.

Stable engraftment of human microbiota into mice with a single oral gavage following antibiotic conditioning.

PubMed

Staley, Christopher; Kaiser, Thomas; Beura, Lalit K; Hamilton, Matthew J; Weingarden, Alexa R; Bobr, Aleh; Kang, Johnthomas; Masopust, David; Sadowsky, Michael J; Khoruts, Alexander

2017-08-01

Human microbiota-associated (HMA) animal models relying on germ-free recipient mice are being used to study the relationship between intestinal microbiota and human disease. However, transfer of microbiota into germ-free animals also triggers global developmental changes in the recipient intestine, which can mask disease-specific attributes of the donor material. Therefore, a simple model of replacing microbiota into a developmentally mature intestinal environment remains highly desirable. Here we report on the development of a sequential, three-course antibiotic conditioning regimen that allows sustained engraftment of intestinal microorganisms following a single oral gavage with human donor microbiota. SourceTracker, a Bayesian, OTU-based algorithm, indicated that 59.3 ± 3.0% of the fecal bacterial communities in treated mice were attributable to the donor source. This overall degree of microbiota engraftment was similar in mice conditioned with antibiotics and germ-free mice. Limited surveys of systemic and mucosal immune sites did not show evidence of immune activation following introduction of human microbiota. The antibiotic treatment protocol described here followed by a single gavage of human microbiota may provide a useful, complimentary HMA model to that established in germ-free facilities. The model has the potential for further in-depth translational investigations of microbiota in a variety of human disease states.

Primary Culture System for Germ Cells from Caenorhabditis elegans Tumorous Germline Mutants

PubMed Central

Vagasi, Alexandra S.; Rahman, Mohammad M.; Chaudhari, Snehal N.; Kipreos, Edward T.

2017-01-01

The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016. PMID:28868332

MASTL is essential for anaphase entry of proliferating primordial germ cells and establishment of female germ cells in mice

PubMed Central

Risal, Sanjiv; Zhang, Jingjing; Adhikari, Deepak; Liu, Xiaoman; Shao, Jingchen; Hu, Mengwen; Busayavalasa, Kiran; Tu, Zhaowei; Chen, Zijiang; Kaldis, Philipp; Liu, Kui

2017-01-01

In mammals, primordial germ cells (PGCs) are the embryonic cell population that serve as germ cell precursors in both females and males. During mouse embryonic development, the majority of PGCs are arrested at the G2 phase when they migrate into the hindgut at 7.75–8.75 dpc (days post coitum). It is after 9.5 dpc that the PGCs undergo proliferation with a doubling time of 12.6 h. The molecular mechanisms underlying PGC proliferation are however not well studied. In this work. Here we studied how MASTL (microtubule-associated serine/threonine kinase-like)/Greatwall kinase regulates the rapid proliferation of PGCs. We generated a mouse model where we specifically deleted Mastl in PGCs and found a significant loss of PGCs before the onset of meiosis in female PGCs. We further revealed that the deletion of Mastl in PGCs did not prevent mitotic entry, but led to a failure of the cells to proceed beyond metaphase-like stage, indicating that MASTL-mediated molecular events are indispensable for anaphase entry in PGCs. These mitotic defects further led to the death of Mastl-null PGCs by 12.5 dpc. Moreover, the defect in mitotic progression observed in the Mastl-null PGCs was rescued by simultaneous deletion of Ppp2r1a (α subunit of PP2A). Thus, our results demonstrate that MASTL, PP2A, and therefore regulated phosphatase activity have a fundamental role in establishing female germ cell population in gonads by controlling PGC proliferation during embryogenesis. PMID:28224044

Sex, stem cells and tumors in the Drosophila ovary.

PubMed

Salz, Helen K

2013-01-01

The Drosophila Sex-lethal (Sxl) gene encodes a female-specific RNA binding protein that in somatic cells globally regulates all aspects of female-specific development and behavior. Sxl also has a critical, but less well understood, role in female germ cells. Germ cells without Sxl protein can adopt a stem cell fate when housed in a normal ovary, but fail to successfully execute the self-renewal differentiation fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation, and formation of a tumor. The findings in Chau et al., (2012) identify the germline stem cell maintenance factor nanos as one of its target genes, and suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional downregulation of nanos expression. These studies provide the basis for a new model in which Sxl directly couples sexual identity with the self-renewal differentiation decision and raises several interesting questions about the genesis of the tumor phenotype.

Generating Germ-Free Drosophila to Study Gut-Microbe Interactions: Protocol to Rear Drosophila Under Axenic Conditions.

PubMed

Kietz, Christa; Pollari, Vilma; Meinander, Annika

2018-06-22

As several diseases have been linked to dysbiosis of the human intestinal microflora, manipulation of the microbiota has emerged as an exciting new strategy for potentially treating and preventing diseases. However, the human microbiota consists of a plethora of different species, and distinguishing the impact of a specific bacterial species on human health is challenging. In tackling this challenge, the fruit fly Drosophila melanogaster, with its far simpler microbial composition, has emerged as a powerful model for unraveling host-microbe interactions. To study the interplay between the resident commensal microbiome and the host, flies can be made germ-free, or axenic. To elucidate the impact of specific bacteria, axenic flies can then be re-introduced to specific microbial species. In this unit, we provide a step-by-step protocol on how to rear Drosophila melanogaster under axenic conditions and confirm the axenity of flies. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Simplified adsorption method for detection of antibodies to Candida albicans germ tubes.

PubMed Central

Ponton, J; Quindos, G; Arilla, M C; Mackenzie, D W

1994-01-01

Two modifications that simplify and shorten a method for adsorption of the antibodies against the antigens expressed on both blastospore and germ tube cell wall surfaces (methods 2 and 3) were compared with the original method of adsorption (method 1) to detect anti-Candida albicans germ tube antibodies in 154 serum specimens. Adsorption of the sera by both modified methods resulted in titers very similar to those obtained by the original method. Only 5.2% of serum specimens tested by method 2 and 5.8% of serum specimens tested by method 3 presented greater than one dilution discrepancies in the titers with respect to the titer observed by method 1. When a test based on method 2 was evaluated with sera from patients with invasive candidiasis, the best discriminatory results (sensitivity, 84.6%; specificity, 87.9%; positive predictive value, 75.9%; negative predictive value, 92.7%; efficiency, 86.9%) were obtained when a titer of > or = 1:160 was considered positive. PMID:8126184

Physiological and physiopathological aspects of connexins and communicating gap junctions in spermatogenesis

PubMed Central

Pointis, Georges; Gilleron, Jérome; Carette, Diane; Segretain, Dominique

2010-01-01

Spermatogenesis is a highly regulated process of germ cell proliferation and differentiation, starting from spermatogonia to spermatocytes and giving rise to spermatids, the future spermatozoa. In addition to endocrine regulation, testicular cell–cell interactions are essential for spermatogenesis. This precise control is mediated through paracrine/autocrine pathways, direct intercellular contacts and through intercellular communication channels, consisting of gap junctions and their constitutive proteins, the connexins. Gap junctions are localized between adjacent Leydig cells, between Sertoli cells and between Sertoli cells and specific germ cells. This review focuses on the distribution of connexins within the seminiferous epithelium, their participation in gap junction channel formation, the control of their expression and the physiological relevance of these junctions in both the Sertoli–Sertoli cell functional synchronization and the Sertoli–germ cell dialogue. In this review, we also discuss the potential implication of disrupted connexin in testis cancer, since impaired expression of connexin has been described as a typical feature of tumoral proliferation. PMID:20403873

Quantitative Differences in a Single Maternal Factor Determine Survival Probabilities among Drosophila Germ Cells.

PubMed

Slaidina, Maija; Lehmann, Ruth

2017-01-23

Germ cell death occurs in many species [1-3] and has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. We find that the decision to die is a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Geochemical Earth Reference Model (GERM)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Staudigel, H.; Albarede, F.; Shaw, H.

The Geochemical Earth Reference Model (GERM) initiative is a grass- roots effort with the goal of establishing a community consensus on a chemical characterization of the Earth, its major reservoirs, and the fluxes between them. Long term goal of GERM is a chemical reservoir characterization analogous to the geophysical effort of the Preliminary Reference Earth Model (PREM). Chemical fluxes between reservoirs are included into GERM to illuminate the long-term chemical evolution of the Earth and to characterize the Earth as a dynamic chemical system. In turn, these fluxes control geological processes and influence hydrosphere-atmosphere-climate dynamics. While these long-term goals aremore » clearly the focus of GERM, the process of establishing GERM itself is just as important as its ultimate goal. The GERM initiative is developed in an open community discussion on the World Wide Web (GERM home page is at http://www-ep.es.llnl. gov/germ/germ-home.html) that is mediated by a series of editors with responsibilities for distinct reservoirs and fluxes. Beginning with the original workshop in Lyons (March 1996) GERM is continued to be developed on the Internet, punctuated by workshops and special sessions at professional meetings. It is planned to complete the first model by mid-1997, followed by a call for papers for a February 1998 GERM conference in La Jolla, California.« less

Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

PubMed

Piao, Yulan; Hung, Sandy Shen-Chi; Lim, Shiang Y; Wong, Raymond Ching-Bong; Ko, Minoru S H

2014-07-01

Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes. ©AlphaMed Press.

An analysis of the teratagenic effects that could possibly be due to alcohol consumption by pregnant mothers.

PubMed

Chaudhuri, J D

2000-10-01

It can be concluded that alcohol is definitely harmful to the developing fetus. The effect can manifest in various ways, the most extreme of which is a condition called Fetal Alcohol Syndrome (FAS). The diagnosis of maternal alcoholism leading onto cases of FAS is difficult due to absence of accurate diagnostic tests. The diagnosis of FAS in a child is easier by a proper examination. There is no specific treatment of FAS in a child. The only management is by institution of corrective and rehabilitative measures. The exact mechanism of the teratogenic action of alcohol is not known. It is probably due to the harmful effect of alcohol on the epiblast layer of the bilaminar germ disc. In the absence of adequate knowledge regarding FAS, not much can be done to remedy the deleterious effects of alcohol. Hence, a word of advice to all pregnant women is to avoid drinking during pregnancy.

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

Imaging transcription factors dynamics with advanced fluorescence microscopy methods.

PubMed

Verneri, Paula; Romero, Juan José; De Rossi, María Cecilia; Alvarez, Yanina; Oses, Camila; Guberman, Alejandra; Levi, Valeria

2018-05-10

Pluripotent stem cells (PSCs) are capable of self-renewing and producing all cell types derived from the three germ layers in response to developmental cues, constituting an important promise for regenerative medicine. Pluripotency depends on specific transcription factors (TFs) that induce genes required to preserve the undifferentiated state and repress other genes related to differentiation. The transcription machinery and regulatory components such as TFs are recruited dynamically on their target genes making it essential exploring their dynamics in living cells to understand the transcriptional output. Non-invasive and very sensitive fluorescence microscopy methods are making it possible visualizing the dynamics of TFs in living specimens, complementing the information extracted from studies in fixed specimens and bulk assays. In this work, we briefly describe the basis of these microscopy methods and review how they contributed to our knowledge of the function of TFs relevant to embryo development and cell differentiation in a variety of systems ranging from single cells to whole organisms. Copyright © 2017. Published by Elsevier B.V.

Development of space-fertilized eggs and formation of primordial germ cells in the embryos of medaka fish

NASA Astrophysics Data System (ADS)

Ijiri, K.

In the second International Microgravity Laboratory (IML-2) mission in 1994, four small Japanese killifish (Medaka, Oryzias latipes) made a space travel of 15 days aboard a space shuttle. These four adult Medaka fish successfully mated in space for the first time among vertebrate animals. Moreover, the eggs they laid developed normally, at least in their external appearance, hatching as fry (baby fish) in space. Fish mated and laid eggs every day during the first week. Near the end of the mission most of the eggs had a well-developed body with two pigmented eyes. In total, 43 eggs were laid (detected), out of which 8 fry hatched in space, as truly `space-originated' babies. A further 30 fry hatched within 3 days after landing. This is the normal hatching rate, compared with the ground-based data. Among the 8 space-originated fry, four were killed for histological sections, and germ cells at the gonadal region were counted for each fry. Their numbers were in the range of the germ cells of the normal control fry (ground-kept samples). Thus, as embryos developed normally in their external appearance, inside the embryos the formation of primordial germ cells took place normally in space, and their migration to the genital ridges was not hindered by microgravity. The two of the remaining space-originated fry have grown up and been creating their offspring in the laboratory. This proved that the primordial germ cells formed in space were also normal from a functional point of view. The four space-travelled adult fish re-started mating and laying eggs on the 7th day after landing and continued to do so every day afterward. Fertilization rate and hatchability of these eggs were as high as the eggs laid by the laboratory-kept fish. This fact implies that in gametogenesis of adult fish, there are no specific stages of germ cells extremely susceptible to microgravity.

Treatment Option Overview (Extragonadal Germ Cell Tumors)

MedlinePlus

... Professional Extragonadal Germ Cell Tumors Treatment Extragonadal Germ Cell Tumors Treatment (PDQ®)–Patient Version General Information About Extragonadal Germ Cell Tumors Go to Health Professional Version Key Points ...

HoxBlinc RNA Recruits Set1/MLL Complexes to Activate Hox Gene Expression Patterns and Mesoderm Lineage Development.

PubMed

Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Naohiro; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

2016-01-05

Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1(+) mesoderm and then promotes hematopoietic differentiation through regulation of hoxb pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated knockdown or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1(+) precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1(+) precursors and differentiation of Flk1(+) cells into hematopoietic lineages. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

HoxBlinc RNA recruits Set1/MLL complexes to activate Hox gene expression patterns and mesoderm lineage development

PubMed Central

Deng, Changwang; Li, Ying; Zhou, Lei; Cho, Joonseok; Patel, Bhavita; Terada, Nao; Li, Yangqiu; Bungert, Jörg; Qiu, Yi; Huang, Suming

2015-01-01

Summary Trithorax proteins and long-intergenic noncoding RNAs are critical regulators of embryonic stem cell pluripotency; however, how they cooperatively regulate germ layer mesoderm specification remains elusive. We report here that HoxBlinc RNA first specifies Flk1+ mesoderm and then promotes hematopoietic differentiation through regulating hoxb gene pathways. HoxBlinc binds to the hoxb genes, recruits Setd1a/MLL1 complexes, and mediates long-range chromatin interactions to activate transcription of the hoxb genes. Depletion of HoxBlinc by shRNA-mediated KD or CRISPR-Cas9-mediated genetic deletion inhibits expression of hoxb genes and other factors regulating cardiac/hematopoietic differentiation. Reduced hoxb gene expression is accompanied by decreased recruitment of Set1/MLL1 and H3K4me3 modification, as well as by reduced chromatin loop formation. Re-expression of hoxb2-b4 genes in HoxBlinc-depleted embryoid bodies rescues Flk1+ precursors that undergo hematopoietic differentiation. Thus, HoxBlinc plays an important role in controlling hoxb transcription networks that mediate specification of mesoderm-derived Flk1+ precursors and differentiation of Flk1+ cells into hematopoietic lineages. PMID:26725110

The functional performance of microencapsulated human pancreatic islet-derived precursor cells.

PubMed

Montanucci, Pia; Pennoni, Ilaria; Pescara, Teresa; Blasi, Paolo; Bistoni, Giovanni; Basta, Giuseppe; Calafiore, Riccardo

2011-12-01

We have examined long-term cultured, human islet-derived stem/precursor cells (hIPC). Whole human islets (HI) were obtained by multi-enzymatic digestion of cadaveric donor pancreases, plated on tissue flasks, and allowed to adhere and expand for several in vitro passages, in order to obtain hIPC. We detected specific stem cell markers (Oct-4, Sox-2, Nanog, ABCG2, Klf-4, CD117) in both intact HI and hIPC. Moreover, hIPC while retaining the expression of Glut-2, Pdx-1, CK-19, and ICA-512, started re-expressing Ngn3, thereby indicating acquisition of a specific pancreatic islet beta cell-oriented phenotype identity. The intrinsic plasticity of hIPC was documented by their ability to differentiate into various germ layer-derived cell phenotypes (ie, osteocytic, adipocytic and neural), including endocrine cells associated with insulin secretory capacity. To render hIPC suitable for transplantation we have enveloped them within our highly purified, alginate-based microcapsules. Upon intraperitoneal graft in NOD/SCID mice we have observed that the microcapsules acted as three-dimensional niches favouring post-transplant hIPC differentiation and acquisition of beta cell-like functional competence. Copyright © 2011 Elsevier Ltd. All rights reserved.

RBP-Jκ-Dependent Notch Signaling Is Dispensable for Mouse Early Embryonic Development

PubMed Central

Souilhol, Céline; Cormier, Sarah; Tanigaki, Kenji; Babinet, Charles; Cohen-Tannoudji, Michel

2006-01-01

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jκ-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. PMID:16782866

RBP-Jkappa-dependent notch signaling is dispensable for mouse early embryonic development.

PubMed

Souilhol, Céline; Cormier, Sarah; Tanigaki, Kenji; Babinet, Charles; Cohen-Tannoudji, Michel

2006-07-01

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion.

Isogenic mice exhibit sexually-dimorphic DNA methylation patterns across multiple tissues.

PubMed

McCormick, Helen; Young, Paul E; Hur, Suzy S J; Booher, Keith; Chung, Hunter; Cropley, Jennifer E; Giannoulatou, Eleni; Suter, Catherine M

2017-12-13

Cytosine methylation is a stable epigenetic modification of DNA that plays an important role in both normal physiology and disease. Most diseases exhibit some degree of sexual dimorphism, but the extent to which epigenetic states are influenced by sex is understudied and poorly understood. To address this deficit we studied DNA methylation patterns across multiple reduced representation bisulphite sequencing datasets (from liver, heart, brain, muscle and spleen) derived from isogenic male and female mice. DNA methylation patterns varied significantly from tissue to tissue, as expected, but they also varied between the sexes, with thousands of sexually dimorphic loci identified. The loci affected were largely autonomous to each tissue, even within tissues derived from the same germ layer. At most loci, differences between genders were driven by females exhibiting hypermethylation relative to males; a proportion of these differences were independent of the presence of testosterone in males. Loci harbouring gender differences were clustered in ontologies related to tissue function. Our findings suggest that gender is underwritten in the epigenome in a tissue-specific and potentially sex hormone-independent manner. Gender-specific epigenetic states are likely to have important implications for understanding sexually dimorphic phenotypes in health and disease.

Extraction and demulsification of oil from wheat germ, barley germ, and rice bran using an aqueous enzymatic method

USDA-ARS?s Scientific Manuscript database

An aqueous enzymatic method was developed to extract oil from wheat germ. The parameters that influence oil yield were investigated, including wheat germ pretreatment, comparison of various industrial enzymes, pH, ratio of wheat germ to water, reaction time and demulsification. Pretreatment at 180ºC...

Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

PubMed

Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

2016-09-07

Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Biology of the Germ line in Echinoderms

PubMed Central

Wessel, Gary M.; Brayboy, Lynae; Fresques, Tara; Gustafson, Eric A.; Oulhen, Nathalie; Ramos, Isabela; Reich, Adrian; Swartz, S. Zachary; Yajima, Mamiko; Zazueta, Vanessa

2014-01-01

SUMMARY The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed. PMID:23900765

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution.

PubMed

Johnson, Andrew D; Drum, Matthew; Bachvarova, Rosemary F; Masi, Thomas; White, Mary E; Crother, Brian I

2003-01-01

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.

Ovarian Germ Cell Tumors Symptoms, Tests, Prognosis, and Stages (PDQ®)—Patient Version

Cancer.gov

Ovarian germ cell tumors form in germ (egg) cells in the ovary. Ovarian germ cell tumors usually occur in teenage girls or young women and most often affect just one ovary. They are usually cured if found and treated early. Learn about signs and symptoms, tests to diagnose, and stages of ovarian germ cell tumors.

Quantification of healthy and atretic germ cells and follicles in the developing and post-natal ovary of the South American plains vizcacha, Lagostomus maximus: evidence of continuous rise of the germinal reserve.

PubMed

Inserra, P I F; Leopardo, N P; Willis, M A; Freysselinard, A L; Vitullo, A D

2014-02-01

The female germ line in mammals is subjected to massive cell death that eliminates 60-85% of the germinal reserve by birth and continues from birth to adulthood until the exhaustion of the germinal pool. Germ cell demise occurs mainly through apoptosis by means of a biased expression in favour of pro-apoptotic members of the BCL2 gene family. By contrast, the South American plains vizcacha, Lagostomus maximus, exhibits sustained expression of the anti-apoptotic BCL2 gene throughout gestation and a low incidence of germ cell apoptosis. This led to the proposal that, in the absence of death mechanisms other than apoptosis, the female germ line should increase continuously from foetal life until after birth. In this study, we quantified all healthy germ cells and follicles in the ovaries of L. maximus from early foetal life to day 60 after birth using unbiased stereological methods and detected apoptosis by labelling with TUNEL assay. The healthy germ cell population increased continuously from early-developing ovary reaching a 50 times higher population number by the end of gestation. TUNEL-positive germ cells were <0.5% of the germ cell number, except at mid-gestation (3.62%). Mitotic proliferation, entrance into prophase I stage and primordial follicle formation occurred as overlapping processes from early pregnancy to birth. Germ cell number remained constant in early post-natal life, but a remnant population of non-follicular VASA- and PCNA-positive germ cells still persisted at post-natal day 60. L. maximus is the first mammal so far described in which female germ line develops in the absence of constitutive massive germ cell elimination.

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

PubMed Central

Western, Patrick S.; Ralli, Rachael A.; Wakeling, Stephanie I.; Lo, Camden; van den Bergen, Jocelyn A.; Miles, Denise C.; Sinclair, Andrew H.

2011-01-01

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility. PMID:21674058

An in vivo proteomic analysis of the Me31B interactome in Drosophila germ granules.

PubMed

DeHaan, Hunter; McCambridge, Aidan; Armstrong, Brittany; Cruse, Carlie; Solanki, Dhruv; Trinidad, Jonathan C; Arkov, Alexey L; Gao, Ming

2017-11-01

Drosophila Me31B is a conserved protein of germ granules, ribonucleoprotein complexes essential for germ cell development. Me31B post-transcriptionally regulates mRNAs by interacting with other germ granule proteins. However, a Me31B interactome is lacking. Here, we use an in vivo proteomics approach to show that the Me31B interactome contains polypeptides from four functional groups: RNA regulatory proteins, glycolytic enzymes, cytoskeleton/motor proteins, and germ plasm components. We further show that Me31B likely colocalizes with the germ plasm components Tudor (Tud), Vasa, and Aubergine in the nuage and germ plasm and provide evidence that Me31B may directly bind to Tud in a symmetrically dimethylated arginine-dependent manner. Our study supports the role of Me31B in RNA regulation and suggests its novel roles in germ granule assembly and function. © 2017 Federation of European Biochemical Societies.

Intratubular germ cell neoplasia of the human testis: heterogeneous protein expression and relation to invasive potential

PubMed Central

Mitchell, Rod T; Camacho-Moll, Maria; Macdonald, Joni; Anderson, Richard A; Kelnar, Christopher JH; O’Donnell, Marie; Sharpe, Richard M; Smith, Lee B; Grigor, Ken M; Wallace, W Hamish B; Stoop, Hans; Wolffenbuttel, Katja P; Donat, Roland

2014-01-01

Testicular germ cell cancer develops from pre-malignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4+/ MAGEA4−) into pre-spermatogonia (OCT4−/MAGEA4+). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesised that cells expressing an immature (OCT4+/MAGEA4−) germ cell profile would exhibit an increased proliferation rate compared to those with a mature profile (OCT4+/ MAGEA4+). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with pre-invasive disease, seminoma and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4+/MAGEA4- cells showed a significantly increased rate of proliferation compared with the OCT4+/MAGEA4+ population (12.8 v 3.4%, p<0.0001) irrespective of histological tumour type, reflected in the predominance of OCT4+/MAGEA4− cells in the invasive tumour component. Surprisingly, OCT4+/MAGEA4− cells in patients with pre-invasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 v 10.2 v 7.2%, p<0.05 respectively). In conclusion, this study has demonstrated that OCT4+/MAGEA4− cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas. PMID:24457464

Skin-derived mesenchymal stem cells help restore function to ovaries in a premature ovarian failure mouse model.

PubMed

Lai, Dongmei; Wang, Fangyuan; Dong, Zhangli; Zhang, Qiuwan

2014-01-01

Skin-derived mesenchymal stem cells (SMSCs) can differentiate into the three embryonic germ layers. For this reason, they are considered a powerful tool for therapeutic cloning and offer new possibilities for tissue therapy. Recent studies showed that skin-derived stem cells can differentiate into cells expressing germ-cell specific markers in vitro and form oocytes in vivo. The idea that SMSCs may be suitable for the treatment of intractable diseases or traumatic tissue damage has attracted attention. To determine the ability of SMSCs to reactivate injured ovaries, a mouse model with ovaries damaged by busulfan and cyclophosphamide was developed and is described here. Female skin-derived mesenchymal stem cells (F-SMSCs) and male skin-derived mesenchymal stem cells (M-SMSCs) from red fluorescence protein (RFP) transgenic adult mice were used to investigate the restorative effects of SMSCs on ovarian function. Significant increases in total body weight and the weight of reproductive organs were observed in the treated animals. Both F-SMSCs and M-SMSCs were shown to be capable of partially restoring fertility in chemotherapy-treated females. Immunostaining with RFP and anti-Müllerian hormone (AMH) antibodies demonstrated that the grafted SMSCs survived, migrated to the recipient ovaries. After SMSCs were administered to the treated mice, real-time PCR showed that the expression levels of pro-inflammatory cytokines TNF-α, TGF-β, IL-8, IL-6, IL-1β, and IFNγ were significantly lower in the ovaries than in the untreated controls. Consistent with this observation, expression of oogenesis marker genes Nobox, Nanos3, and Lhx8 increased in ovaries of SMSCs-treated mice. These findings suggest that SMSCs may play a role within the ovarian follicle microenvironment in restoring the function of damaged ovaries and could be useful in reproductive health.

Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

PubMed Central

Harris, Rebecca M.; Weiss, Jeffrey

2011-01-01

The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

PubMed

Kero, Darko; Vukojevic, Katarina; Stazic, Petra; Sundov, Danijela; Mardesic Brakus, Snjezana; Saraga-Babic, Mirna

2017-10-02

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19 INK4d . p19 INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19 INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19 INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19 INK4d throughout the investigated period indicates that p19 INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19 INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

A Comparative Proteome Profile of Female Mouse Gonads Suggests a Tight Link between the Electron Transport Chain and Meiosis Initiation.

PubMed

Shen, Cong; Li, Mingrui; Zhang, Pan; Guo, Yueshuai; Zhang, Hao; Zheng, Bo; Teng, Hui; Zhou, Tao; Guo, Xuejiang; Huo, Ran

2018-01-01

Generation of haploid gametes by meiosis is a unique property of germ cells and is critical for sexual reproduction. Leaving mitosis and entering meiosis is a key step in germ cell development. Several inducers or intrinsic genes are known to be important for meiotic initiation, but the regulation of meiotic initiation, especially at the protein level, is still not well understood. We constructed a comparative proteome profile of female mouse fetal gonads at specific time points (11.5, 12.5, and 13.5 days post coitum), spanning a critical window for initiation of meiosis in female germ cells. We identified 3666 proteins, of which 473 were differentially expressed. Further bioinformatics analysis showed that these differentially expressed proteins were enriched in the mitochondria, especially in the electron transport chain and, notably, 9 proteins in electron transport chain Complex I were differentially expressed. We disrupted the mitochondrial electron transport chain function by adding the complex I inhibitor, rotenone to 11.5 days post coitum female gonads cultured in vitro. This treatment resulted in a decreased proportion of meiotic germ cells, as assessed by staining for histone γH2AX. Rotenone treatment also caused decreased ATP levels, increased reactive oxygen species levels and failure of the germ cells to undergo premeiotic DNA replication. These effects were partially rescued by adding Coenzyme Q10. Taken together, our results suggested that a functional electron transport chain is important for meiosis initiation. Our characterization of the quantitative proteome of female gonads provides an inventory of proteins, useful for understanding the mechanisms of meiosis initiation and female fertility. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Defatted wheat germ application: Influence on cookies' properties with regard to its particle size and dough moisture content.

PubMed

Petrović, Jovana; Rakić, Dušan; Fišteš, Aleksandar; Pajin, Biljana; Lončarević, Ivana; Tomović, Vladimir; Zarić, Danica

2017-10-01

The introduction of agro-food industry by-products rich in bioactive compounds represents major challenge in food industry sector. The influence of wheat germ particle size (<150 µm, 150-1000 µm, and 800-2000 µm), wheat germ content (5, 10, and 15%), and dough moisture content (20, 22, and 24%) on chemical, textural, and sensory characteristics of cookies was investigated using the Box-Behnken experimental design. The substitution of wheat flour with wheat germ increased the protein, fat, mineral, and fiber content of the cookies. The particle size of wheat germ affected the textural properties of cookies. As the particle size of wheat germ increased, the hardness of cookies decreased. The color of the cookie was most influenced by the interaction of dough moisture content and wheat germ particle size. Wheat germ level up to 15% had no significant effect on the sensory characteristics of cookies. A suitable combination of defatted wheat germ level, its particle size, and dough moisture content can improve the nutritional value of cookies, without causing a negative effect on the cookies' sensory characteristics.

Identification of Grandchildless Loci Whose Products Are Required for Normal Germ-Line Development in the Nematode Caenorhabditis Elegans

PubMed Central

Capowski, E. E.; Martin, P.; Garvin, C.; Strome, S.

1991-01-01

To identify genes that encode maternal components required for development of the germ line in the nematode Caenorhabditis elegans, we have screened for mutations that confer a maternal-effect sterile or ``grandchildless'' phenotype: homozygous mutant hermaphrodites produced by heterozygous mothers are themselves fertile, but produce sterile progeny. Our screens have identified six loci, defined by 21 mutations. This paper presents genetic and phenotypic characterization of four of the loci. The majority of mutations, those in mes-2, mes-3 and mes-4, affect postembryonic germ-line development; the progeny of mutant mothers undergo apparently normal embryogenesis but develop into agametic adults with 10-1000-fold reductions in number of germ cells. In contrast, mutations in mes-1 cause defects in cytoplasmic partitioning during embryogenesis, and the resulting larvae lack germ-line progenitor cells. Mutations in all of the mes loci primarily affect the germ line, and none disrupt the structural integrity of germ granules. This is in contrast to grandchildless mutations in Drosophila melanogaster, all of which disrupt germ granules and affect abdominal as well as germ-line development. PMID:1783292

The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline.

PubMed

Calvopina, Joseph Hargan; Cook, Helene; Vincent, John J; Nee, Kevin; Clark, Amander T

2015-07-01

Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

Foxi3 deficiency compromises hair follicle stem cell specification and activation

PubMed Central

Shirokova, Vera; Biggs, Leah C.; Jussila, Maria; Ohyama, Takahiro; Groves, Andrew K.; Mikkola, Marja L.

2017-01-01

The hair follicle is an ideal system to study stem cell specification and homeostasis due to its well characterized morphogenesis and stereotypic cycles of stem cell activation upon each hair cycle to produce a new hair shaft. The adult hair follicle stem cell niche consists of two distinct populations, the bulge and the more activation-prone secondary hair germ. Hair follicle stem cells are set aside during early stages of morphogenesis. This process is known to depend on the Sox9 transcription factor, but otherwise the establishment of the hair follicle stem cell niche is poorly understood. Here we show that that mutation of Foxi3, a Forkhead family transcription factor mutated in several hairless dog breeds, compromises stem cell specification. Further, loss of Foxi3 impedes hair follicle downgrowth and progression of the hair cycle. Genome-wide profiling revealed a number of downstream effectors of Foxi3 including transcription factors with a recognized function in hair follicle stem cells such as Lhx2, Runx1, and Nfatc1, suggesting that the Foxi3 mutant phenotype results from simultaneous downregulation of several stem cell signature genes. We show that Foxi3 displays a highly dynamic expression pattern during hair morphogenesis and cycling, and identify Foxi3 as a novel secondary hair germ marker. Absence of Foxi3 results in poor hair regeneration upon hair plucking, and a sparse fur phenotype in unperturbed mice that exacerbates with age, caused by impaired secondary hair germ activation leading to progressive depletion of stem cells. Thus, Foxi3 regulates multiple aspects of hair follicle development and homeostasis. PMID:26992132

Production of transgenic chickens using an avian retroviral vector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopchick, J.; Mills, E.; Rosenblum C.

1987-05-01

The authors efforts to insert genes into the chicken germ line are dependent upon the ability of exogenous avian retroviruses to infect chicken germ cells. They have used a transformation defective Schmidt Ruppin A strain of Rous Sarcoma Virus (RSV-SRA) in their initial experiments. The general protocol involved generating RSV-SRA viremic female chickens (Go), which shed exogenous virus via the oviduct. As the fertilized egg passes through the oviduct, embryonic cells are exposed to the virus. If the germ cell precursors are infected by the virus, offspring (G1) should be generated which are capable of passing the viral DNA tomore » the next generation (G2). Fifteen viremic G1 males were selected for breeding and progeny testing. Since male chickens do not congenitally pass retroviruses through semen, production of viremic G2 offspring indicates germ line DNA transmission. This is confirmed by DNA analysis of the experimental chickens. Using a specific probe for exogenous retrovirus, they have detected the presence of RSV-SRA DNA in viremic chickens. Southern DNA analysis revealed junction fragments for RSV-SRA DNA in viremic G2 chickens, but not in non-viremic siblings. Furthermore, DNA isolated from various tissues of a viremic G2 chicken showed an identical DNA junction fragment pattern, indicating all tissues were derived from the same embryonic cell which contained integrated provirus. To date they have generated 50 transgenic chickens.« less

Differential expression of conserved germ line markers and delayed segregation of male and female primordial germ cells in a hermaphrodite, the leech helobdella.

PubMed

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A

2014-02-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals.

Differential Expression of Conserved Germ Line Markers and Delayed Segregation of Male and Female Primordial Germ Cells in a Hermaphrodite, the Leech Helobdella

PubMed Central

Cho, Sung-Jin; Vallès, Yvonne; Weisblat, David A.

2014-01-01

In sexually reproducing animals, primordial germ cells (PGCs) are often set aside early in embryogenesis, a strategy that minimizes the risk of genomic damage associated with replication and mitosis during the cell cycle. Here, we have used germ line markers (piwi, vasa, and nanos) and microinjected cell lineage tracers to show that PGC specification in the leech genus Helobdella follows a different scenario: in this hermaphrodite, the male and female PGCs segregate from somatic lineages only after more than 20 rounds of zygotic mitosis; the male and female PGCs share the same (mesodermal) cell lineage for 19 rounds of zygotic mitosis. Moreover, while all three markers are expressed in both male and female reproductive tissues of the adult, they are expressed differentially between the male and female PGCs of the developing embryo: piwi and vasa are expressed preferentially in female PGCs at a time when nanos is expressed preferentially in male PGCs. A priori, the delayed segregation of male and female PGCs from somatic tissues and from one another increases the probability of mutations affecting both male and female PGCs of a given individual. We speculate that this suite of features, combined with a capacity for self-fertilization, may contribute to the dramatically rearranged genome of Helobdella robusta relative to other animals. PMID:24217283

Testis sparing surgery for treatment of small testicular lesions: Is it feasible even in germ cell tumors?

PubMed

Bojanic, Nebojsa; Bumbasirevic, Uros; Bojanic, Gordana; Vukovic, Ivan; Milojevic, Bogomir; Pekmezovic, Tatjana

2017-03-01

To evaluate the results of testis-sparing surgery (TSS) in patients, with small testicular lesions and a normal contralateral testicle. In all, 28 patients were treated with TSS for small testicular lesions and a normal contralateral testicle. TSS was considered in patients with testicular lesions smaller than 2 cm and no evidence of metastatic disease. The mean age of patients was 35.3 ± 7.3 years, while the mean diameter of the testicular lesions was 11.4 ± 3.7 mm. After pathological examination, 18 patients (64.3%) were diagnosed with stromal tumors and miscellaneous lesions, while 10 (35.7%) had a germ cell tumor. The median follow-up time for the former group was 33 months and no recurrences were observed. In one patient with germ cell tumor, immediate orchiectomy was performed, while the remaining nine were followed-up (median time, 45 months). One patient developed local recurrence after 39 months. Excellent outcomes for benign lesions could be achieved using TSS. TSS could be offered safely in highly selected patients with germ cell tumors, specifically within a clinical trial but there is more data needed regarding the potential risks and benefits. J. Surg. Oncol. 2017;115:287-290. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

PubMed

Nagai, N; Nakano, K; Sado, Y; Naito, I; Gunduz, M; Tsujigiwa, H; Nagatsuka, H; Ninomiya, Y; Siar, C H

2001-10-01

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.

Exposure to Brefeldin A promotes initiation of meiosis in murine female germ cells.

PubMed

Zhang, Lian-Jun; Chen, Bo; Feng, Xin-Lei; Ma, Hua-Gang; Sun, Li-Lan; Feng, Yan-Min; Liang, Gui-Jin; Cheng, Shun-Feng; Li, Lan; Shen, Wei

2015-01-01

In mammals, ontogenesis starts from a fusion of spermatozoon and oocyte, which are produced by reductive nuclear division of a diploid germ cell in a specialised but complex biological process known as meiosis. However, little is known about the mechanism of meiotic initiation in germ cells, although many factors may be responsible for meiosis both in male and female gonads. In this study, 11.5 days post coitum (dpc) female fetal mouse genital ridges were cultured in vitro with exposure to Brefeldin A (BFA) for 6h, and the changes in meiosis were detected. Synaptonemal-complex analysis implied that BFA played a positive role in meiosis initiation and this hypothesis was confirmed by quantitative PCR of meiosis-specific genes: stimulated by retinoic acid gene 8 (Stra8) and deleted in a zoospermia-like (DAZL). At the same time, mRNA expression of retinoic acid synthetase (Raldh2) and retinoic acid (RA) receptors increased in female gonads with in vitro exposure to BFA. Transplanting genital ridges treated with BFA into the kidney capsule of immunodeficient mice demonstrated that the development capacity of female germ cells was normal, while formation of primordial follicles was seen to be a result of accelerated meiosis after exposure to BFA. In conclusion, the study indicated that BFA stimulated meiosis initiation partly by RA signalling and then promoted the development of follicles.

Characterization of immortalized dairy goat male germline stem cells (mGSCs).

PubMed

Zhu, Haijing; Ma, Jing; Du, Rui; Zheng, Liming; Wu, Jiang; Song, Wencong; Niu, Zhiwei; He, Xin; Du, Enqi; Zhao, Shanting; Hua, Jinlian

2014-09-01

Male germline stem cells (mGSCs), in charge for the fertility in male testis, are the only kind of adult stem cells that transmit genetic information to next generation, with promising prospects in germplasm resources preservation and optimization, and production of transgenic animals. Mouse male germline stem cell lines have been established and are valuable for studying the mechanisms of spermatogenesis. However, there is a lack of stable mGSC cell lines in livestock, which restricts the progress of transgenic research and related biotechnology. Here, we firstly established an immortalized dairy goat mGSC cell line to study the biological properties and the signaling pathways associated with mGSCs self-renewal and differentiation. The ectopic factors SV40 large T antigen and Bmi1 genes were transduced into dairy goat mGSCs, and the results showed that the proliferation of these cells that were named mGSCs-I-SB was improved significantly. They maintained the typical characteristics including the expression of mGSC markers, and the potential to differentiate into all three germ layers, sperm-like cells in vitro. Additionally, mGSCs-I-SB survived and differentiated into three germ layer cell types when they were transplanted into chicken embryos. Importantly, the cells also survived in mouse spermatogenesis deficiency model testis which seemed to be the golden standard to examine mGSCs. Conclusively, our results demonstrate that mGSCs-I-SB present the characteristics of mGSCs and may promote the future study on goat mGSCs. © 2014 Wiley Periodicals, Inc.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

PubMed

Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

2015-01-01

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

PubMed

Wei, Ruxue; Zhao, Xueming; Hao, Haisheng; Du, Weihua; Zhu, Huabin

2016-11-01

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10 -7  M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Expansion of Genes Encoding piRNA-Associated Argonaute Proteins in the Pea Aphid: Diversification of Expression Profiles in Different Plastic Morphs

PubMed Central

Lu, Hsiao-ling; Tanguy, Sylvie; Rispe, Claude; Gauthier, Jean-Pierre; Walsh, Tom; Gordon, Karl; Edwards, Owain; Tagu, Denis; Chang, Chun-che; Jaubert-Possamai, Stéphanie

2011-01-01

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are “specialised” in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction. PMID:22162754

Insights into female germ cell biology: from in vivo development to in vitro derivations.

PubMed

Jung, Dajung; Kee, Kehkooi

2015-01-01

Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments: namely, primordial germ cell formation, meiotic initiation, and folliculogenesis.

C-X-C motif chemokine ligand 10 produced by mouse Sertoli cells in response to mumps virus infection induces male germ cell apoptosis

PubMed Central

Jiang, Qian; Wang, Fei; Shi, Lili; Zhao, Xiang; Gong, Maolei; Liu, Weihua; Song, Chengyi; Li, Qihan; Chen, Yongmei; Wu, Han; Han, Daishu

2017-01-01

Mumps virus (MuV) infection usually results in germ cell degeneration in the testis, which is an etiological factor for male infertility. However, the mechanisms by which MuV infection damages male germ cells remain unclear. The present study showed that C-X-C motif chemokine ligand 10 (CXCL10) is produced by mouse Sertoli cells in response to MuV infection, which induces germ cell apoptosis through the activation of caspase-3. CXC chemokine receptor 3 (CXCR3), a functional receptor of CXCL10, is constitutively expressed in male germ cells. Neutralizing antibodies against CXCR3 and an inhibitor of caspase-3 activation significantly inhibited CXCL10-induced male germ cell apoptosis. Furthermore, the tumor necrosis factor-α (TNF-α) upregulated CXCL10 production in Sertoli cells after MuV infection. The knockout of either CXCL10 or TNF-α reduced germ cell apoptosis in the co-cultures of germ cells and Sertoli cells in response to MuV infection. Local injection of MuV into the testes of mice confirmed the involvement of CXCL10 in germ cell apoptosis in vivo. These results provide novel insights into MuV-induced germ cell apoptosis in the testis. PMID:29072682

R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation.

PubMed

Kocer, Ayhan; Pinheiro, Iris; Pannetier, Maëlle; Renault, Lauriane; Parma, Pietro; Radi, Orietta; Kim, Kyung-Ah; Camerino, Giovanna; Pailhoux, Eric

2008-04-02

Up to now, two loci have been involved in XX sex-reversal in mammals following loss-of-function mutations, PIS (Polled Intersex Syndrome) in goats and R-spondin1 (RSPO1) in humans. Here, we analyze the possible interaction between these two factors during goat gonad development. Furthermore, since functional redundancy between different R-spondins may influence gonad development, we also studied the expression patterns of RSPO2, 3 and 4. Similarly to the mouse, RSPO1 shows a sex-dimorphic expression pattern during goat gonad development with higher levels in the ovaries. Interestingly, the PIS mutation does not seem to influence its level of expression. Moreover, using an RSPO1 specific antibody, the RSPO1 protein was localized in the cortical area of early differentiating ovaries (36 and 40 dpc). This cortical area contains the majority of germ cell that are surrounded by FOXL2 negative somatic cells. At latter stages (50 and 60 dpc) RSPO1 protein remains specifically localized on the germ cell membranes. Interestingly, a time-specific relocation of RSPO1 on the germ cell membrane was noticed, moving from a uniform distribution at 40 dpc to a punctuated staining before and during meiosis (50 and 60 dpc respectively). Interestingly, also RSPO2 and RSPO4 show a sex-dimorphic expression pattern with higher levels in the ovaries. Although RSPO4 was found to be faintly and belatedly expressed, the expression of RSPO2 increases at the crucial 36 dpc stage, as does that of FOXL2. Importantly, RSPO2 expression appears dramatically decreased in XX PIS-/- gonads at all three tested stages (36, 40 and 50 dpc). During goat ovarian development, the pattern of expression of RSPO1 is in agreement with its possible anti-testis function but is not influenced by the PIS mutation. Moreover, our data suggest that RSPO1 may be associated with germ cell development and meiosis. Interestingly, another RSPO gene, RSPO2 shows a sex-dimorphic pattern of expression that is dramatically influenced by the PIS mutation.

R-spondin1 and FOXL2 act into two distinct cellular types during goat ovarian differentiation

PubMed Central

Kocer, Ayhan; Pinheiro, Iris; Pannetier, Maëlle; Renault, Lauriane; Parma, Pietro; Radi, Orietta; Kim, Kyung-Ah; Camerino, Giovanna; Pailhoux, Eric

2008-01-01

Background Up to now, two loci have been involved in XX sex-reversal in mammals following loss-of-function mutations, PIS (Polled Intersex Syndrome) in goats and R-spondin1 (RSPO1) in humans. Here, we analyze the possible interaction between these two factors during goat gonad development. Furthermore, since functional redundancy between different R-spondins may influence gonad development, we also studied the expression patterns of RSPO2, 3 and 4. Results Similarly to the mouse, RSPO1 shows a sex-dimorphic expression pattern during goat gonad development with higher levels in the ovaries. Interestingly, the PIS mutation does not seem to influence its level of expression. Moreover, using an RSPO1 specific antibody, the RSPO1 protein was localized in the cortical area of early differentiating ovaries (36 and 40 dpc). This cortical area contains the majority of germ cell that are surrounded by FOXL2 negative somatic cells. At latter stages (50 and 60 dpc) RSPO1 protein remains specifically localized on the germ cell membranes. Interestingly, a time-specific relocation of RSPO1 on the germ cell membrane was noticed, moving from a uniform distribution at 40 dpc to a punctuated staining before and during meiosis (50 and 60 dpc respectively). Interestingly, also RSPO2 and RSPO4 show a sex-dimorphic expression pattern with higher levels in the ovaries. Although RSPO4 was found to be faintly and belatedly expressed, the expression of RSPO2 increases at the crucial 36 dpc stage, as does that of FOXL2. Importantly, RSPO2 expression appears dramatically decreased in XX PIS-/- gonads at all three tested stages (36, 40 and 50 dpc). Conclusion During goat ovarian development, the pattern of expression of RSPO1 is in agreement with its possible anti-testis function but is not influenced by the PIS mutation. Moreover, our data suggest that RSPO1 may be associated with germ cell development and meiosis. Interestingly, another RSPO gene, RSPO2 shows a sex-dimorphic pattern of expression that is dramatically influenced by the PIS mutation. PMID:18384673

Changes in Brain Function in Patients With Stage I, Stage II, Stage III, or Stage IV Ovarian, Primary Peritoneal, or Fallopian Tube Cancer Who Are Receiving Chemotherapy

ClinicalTrials.gov

2018-04-11

Cognitive Side Effects of Cancer Therapy; Malignant Ovarian Epithelial Tumor; Ovarian Brenner Tumor; Ovarian Carcinosarcoma; Ovarian Choriocarcinoma; Ovarian Clear Cell Cystadenocarcinoma; Ovarian Dysgerminoma; Ovarian Embryonal Carcinoma; Ovarian Endometrioid Adenocarcinoma; Ovarian Mixed Germ Cell Tumor; Ovarian Mucinous Cystadenocarcinoma; Ovarian Polyembryoma; Ovarian Sarcoma; Ovarian Seromucinous Carcinoma; Ovarian Serous Cystadenocarcinoma; Ovarian Teratoma; Ovarian Yolk Sac Tumor; Stage I Ovarian Cancer; Stage IA Fallopian Tube Cancer; Stage IA Ovarian Cancer; Stage IA Ovarian Germ Cell Tumor; Stage IB Fallopian Tube Cancer; Stage IB Ovarian Cancer; Stage IB Ovarian Germ Cell Tumor; Stage IC Fallopian Tube Cancer; Stage IC Ovarian Cancer; Stage IC Ovarian Germ Cell Tumor; Stage II Ovarian Cancer; Stage IIA Fallopian Tube Cancer; Stage IIA Ovarian Cancer; Stage IIA Ovarian Germ Cell Tumor; Stage IIB Fallopian Tube Cancer; Stage IIB Ovarian Cancer; Stage IIB Ovarian Germ Cell Tumor; Stage IIC Fallopian Tube Cancer; Stage IIC Ovarian Cancer; Stage IIC Ovarian Germ Cell Tumor; Stage IIIA Fallopian Tube Cancer; Stage IIIA Ovarian Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIA Primary Peritoneal Cancer; Stage IIIB Fallopian Tube Cancer; Stage IIIB Ovarian Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIB Primary Peritoneal Cancer; Stage IIIC Fallopian Tube Cancer; Stage IIIC Ovarian Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IIIC Primary Peritoneal Cancer; Stage IV Fallopian Tube Cancer; Stage IV Ovarian Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Primary Peritoneal Cancer; Undifferentiated Ovarian Carcinoma

Palliative Care in Improving Quality of Life and Symptoms in Patients With Stage III-IV Pancreatic or Ovarian Cancer

ClinicalTrials.gov

2014-12-18

Recurrent Ovarian Epithelial Cancer; Recurrent Ovarian Germ Cell Tumor; Recurrent Pancreatic Cancer; Stage III Pancreatic Cancer; Stage IIIA Ovarian Epithelial Cancer; Stage IIIA Ovarian Germ Cell Tumor; Stage IIIB Ovarian Epithelial Cancer; Stage IIIB Ovarian Germ Cell Tumor; Stage IIIC Ovarian Epithelial Cancer; Stage IIIC Ovarian Germ Cell Tumor; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Stage IV Pancreatic Cancer

Extracranial Germ Cell Tumors—Health Professional Version

Cancer.gov

Extracranial germ cell tumors (GCTs) arise from primordial germ cells, which migrate during embryogenesis from the yolk sac to the gonads. Childhood extracranial GCTs can be divided into the following two types: gonadal, and extragonadal. Find evidence-based information on extracranial germ cell tumors treatment.

Identification of Potential Germ-Cell Mutagens

EPA Science Inventory

The existence of agents that can induce germ-cell mutations in experimental systems has been recognized since 1927 with the discovery of the ability of X-rays to induce such mutations in Drosophila. Various rodent-based germ-cell mutation assays have been developed, and ~50 germ...

Maternal dazap2 Regulates Germ Granules by Counteracting Dynein in Zebrafish Primordial Germ Cells.

PubMed

Forbes, Meredyth M; Rothhämel, Sophie; Jenny, Andreas; Marlow, Florence L

2015-07-07

Primordial germ cells (PGCs) are the stem cells of the germline. Generally, germline induction occurs via zygotic factors or the inheritance of maternal determinants called germ plasm (GP). GP is packaged into ribonucleoprotein complexes within oocytes and later promotes the germline fate in embryos. Once PGCs are specified by either mechanism, GP components localize to perinuclear granular-like structures. Although components of zebrafish PGC germ granules have been studied, the maternal factors regulating their assembly and contribution to germ cell development are unknown. Here, we show that the scaffold protein Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and GP assembly, and colocalizes with the GP in oocytes and in PGCs. Mutational analysis revealed a requirement for maternal Dazap2 (MDazap2) in germ-granule maintenance. Through molecular epistasis analyses, we show that MDazap2 is epistatic to Tdrd7 and maintains germ granules in the embryonic germline by counteracting Dynein activity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Extragonadal Germ Cell Tumors—Health Professional Version

Cancer.gov

Extragonadal germ cell tumors are rare and account for only a small percentage of all germ cell tumors. However, the true incidence of these tumors may be higher than originally thought because of failure to diagnose them properly. Find evidence-based information on extragonadal germ cell tumors treatment.

Extraction and characterization of corn germ proteins

USDA-ARS?s Scientific Manuscript database

Our study was conducted to develop methods to extract corn germ protein economically and characterize and identify potential applications of the recovered protein. Protein was extracted from both wet germ and finished (dried) germ using 0.1M NaCl as solvent. The method involved homogenization, sti...

Improvement of dry fractionation ethanol fermentation by partial germ supplementation

USDA-ARS?s Scientific Manuscript database

Ethanol fermentation of dry fractionated grits (corn endosperm pieces) containing different levels of germ was studied using the dry grind process. Partial removal of germ fraction allows for marketing the germ fraction and potentially more efficient fermentation. Grits obtained from a dry milling p...

Propranolol Hydrochloride in Treating Patients With Locally Recurrent or Metastatic Solid Tumors That Cannot Be Removed By Surgery

ClinicalTrials.gov

2017-11-27

Male Breast Cancer; Recurrent Melanoma; Stage IV Breast Cancer; Stage IV Melanoma; Stage IV Ovarian Epithelial Cancer; Stage IV Ovarian Germ Cell Tumor; Unspecified Adult Solid Tumor, Protocol Specific; Hepatocellular Carcinoma

The Endocannabinoid System and Spermatogenesis

PubMed Central

Grimaldi, Paola; Di Giacomo, Daniele; Geremia, Raffaele

2013-01-01

Spermatogenesis is a complex process in which male germ cells undergo a mitotic phase followed by meiosis and by a morphogenetic process to form mature spermatozoa. Spermatogenesis is under the control of gonadotropins, steroid hormones and it is modulated by a complex network of autocrine and paracrine factors. These modulators ensure the correct progression of germ cell differentiation to form mature spermatozoa. Recently, it has been pointed out the relevance of endocannabinoids as critical modulators of male reproduction. Endocannabinoids are natural lipids able to bind to cannabinoid receptors and whose levels are regulated by specific biosynthetic and degradative enzymes. Together with their receptors and metabolic enzymes, they form the “endocannabinoid system” (ECS). In male reproductive tracts, they affect Sertoli cell activities, Leydig cell proliferation, germ cell differentiation, sperm motility, capacitation, and acrosome reaction. The ECS interferes with the pituitary-gonadal axis, and an intricate crosstalk between ECS and steroid hormones has been highlighted. This mini-review will focus on the involvement of the ECS in the control of spermatogenesis and on the interaction between ECS and steroid hormones. PMID:24379805

Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells.

PubMed

Shishido, Yurina; Baba, Takashi; Sato, Tetsuya; Shima, Yuichi; Miyabayashi, Kanako; Inoue, Miki; Akiyama, Haruhiko; Kimura, Hiroshi; Kanai, Yoshiakira; Ishihara, Yasuhiro; Haraguchi, Shogo; Miyazaki, Akira; Rozman, Damjana; Yamazaki, Takeshi; Choi, Man-Ho; Ohkawa, Yasuyuki; Suyama, Mikita; Morohashi, Ken-Ichirou

2017-02-02

SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that down-regulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells.

Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells

PubMed Central

Shishido, Yurina; Baba, Takashi; Sato, Tetsuya; Shima, Yuichi; Miyabayashi, Kanako; Inoue, Miki; Akiyama, Haruhiko; Kimura, Hiroshi; Kanai, Yoshiakira; Ishihara, Yasuhiro; Haraguchi, Shogo; Miyazaki, Akira; Rozman, Damjana; Yamazaki, Takeshi; Choi, Man-Ho; Ohkawa, Yasuyuki; Suyama, Mikita; Morohashi, Ken-ichirou

2017-01-01

SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that down-regulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells. PMID:28150810

Extraordinary genome stability in the ciliate Paramecium tetraurelia

PubMed Central

Sung, Way; Tucker, Abraham E.; Doak, Thomas G.; Choi, Eunjin; Thomas, W. Kelley; Lynch, Michael

2012-01-01

Mutation plays a central role in all evolutionary processes and is also the basis of genetic disorders. Established base-substitution mutation rates in eukaryotes range between ∼5 × 10−10 and 5 × 10−8 per site per generation, but here we report a genome-wide estimate for Paramecium tetraurelia that is more than an order of magnitude lower than any previous eukaryotic estimate. Nevertheless, when the mutation rate per cell division is extrapolated to the length of the sexual cycle for this protist, the measure obtained is comparable to that for multicellular species with similar genome sizes. Because Paramecium has a transcriptionally silent germ-line nucleus, these results are consistent with the hypothesis that natural selection operates on the cumulative germ-line replication fidelity per episode of somatic gene expression, with the germ-line mutation rate per cell division evolving downward to the lower barrier imposed by random genetic drift. We observe ciliate-specific modifications of widely conserved amino acid sites in DNA polymerases as one potential explanation for unusually high levels of replication fidelity. PMID:23129619

Sexual dimorphic expression of DMRT1 and Sox9a during gonadal differentiation and hormone-induced sex reversal in the teleost fish Nile tilapia (Oreochromis niloticus).

PubMed

Kobayashi, Tohru; Kajiura-Kobayashi, Hiroko; Guan, Guijun; Nagahama, Yoshitaka

2008-01-01

We examined the expression profiles of tDMRT1 and Sox9a during gonadal sex differentiation and hormone-induced sex reversal. tDMRT1 was detected in the gonial germ-cell-surrounding cells in XY fry specifically before the appearance of any signs of morphological sex differentiation, that is, sex differences in germ cell number and histogenesis, such as differentiation into intratesticular efferent duct or ovarian cavity. The signals became localized in the Sertoli and epithelial cells comprising the efferent duct during gonadal differentiation. After the induction of XY sex reversal with estrogen, tDMRT1 decreased and then disappeared completely. In contrast, tDMRT1 was expressed in the germ-cell-surrounding cells in XX sex reversal with androgen. On the other hand, Sox9a did not show sexual dimorphism before the appearance of sex differences in histogenesis and was not expressed in the efferent duct in the testis. These results suggest that tDMRT1 is a superior testicular differentiation marker in tilapia.

Vorinostat and Bortezomib in Treating Young Patients With Refractory or Recurrent Solid Tumors, Including Central Nervous System Tumors and Lymphoma

ClinicalTrials.gov

2013-07-01

Childhood Burkitt Lymphoma; Childhood Central Nervous System Choriocarcinoma; Childhood Central Nervous System Germ Cell Tumor; Childhood Central Nervous System Germinoma; Childhood Central Nervous System Mixed Germ Cell Tumor; Childhood Central Nervous System Teratoma; Childhood Central Nervous System Yolk Sac Tumor; Childhood Choroid Plexus Tumor; Childhood Craniopharyngioma; Childhood Diffuse Large Cell Lymphoma; Childhood Immunoblastic Large Cell Lymphoma; Childhood Medulloepithelioma; Childhood Meningioma; Childhood Mixed Glioma; Childhood Nasal Type Extranodal NK/T-cell Lymphoma; Childhood Oligodendroglioma; Recurrent Childhood Anaplastic Large Cell Lymphoma; Recurrent Childhood Brain Stem Glioma; Recurrent Childhood Central Nervous System Embryonal Tumor; Recurrent Childhood Cerebellar Astrocytoma; Recurrent Childhood Cerebral Astrocytoma; Recurrent Childhood Ependymoma; Recurrent Childhood Grade III Lymphomatoid Granulomatosis; Recurrent Childhood Large Cell Lymphoma; Recurrent Childhood Lymphoblastic Lymphoma; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Childhood Medulloblastoma; Recurrent Childhood Pineoblastoma; Recurrent Childhood Small Noncleaved Cell Lymphoma; Recurrent Childhood Subependymal Giant Cell Astrocytoma; Recurrent Childhood Supratentorial Primitive Neuroectodermal Tumor; Recurrent Childhood Visual Pathway and Hypothalamic Glioma; Recurrent Childhood Visual Pathway Glioma; Recurrent/Refractory Childhood Hodgkin Lymphoma; Unspecified Childhood Solid Tumor, Protocol Specific

Effects of some plant lectins on hydrogen peroxide release from macrophages induced with streptococcal preparation OK-432.

PubMed Central

Tomioka, H; Saito, H

1980-01-01

Concanavalin A and phytohemagglutinin were found to cause marked inhibition of H2O2 release from macrophages induced with killed streptococci (preparation OK-432). The inhibitory effect of these two lectins on the H2O2 release from macrophages was observed with spontaneous and wheat germ lectin-triggered H2O2 release. This suggests that the lectins act directly on the macrophage H2O2-releasing function, per se, but not on the wheat germ lectin-H2O2 release-enhancing process. Concanavalin A exhibited its inhibitory action on macrophage H2O2 release by specific binding to D-mannopyranoside receptor sites on the macrophage cell surface. Galactose-binding lectins, peanut agglutinin, and soybean agglutinin failed to inhibit, but, on the other hand, slightly enhanced macrophage H2O2 release. The effect of these five lectins on the phagocytosis of latex particles by macrophages was tested. Wheat germ lectin, concanavalin A, and phytohemagglutinin significantly depressed the macrophage phagocytosis, whereas peanut agglutinin and soybean agglutinin failed to show any inhibitory action. PMID:7399666

Constructing and dismantling frameworks of disease etiology: the rise and fall of sewer gas in America, 1870-1910.

PubMed Central

An, Perry G.

2004-01-01

For roughly forty years, from 1870 to 1910, Americans recognized and feared gases emanating from sewers, believing that they were responsible for causing an array of diseases. Fears of sewer gas arose from deeper anxieties toward contact with decomposing organic matter and the vapors emitted from such refuse. These anxieties were exacerbated by the construction of sewers across the country during the mid-to-late-nineteenth century, which concentrated waste emanations and connected homes to one another. The result was the birth of sewer gas and the attribution of sickness and death to it, as well as the development of a host of plumbing devices and, especially, bathroom fixtures, to combat sewer gas. The rise of the germ theory, laboratory science, and belief in disease specificity, however, transformed the threat of sewer gas, eventually replacing it (and the larger fear of miasmas) with the threat of germs. The germ theory framework, by 1910, proved more suitable than the sewer gas framework in explaining disease causation; it is this suitability that often shapes the relationship between science and society. PMID:15829149

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

PubMed Central

Goodhardt, M; Babinet, C; Lutfalla, G; Kallenbach, S; Cavelier, P; Rougeon, F

1989-01-01

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo. Images PMID:2508061

From Young Children's Ideas about Germs to Ideas Shaping a Learning Environment

NASA Astrophysics Data System (ADS)

Ergazaki, Marida; Saltapida, Konstantina; Zogza, Vassiliki

2010-11-01

This paper is concerned with highlighting young children’s ideas about the nature, location and appearance of germs, as well as their reasoning strands about germs’ ontological category and biological functions. Moreover, it is concerned with exploring how all these could be taken into account for shaping a potentially fruitful learning environment. Conducting individual, semi-structured interviews with 35 preschoolers (age 4.5-5.5) of public kindergartens in the broader area of Patras, we attempted to trace their ideas about what germs are, where they may be found, whether they are good or bad and living or non-living and how they might look like in a drawing. Moreover, children were required to attribute a series of biological functions to dogs, chairs and germs, and finally to create a story with germs holding a key-role. The analysis of our qualitative data within the “NVivo” software showed that the informants make a strong association of germs with health and hygiene issues, locate germs mostly in our body and the external environment, are not familiar with the ‘good germs’-idea, and draw germs as ‘human-like’, ‘animal-like’ or ‘abstract’ entities. Moreover, they have significant difficulties not only in employing biological functions as criteria for classifying germs in the category of ‘living’, but also in just attributing such functions to germs using a warrant. Finally, the shift from our findings to a 3-part learning environment aiming at supporting preschoolers in refining their initial conceptualization of germs is thoroughly discussed in the paper.

0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Heng; Denhard, Leslie A.; Zhou Huaxin

Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and roundmore » spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis.« less

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

PubMed

Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate

2007-09-26

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

Extracranial Germ Cell Tumors—Patient Version

Cancer.gov

Extracranial germ cell tumors are tumors that develop from germ cells (fetal cells that give rise to sperm and eggs) and can form in many parts of the body. They are most common in teenagers and can often be cured. Start here to find information on extracranial germ cell tumors treatment.

DNA Analysis in Samples From Younger Patients With Germ Cell Tumors and Their Parents or Siblings

ClinicalTrials.gov

2017-10-05

Childhood Malignant Ovarian Germ Cell Tumor; Childhood Malignant Testicular Germ Cell Tumor; Ovarian Choriocarcinoma; Ovarian Embryonal Carcinoma; Ovarian Mixed Germ Cell Tumor; Ovarian Teratoma; Ovarian Yolk Sac Tumor; Testicular Choriocarcinoma; Testicular Embryonal Carcinoma; Testicular Seminoma; Testicular Teratoma; Testicular Yolk Sac Tumor

Metabolism of 1-nitropyrene in germ-free and conventional rats.

PubMed

Kinouchi, T; Morotomi, M; Mutai, M; Fifer, E K; Beland, F A; Ohnishi, Y

1986-04-01

The distribution, covalent binding and metabolism of radioactive 1-nitropyrene (1-NP) were examined following its oral administration to conventional and germ-free male Wistar rats. With both groups of animals, the liver, kidney, bladder, adipose tissue and gastrointestinal tract had the highest specific radioactivity. However, the maximum concentration of radioactivity occurred at 12 hr in conventional rats as compared to 24 hr in germ-free animals. This difference may be due to the faster transit time of the intestinal contents through conventional rats. At 48 hr after treatment, the covalent binding of 1-NP metabolites was greatest in liver and kidney of conventional rats, while in germ-free rats, substantial binding was also found in the gastrointestinal tract. The mutagenic activity in Salmonella typhimurium TA98 of fecal extracts and urine from conventional rats was greater in the presence of an S9 mix, whereas similar extracts from germ-free animals were more mutagenic in the absence of S9. The major fecal metabolites in germ-free rats were (in order of decreasing concentration): 3-nitropyrenol greater than 1-NP greater than 4,5-dihydroxy-4,5-dihydro-1-NP greater than 6-nitropyrenol greater than 8-nitropyrenol. With the exception of 1-NP, similar metabolites were found in the urine as their glucuronide conjugates. In the feces from conventional rats, substantial nitro reduction and N-acetylation occurred with the major metabolites being: 1-NP greater than 1-aminopyrene greater than 8-acetylaminopyrenol greater than 6-acetylaminopyrenol greater than 3-acetylaminopyrenol. The major metabolites identified in the urine from conventional rats were glucuronide conjugates of 6- and 8-acetylaminopyrenol, while the major biliary conjugates identified were glucuronide conjugates of 4,5-dihydroxy-4,5-dihydro-1-NP and 3-, 6-, and 8-nitropyrenol, although the relative proportion of glucuronide conjugates of 6- and 8-aminopyrenol and 6- and 8-acetylaminopyrenol increased in later stages of the biliary excretion. The polar and beta-glucuronidase-refractory metabolites, which may be sulfate and glutathione conjugates, remain to be identified.

Retinoic Acid Metabolic Genes, Meiosis, and Gonadal Sex Differentiation in Zebrafish

PubMed Central

Rodríguez-Marí, Adriana; Cañestro, Cristian; BreMiller, Ruth A.; Catchen, Julian M.; Yan, Yi-Lin; Postlethwait, John H.

2013-01-01

To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish. PMID:24040125

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

PubMed Central

Rijlaarsdam, Martin A.; Tax, David M. J.; Gillis, Ad J. M.; Dorssers, Lambert C. J.; Koestler, Devin C.; de Ridder, Jeroen; Looijenga, Leendert H. J.

2015-01-01

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809). PMID:25859847

Ectoderm gene activation in sea urchin embryos mediated by the CCAAT-binding factor.

PubMed

Li, Xiaotao; Bhattacharya, Chitralekha; Dayal, Sandeep; Maity, Sankar; Klein, William H

2002-05-01

Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

Cell type-specific translational repression of Cyclin B during meiosis in males.

PubMed

Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

2015-10-01

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

Resolving early mesoderm diversification through single-cell expression profiling.

PubMed

Scialdone, Antonio; Tanaka, Yosuke; Jawaid, Wajid; Moignard, Victoria; Wilson, Nicola K; Macaulay, Iain C; Marioni, John C; Göttgens, Berthold

2016-07-14

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.

Extraction and functional properties of non-zein proteins in corn germ from wet-milling

USDA-ARS?s Scientific Manuscript database

This study was conducted to develop methods of extracting corn germ protein and characterize and identify potential applications of the recovered protein. Protein was extracted from both wet germ and finished (dried) germ using 0.1M NaCl as solvent. The method involved homogenization, stirring, cent...

Retrotransposons Mimic Germ Plasm Determinants to Promote Transgenerational Inheritance.

PubMed

Tiwari, Bhavana; Kurtz, Paula; Jones, Amanda E; Wylie, Annika; Amatruda, James F; Boggupalli, Devi Prasad; Gonsalvez, Graydon B; Abrams, John M

2017-10-09

Retrotransposons are a pervasive class of mobile elements present in the genomes of virtually all forms of life [1, 2]. In metazoans, these are preferentially active in the germline, which, in turn, mounts defenses that restrain their activity [3, 4]. Here we report that certain classes of retrotransposons ensure transgenerational inheritance by invading presumptive germ cells before they are formed. Using sensitized Drosophila and zebrafish models, we found that diverse classes of retrotransposons migrate to the germ plasm, a specialized region of the oocyte that prefigures germ cells and specifies the germline of descendants in the fertilized egg. In Drosophila, we found evidence for a "stowaway" model, whereby Tahre retroelements traffic to the germ plasm by mimicking oskar RNAs and engaging the Staufen-dependent active transport machinery. Consistent with this, germ plasm determinants attracted retroelement RNAs even when these components were ectopically positioned in bipolar oocytes. Likewise, vertebrate retrotransposons similarly migrated to the germ plasm in zebrafish oocytes. Together, these results suggest that germ plasm targeting represents a fitness strategy adopted by some retrotransposons to ensure transgenerational propagation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation of the germ-cell potential of human bone marrow-derived cells by a chemical carcinogen

PubMed Central

Liu, Chunfang; Ma, Zhan; Xu, Songtao; Hou, Jun; Hu, Yao; Yu, Yinglu; Liu, Ruilai; Chen, Zhihong; Lu, Yuan

2014-01-01

Embryonic/germ cell traits are common in malignant tumors and are thought to be involved in malignant tumor behaviors. The reasons why tumors show strong embryonic/germline traits (displaced germ cells or gametogenic programming reactivation) are controversial. Here, we show that a chemical carcinogen, 3-methyl-cholanthrene (3-MCA), can trigger the germ-cell potential of human bone marrow-derived cells (hBMDCs). 3-MCA promoted the generation of germ cell-like cells from induced hBMDCs that had undergone malignant transformation, whereas similar results were not observed in the parallel hBMDC culture at the same time point. The malignant transformed hBMDCs spontaneously and more efficiently generated into germ cell-like cells even at the single-cell level. The germ cell-like cells from induced hBMDCs were similar to natural germ cells in many aspects, including morphology, gene expression, proliferation, migration, further development, and teratocarcinoma formation. Therefore, our results demonstrate that a chemical carcinogen can reactivate the germline phenotypes of human somatic tissue-derived cells, which might provide a novel idea to tumor biology and therapy. PMID:24998261

The making of a germ panic, then and now.

PubMed Central

Tomes, N

2000-01-01

Over the last 2 decades, a heightened interest in germs has been evident in many aspects of American popular culture, including news coverage, advertisements, and entertainment media. Although clearly a response to the AIDS epidemic and other recent disease outbreaks, current obsessions with germs have some striking parallels with a similar period of intense anxiety about disease germs that occurred between 1900 and 1940. A comparison of these 2 periods of germ "panic" suggests some of the long-term cultural trends that contributed to their making. Both germ panics reflected anxieties about societal incorporation, associated with expanding markets, transportation networks, and mass immigration. They were also shaped by new trends in public health education, journalism, advertising, and entertainment media. In comparison to the first germ panic, the current discourse about the "revenge of the superbugs" is considerably more pessimistic because of increasing worries about the environment, suspicions of governmental authority, and distrust of expert knowledge. Yet, as popular anxieties about infectious disease have increased, public health scientists have been attracting favorable coverage in their role as "medical detectives" on the trail of the "killer germ." PMID:10667179

Chromatin associated Sin3A is essential for male germ cell lineage in the mouse

PubMed Central

Pellegrino, Jessica; Castrillon, Diego H.; David, Gregory

2012-01-01

Spermatogenesis is a complex process that requires coordinated proliferation and differentiation of male germ cells. The molecular events that dictate this process are largely unknown, but are likely to involve highly regulated transcriptional control. In this study, we investigate the contribution of chromatin associated Sin3A in mouse germ cell lineage development. Genetic inactivation of Sin3A in the male germline leads to sterility that results from the early and penetrant apoptotic death observed in Sin3A-deleted germ cells, coincident with the reentry in mitosis. Sin3A-deleted testes exhibit a Sertoli-cell only phenotype, consistent with the absolute requirement for Sin3A in germ cells’ development and/or viability. Interestingly, transcripts analysis revealed that the expression program of Sertoli cells is altered upon inactivation of Sin3A in germ cells. These studies identified a central role for the mammalian Sin3-HDAC complex in the germ cell lineage, and point to an exquisite transcriptional crosstalk between germ cells and their niche to support fertility in mammals. PMID:22820070

RSPO1/β-Catenin Signaling Pathway Regulates Oogonia Differentiation and Entry into Meiosis in the Mouse Fetal Ovary

PubMed Central

Chassot, Anne-Amandine; Gregoire, Elodie P.; Lavery, Rowena; Taketo, Makoto M.; de Rooij, Dirk G.; Adams, Ian R.; Chaboissier, Marie-Christine

2011-01-01

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1−/− gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm. PMID:21991325

Cellular mechanics of germ band retraction in Drosophila.

PubMed

Lynch, Holley E; Crews, Sarah M; Rosenthal, Brett; Kim, Elliott; Gish, Robert; Echiverri, Karl; Hutson, M Shane

2013-12-15

Germ band retraction involves a dramatic rearrangement of the tissues on the surface of the Drosophila embryo. As germ band retraction commences, one tissue, the germ band, wraps around another, the amnioserosa. Through retraction the two tissues move cohesively as the highly elongated cells of the amnioserosa contract and the germ band moves so it is only on one side of the embryo. To understand the mechanical drivers of this process, we designed a series of laser ablations that suggest a mechanical role for the amnioserosa. First, we find that during mid retraction, segments in the curve of the germ band are under anisotropic tension. The largest tensions are in the direction in which the amnioserosa contracts. Second, ablating one lateral flank of the amnioserosa reduces the observed force anisotropy and leads to retraction failures. The other intact flank of amnioserosa is insufficient to drive retraction, but can support some germ band cell elongation and is thus not a full phenocopy of ush mutants. Another ablation-induced failure in retraction can phenocopy mys mutants, and does so by targeting amnioserosa cells in the same region where the mutant fails to adhere to the germ band. We conclude that the amnioserosa must play a key, but assistive, mechanical role that aids uncurling of the germ band. © 2013 Elsevier Inc. All rights reserved.

Cellular Mechanics of Germ Band Retraction in Drosophila

PubMed Central

Lynch, Holley E.; Crews, Sarah M.; Rosenthal, Brett; Kim, Elliott; Gish, Robert; Echiverri, Karl; Hutson, M. Shane

2013-01-01

Germ band retraction involves a dramatic rearrangement of the tissues on the surface of the Drosophila embryo. As germ band retraction commences, one tissue, the germ band, wraps around another, the amnioserosa. Through retraction the two tissues move cohesively as the highly elongated cells of the amnioserosa contract and the germ band moves so it is only on one side of the embryo. To understand the mechanical drivers of this process, we designed a series of laser ablations that suggest a mechanical role for the amnioserosa. First, we find that during mid retraction, segments in the curve of the germ band are under anisotropic tension. The largest tensions are in the direction in which the amnioserosa contracts. Second, ablating one lateral flank of the amnioserosa reduces the observed force anisotropy and leads to retraction failures. The other intact flank of amnioserosa is insufficient to drive retraction, but can support some germ band cell elongation and is thus not a full phenocopy of ush mutants. Another ablation-induced failure in retraction can phenocopy mys mutants, and does so by targeting amnioserosa cells in the same region where the mutant fails to adhere to the germ band. We conclude that the amnioserosa must play a key, but assistive, mechanical role that aids uncurling of the germ band. PMID:24135149

Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

PubMed Central

Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

2016-01-01

The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

Cytokeratin expression in mouse lacrimal gland germ epithelium.

PubMed

Hirayama, Masatoshi; Liu, Ying; Kawakita, Tetsuya; Shimmura, Shigeto; Tsubota, Kazuo

2016-05-01

The lacrimal gland secretes tear fluids that protect the ocular surface epithelium, and its dysfunction leads to dry eye disease (DED). The functional restoration of the lacrimal gland by engraftment of a bioengineered lacrimal gland using lacrimal gland germ epithelial cells has been proposed to cure DED in mice. Here, we investigate the expression profile of cytokeratins in the lacrimal gland germ epithelium to clarify their unique characteristics. We performed quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC) analysis to clarify the expression profile of cytokeratin in the lacrimal gland germ epithelium. The mRNA expression of keratin (KRT) 5, KRT8, KRT14, KRT15, and KRT18 in the lacrimal gland germ epithelium was increased compared with that in mouse embryonic stem cells and the lacrimal gland germ mesenchyme, as analyzed by Q-PCR. The expression level of KRT15 increased in the transition from stem cells to lacrimal gland germ epithelium, then decreased as the lacrimal gland matured. IHC revealed that the expression set of these cytokeratins in the lacrimal gland germ epithelium was different from that in the adult lacrimal gland. The expression of KRT15 was observed in the lacrimal gland germ epithelium, and it segmentalized into some of the basal cells in the intercanulated duct in mature gland. We determined the expression profile of cytokeratins in the lacrimal gland epithelium, and identified KRT15 as a candidate unique cellular marker for the lacrimal gland germ epithelium. Copyright © 2015 Elsevier Ltd. All rights reserved.

Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.

PubMed Central

Frebourg, T; Kassel, J; Lam, K T; Gryka, M A; Barbier, N; Andersen, T I; Børresen, A L; Friend, S H

1992-01-01

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance. Images PMID:1631137

Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.

PubMed

Frebourg, T; Kassel, J; Lam, K T; Gryka, M A; Barbier, N; Andersen, T I; Børresen, A L; Friend, S H

1992-07-15

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance.

Identification of genes expressed in the hermaphrodite germ line of C. elegans using SAGE

PubMed Central

Wang, Xin; Zhao, Yongjun; Wong, Kim; Ehlers, Peter; Kohara, Yuji; Jones, Steven J; Marra, Marco A; Holt, Robert A; Moerman, Donald G; Hansen, Dave

2009-01-01

Background Germ cells must progress through elaborate developmental stages from an undifferentiated germ cell to a fully differentiated gamete. Some of these stages include exiting mitosis and entering meiosis, progressing through the various stages of meiotic prophase, adopting either a male (sperm) or female (oocyte) fate, and completing meiosis. Additionally, many of the factors needed to drive embryogenesis are synthesized in the germ line. To increase our understanding of the genes that might be necessary for the formation and function of the germ line, we have constructed a SAGE library from hand dissected C. elegans hermaphrodite gonads. Results We found that 4699 genes, roughly 21% of all known C. elegans genes, are expressed in the adult hermaphrodite germ line. Ribosomal genes are highly expressed in the germ line; roughly four fold above their expression levels in the soma. We further found that 1063 of the germline-expressed genes have enriched expression in the germ line as compared to the soma. A comparison of these 1063 germline-enriched genes with a similar list of genes prepared using microarrays revealed an overlap of 460 genes, mutually reinforcing the two lists. Additionally, we identified 603 germline-enriched genes, supported by in situ expression data, which were not previously identified. We also found >4 fold enrichment for RNA binding proteins in the germ line as compared to the soma. Conclusion Using multiple technological platforms provides a more complete picture of global gene expression patterns. Genes involved in RNA metabolism are expressed at a significantly higher level in the germ line than the soma, suggesting a stronger reliance on RNA metabolism for control of the expression of genes in the germ line. Additionally, the number and expression level of germ line expressed genes on the X chromosome is lower than expected based on a random distribution. PMID:19426519

Tissue-specific time courses of spontaneous mutation frequency and deviations in mutation pattern are observed in middle to late adulthood in Big Blue mice.

PubMed

Hill, Kathleen A; Halangoda, Asanga; Heinmoeller, Petra W; Gonzalez, Kelly; Chitaphan, Chaniga; Longmate, Jeffrey; Scaringe, William A; Wang, Ji-Cheng; Sommer, Steve S

2005-06-01

To better define the time course of spontaneous mutation frequency in middle to late adulthood of the mouse, measurements were made at 10, 14, 17, 23, 25, and 30 months of age in samples of adipose tissue, liver, cerebellum (90% neurons), and the male germline (95% germ cells). A total of 46 million plaque-forming units (pfus) were screened at the six time points and 1,450 circular blue plaques were harvested and sequenced. These data improve resolution and confirm the previously observed occurrence of at least two tissue-specific profiles of spontaneous mutation frequency (elevation with age in adipose tissue and liver, and constancy with age in neurons and male germ cells), a low mutation frequency in the male germline, and a mutation pattern unchanged with age within a tissue. These findings appear to extend to very old age (30 months). Additional findings include interanimal variation in spontaneous mutation frequency is larger in adipose tissues and liver compared with neurons and male germ cells, and subtle but significant differences in the mutation pattern among tissues, consistent with a minor effect of tissue-specific metabolism. The presumptive unaltered balance of DNA damage and repair with age in the male germline has evolutionary consequences. It is of particular interest given the controversy over whether or not increasing germline mutation frequency with paternal age underlies the reports associating older males with a higher incidence of some types of genetic disease. These most detailed measurements available to date regarding the time course of spontaneous mutation frequency and pattern in individual tissues help to constrain hypotheses regarding the role of mutational mechanisms in DNA repair and aging.

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

PubMed Central

Mao, Yanfei; Zhang, Zhengjing; Feng, Zhengyan; Wei, Pengliang; Zhang, Hui; Botella, José Ramón; Zhu, Jian-Kang

2017-01-01

Summary The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population. PMID:26360626

Comparative marker analysis after isolation and culture of testicular cells from the immature marmoset.

PubMed

Albert, Silvia; Wistuba, Joachim; Eildermann, Katja; Ehmcke, Jens; Schlatt, Stefan; Gromoll, Joerg; Kossack, Nina

2012-01-01

The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers - anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) - was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis. Copyright © 2012 S. Karger AG, Basel.

Identification and Migration of Primordial Germ Cells in Atlantic Salmon, Salmo salar: Characterization of Vasa, Dead End, and Lymphocyte Antigen 75 Genes

PubMed Central

Nagasawa, Kazue; Fernandes, Jorge MO; Yoshizaki, Goro; Miwa, Misako; Babiak, Igor

2013-01-01

No information exists on the identification of primordial germ cells (PGCs) in the super-order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full-length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi-quantitative RT-PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole-mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid-blastula stage, vasa-expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp-rt-vasa 3′-UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. PMID:23239145

Exercise and supraphysiological dose of nandrolone decanoate increase apoptosis in spermatogenic cells.

PubMed

Shokri, Saeed; Aitken, Robert John; Abdolvahhabi, Mirabbas; Abolhasani, Farid; Ghasemi, Fahimeh Mohammad; Kashani, Iraj; Ejtemaeimehr, Shahram; Ahmadian, Shahin; Minaei, Bagher; Naraghi, Mohammad Ali; Barbarestani, Mohammad

2010-04-01

Anabolic-androgenic steroids are used at high doses by athletes for improving athletic ability, physical appearance and muscle mass. Unfortunately, the abuse of these agents has significantly increased. It has been established that exercise and high doses of anabolic-androgenic steroids may influence the hypothalamic-pituitary-gonadal axis, which can in turn affect testicular apoptosis. However, the effect of the combination of exercise and high dose of anabolic-androgenic steroids on testicular apoptosis is not known. We investigated the combined effects of exercise and high doses of nandrolone decanoate on apoptosis in the spermatogenic cell lineage. Five groups of male Wistar strain albino rats were treated as follows for 8 weeks: solvent of nandrolone decanoate (peanut oil) as a vehicle (Sham); nandrolone decanoate (10 mg/kg/weekly) (nandrolone decanoate); exercise (1 hr/day, 5 days a week) (exercise); nandrolone decanoate (10 mg/kg/weekly) and exercise (1 hr/day, 5 days a week) (nandrolone decanoate exercise); and sedentary control without any injection or exercise (Control). Apoptosis in the male germ line was characterized by TUNEL, caspase-3 assay and transmission electron microscopy. The weights of the testis and accessory sex organs, as well as sperm parameters significantly decreased in the experimental groups relative to the sham and control groups (p < or = 0.05). Germ cell apoptosis and a significant decrease in the number of germ cell layers in nandrolone decanoate exercise-treated testes were observed (p < or = 0.05). Exercise training seems to increase the extent of apoptotic changes caused by supraphysiological dose of nandrolone decanoate in rats, which in turn affects fertility.

Establishment and optimization of a wheat germ cell-free protein synthesis system and its application in venom kallikrein.

PubMed

Wang, Yunpeng; Xu, Wentao; Kou, Xiaohong; Luo, Yunbo; Zhang, Yanan; Ma, Biao; Wang, Mengsha; Huang, Kunlun

2012-08-01

Wheat germ cell-free protein synthesis systems have the potential to synthesize functional proteins safely and with high accuracy, but the poor energy supply and the instability of mRNA templates reduce the productivity of this system, which restricts its applications. In this report, phosphocreatine and pyruvate were added to the system to supply ATP as a secondary energy source. After comparing the protein yield, we found that phosphocreatine is more suitable for use in the wheat germ cell-free protein synthesis system. To stabilize the mRNA template, the plasmid vector, SP6 RNA polymerase, and Cu(2+) were optimized, and a wheat germ cell-free protein synthesis system with high yield and speed was established. When plasmid vector (30 ng/μl), SP6 RNA polymerase (15 U), phosphocreatine (25 mM), and Cu(2+) (5 mM) were added to the system and incubated at 26°C for 16 h, the yield of venom kallikrein increased from 0.13 to 0.74 mg/ml. The specific activity of the recombinant protein was 1.3 U/mg, which is only slightly lower than the crude venom kallikrein (1.74 U/mg) due to the lack of the sugar chain. In this study, the yield of venom kallikrein was improved by optimizing the system, and a good foundation has been laid for industrial applications and for further studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Germ-line PHD1 and PHD2 mutations detected in patients with pheochromocytoma/paraganglioma-polycythemia.

PubMed

Yang, Chunzhang; Zhuang, Zhengping; Fliedner, Stephanie M J; Shankavaram, Uma; Sun, Michael G; Bullova, Petra; Zhu, Roland; Elkahloun, Abdel G; Kourlas, Peter J; Merino, Maria; Kebebew, Electron; Pacak, Karel

2015-01-01

We have investigated genetic/pathogenetic factors associated with a new clinical entity in patients presenting with pheochromocytoma/paraganglioma (PHEO/PGL) and polycythemia. Two patients without hypoxia-inducible factor 2α (HIF2A) mutations, who presented with similar clinical manifestations, were analyzed for other gene mutations, including prolyl hydroxylase (PHD) mutations. We have found for the first time a germ-line mutation in PHD1 in one patient and a novel germ-line PHD2 mutation in a second patient. Both mutants exhibited reduced protein stability with substantial quantitative protein loss and thus compromised catalytic activities. Due to the unique association of patients' polycythemia with borderline or mildly elevated erythropoietin (EPO) levels, we also performed an in vitro sensitivity assay of erythroid progenitors to EPO and for EPO receptor (EPOR) expression. The results show inappropriate hypersensitivity of erythroid progenitors to EPO in these patients, indicating increased EPOR expression/activity. In addition, the present study indicates that HIF dysregulation due to PHD mutations plays an important role in the pathogenesis of these tumors and associated polycythemia. The PHD1 mutation appears to be a new member contributing to the genetic landscape of this novel clinical entity. Our results support the existence of a specific PHD1- and PHD2-associated PHEO/PGL-polycythemia disorder. • A novel germ-l i n e PHD1 mutation causing heochromocytoma/paraganglioma and polycythemia. • Increased EPOR activity and inappropriate hypersensitivity of erythroid progenitors to EPO.

MEETING REPORT ASSESSING HUMAN GERM-CELL MUTAGENESIS IN THE POST-GENOME ERA: A CELEBRATION OF THE LEGACY OF WILLIAM LAWSON (BILL) RUSSELL

EPA Science Inventory

Although numerous germ-cell mutagens have been identified in animal model systems, to date, no human germ-cell mutagens have been confirmed. Because the genomic integrity of our germ cells is essential for the continuation of the human species, a resolution of this enduring conu...

Distribution of Cytokinin-active Ribonucleosides in Wheat Germ tRNA Species 1

PubMed Central

Struxness, Leslie A.; Armstrong, Donald J.; Gillam, Ian; Tener, Gordon M.; Burrows, William J.; Skoog, Folke

1979-01-01

The distribution of cytokinin activity in wheat (Triticum aestivum) germ tRNA fractionated by BD-cellulose and RPC-5 chromatography has been examined. As in other organisms, the cytokinin moieties in wheat germ tRNA appear to be restricted to tRNA species that would be expected to respond to codons beginning with U. Only a few of the wheat germ tRNA species in this coding group actually contain cytokinin modifications. Cytokinin activity was associated with isoaccepting tRNASer species and with a minor tRNALeu species from wheat germ. All other wheat germ tRNA species corresponding to codons beginning with U were devoid of cytokinin activity in the tobacco callus bioassay. PMID:16660688

Effects of cell wall components on the functionality of wheat gluten.

PubMed

Autio, K

2006-01-01

Normal white wheat flours and especially whole meal flour contain solids from the inner endosperm cell walls, from germ, aleurone layer and the outer layers of cereal grains. These solids can prevent either gluten formation or gas cell structure. The addition of small amounts of pericarp layers (1-2%) to wheat flour had a marked detrimental effect on loaf volume. Microstructural studies indicated that in particular the epicarp hairs appeared to disturb the gas cell structure. The detrimental effects of insoluble cell walls can be prevented by using endoxylanases. It has been shown that some oxidative enzymes, naturally present in flour or added to the dough, will oxidise water-extractable arabinoxylans via ferulic acid bridges, and the resulting arabinoxylan gel will hinder gluten formation. The negative effects of water-unextractable arabinoxylans on gluten yield and rheological properties can be compensated by the addition of ferulic acid. Free ferulic acid can probably prevent arabinoxylan cross-linking via ferulic acid.

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

PubMed

Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells.

PubMed

Liu, Ying; Giannopoulou, Eugenia G; Wen, Duancheng; Falciatori, Ilaria; Elemento, Olivier; Allis, C David; Rafii, Shahin; Seandel, Marco

2016-04-27

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells

PubMed Central

Liu, Ying; Giannopoulou, Eugenia G.; Wen, Duancheng; Falciatori, Ilaria; Elemento, Olivier; Allis, C. David; Rafii, Shahin; Seandel, Marco

2016-01-01

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming. PMID:27117588

Stochastic Seeding Coupled with mRNA Self-Recruitment Generates Heterogeneous Drosophila Germ Granules.

PubMed

Niepielko, Matthew G; Eagle, Whitby V I; Gavis, Elizabeth R

2018-06-18

The formation of ribonucleoprotein assemblies called germ granules is a conserved feature of germline development. In Drosophila, germ granules form at the posterior of the oocyte in a specialized cytoplasm called the germ plasm, which specifies germline fate during embryogenesis. mRNAs, including nanos (nos) and polar granule component (pgc), that function in germline development are localized to the germ plasm through their incorporation into germ granules, which deliver them to the primordial germ cells. Germ granules are nucleated by Oskar (Osk) protein and contain varying combinations and quantities of their constituent mRNAs, which are organized as spatially distinct, multi-copy homotypic clusters. The process that gives rise to such heterogeneous yet organized granules remains unknown. Here, we show that individual nos and pgc transcripts can populate the same nascent granule, and these first transcripts then act as seeds, recruiting additional like transcripts to form homotypic clusters. Within a granule, homotypic clusters grow independently of each other but depend on the simultaneous acquisition of additional Osk. Although granules can contain multiple clusters of a particular mRNA, granule mRNA content is dominated by cluster size. These results suggest that the accumulation of mRNAs in the germ plasm is controlled by the mRNAs themselves through their ability to form homotypic clusters; thus, RNA self-association drives germ granule mRNA localization. We propose that a stochastic seeding and self-recruitment mechanism enables granules to simultaneously incorporate many different mRNAs while ensuring that each becomes enriched to a functional threshold. Copyright © 2018 Elsevier Ltd. All rights reserved.

Germ cell transplantation in an azoospermic Klinefelter bull.

PubMed

Joerg, Hannes; Janett, Fredi; Schlatt, Stefan; Mueller, Simone; Graphodatskaya, Daria; Suwattana, Duangsmorn; Asai, Mika; Stranzinger, Gerald

2003-12-01

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.

Unclassified mixed germ cell-sex cord-stromal tumor with multiple malignant cellular elements in a young woman: a case report and review of the literature.

PubMed

Pang, Shujie; Zhang, Lin; Shi, Yiquan; Liu, Yixin

2014-01-01

Unclassified mixed germ cell-sex cord-stromal tumor composed of germ cells and sex cord derivatives is a rare neoplasm. Approximately 10% of such tumors have malignant germ cell components. We report the case of a 28 year-old female with a right adnexal mass measuring 8 cm in greatest dimension, containing areas with both germ cell and sex cord components. The germ cell portion contained multiple growth patterns with a malignant appearance, while the sex cord element consisted mainly of annular tubules. Within the malignant germ cell elements was a dysgerminoma that accounted for approximately 75% of the tumor volume. Other malignant germ cell elements included yolk sac tumor, embryonal carcinoma, and choriocarcinoma, which comprised about 15% of the tumor volume. The annular tubule structures comprised about 10% of the total tumor volume. To our knowledge, this is the first case reported in the literature of an unclassified mixed germ cell-sex cord-stromal tumor associated with embryonal carcinoma components. The patient had a 46XX karyotype, regular menstrual periods, and no evidence of gross abnormalities in the contralateral ovary. The patient remained clinically well and disease-free 2 years after surgery. In addition to a thorough case description, the literature concerning this entity is reviewed and discussed.

The planarian nanos-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration.

PubMed

Handberg-Thorsager, Mette; Saló, Emili

2007-05-01

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians.

Childhood Central Nervous System Germ Cell Tumors Treatment (PDQ®)—Patient Version

Cancer.gov

Childhood central nervous system (CNS) germ cell tumors form from germ cells (a type of cell that forms as a fetus develops and later becomes sperm in the testicles or eggs in the ovaries). Learn about the signs, tests to diagnose, and treatment of pediatric germ cell tumors in the brain in this expert-reviewed summary.

Mechano-logical model of C. elegans germ line suggests feedback on the cell cycle

PubMed Central

Atwell, Kathryn; Qin, Zhao; Gavaghan, David; Kugler, Hillel; Hubbard, E. Jane Albert; Osborne, James M.

2015-01-01

The Caenorhabditis elegans germ line is an outstanding model system in which to study the control of cell division and differentiation. Although many of the molecules that regulate germ cell proliferation and fate decisions have been identified, how these signals interact with cellular dynamics and physical forces within the gonad remains poorly understood. We therefore developed a dynamic, 3D in silico model of the C. elegans germ line, incorporating both the mechanical interactions between cells and the decision-making processes within cells. Our model successfully reproduces key features of the germ line during development and adulthood, including a reasonable ovulation rate, correct sperm count, and appropriate organization of the germ line into stably maintained zones. The model highlights a previously overlooked way in which germ cell pressure may influence gonadogenesis, and also predicts that adult germ cells might be subject to mechanical feedback on the cell cycle akin to contact inhibition. We provide experimental data consistent with the latter hypothesis. Finally, we present cell trajectories and ancestry recorded over the course of a simulation. The novel approaches and software described here link mechanics and cellular decision-making, and are applicable to modeling other developmental and stem cell systems. PMID:26428008

Lin28a regulates germ cell pool size and fertility

PubMed Central

Shinoda, Gen; de Soysa, T. Yvanka; Seligson, Marc T.; Yabuuchi, Akiko; Fujiwara, Yuko; Huang, Pei Yi; Hagan, John P.; Gregory, Richard I.; Moss, Eric G.; Daley, George Q.

2013-01-01

Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage. PMID:23378032

Ancient connection between NKL genes and the mesoderm? Insights from Tlx expression in a ctenophore.

PubMed

Derelle, Romain; Manuel, Michaël

2007-04-01

In recent years, evo-devo studies on non-bilaterian metazoans have improved our understanding of the early evolution of animal body plans. In particular, works on cnidarians suggested that contrary to classical views, the mesoderm originated far before the emergence of the Bilateria. In this context, a synthesis of genomic and functional data concerning the Antennapedia (Antp) superclass of homeobox genes suggested that early in animal evolution, each of the three germ layers was under the control of one cluster of Antp genes. In particular, the patterning and differentiation of the mesoderm was under the control of the NKL cluster. The ctenophores stand as a key taxon with respect to such issues because unlike other non-bilaterian phyla, their intermediate germ layer satisfies the strict embryological definition of a mesoderm. For that reason, we investigated the only known member of the NKL group in Ctenophora, a gene previously isolated from Pleurobrachia and attributed to the Tlx family. In our analysis of the NKL group, this ctenophore gene branches as the sister-group of bilaterian Tlx genes, but without statistical support. The expression pattern of this gene was revealed by in situ hybridisation in the adult ctenophore. The expression territories of PpiTlx are predominantly ectodermal, in two distinct types of ciliated epidermal cells and in one category of gland cells. We also identified a probable endodermal site of expression. Because we failed to detect any mesodermal expression, the results do not provide support to the hypothesis of an ancient functional association between the NKL group and the mesoderm.

Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

PubMed

Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

2016-01-01

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

Slow Cooling Cryopreservation Optimized to Human Pluripotent Stem Cells.

PubMed

Miyazaki, Takamichi; Suemori, Hirofumi

2016-01-01

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cells that form all three germ layers. Cryopreservation is one of the key processes for successful applications of hPSCs, because it allows semi-permanent preservation of cells and their easy transportation. Most animal cell lines, including mouse embryonic stem cells, are standardly cryopreserved by slow cooling; however, hPSCs have been difficult to preserve and their cell viability has been extremely low whenever cryopreservation has been attempted.Here, we investigate the reasons for failure of slow cooling in hPSC cryopreservation. Cryopreservation involves a series of steps and is not a straightforward process. Cells may die due to various reasons during cryopreservation. Indeed, hPSCs preserved by traditional methods often suffer necrosis during the freeze-thawing stages, and the colony state of hPSCs prior to cryopreservation is a major factor contributing to cell death.It has now become possible to cryopreserve hPSCs using conventional cryopreservation methods without any specific equipment. This review summarizes the advances in this area and discusses the optimization of slow cooling cryopreservation for hPSC storage.

A novel intranuclear RNA vector system for long-term stem cell modification

PubMed Central

Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

2015-01-01

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

Generation of Arbas Cashmere Goat Induced Pluripotent Stem Cells Through Fibroblast Reprogramming.

PubMed

Tai, Dapeng; Liu, Pengxia; Gao, Jing; Jin, Muzi; Xu, Teng; Zuo, Yongchun; Liang, Hao; Liu, Dongjun

2015-08-01

Various factors affect the process of obtaining stable Arbas cashmere goat embryonic stem cells (ESCs), for example, the difficulty in isolating cells at the appropriate stage of embryonic development, the in vitro culture environment, and passage methods. With the emergence of induced pluripotent stem cell (iPSC) technology, it has become possible to use specific genes to induce somatic cell differentiation in PSCs. We transferred OCT4, SOX2, c-MYC, and KLF4 into Arbas cashmere goat fetal fibroblasts, then induced and cultured them using a drug-inducible system to obtain Arbas goat iPSCs that morphologically resembled mouse iPSCs. After identification, the obtained goat iPSCs expressed ESC markers, had a normal karyotype, could differentiate into embryoid bodies in vitro, and could differentiate into three germ layer cell types and form teratomas in vivo. We used microarray gene expression profile analysis to elucidate the reprogramming process. Our results provide the experimental basis for establishing cashmere goat iPSC lines and for future in-depth studies on molecular mechanism of cashmere goat somatic cell reprogramming.

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

PubMed Central

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-01-01

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a−/− embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a−/− embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a−/− ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis. PMID:27026076

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

PubMed

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-03-30

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

Transcriptional control of stem cell fate by E2Fs and pocket proteins

PubMed Central

Julian, Lisa M.; Blais, Alexandre

2015-01-01

E2F transcription factors and their regulatory partners, the pocket proteins (PPs), have emerged as essential regulators of stem cell fate control in a number of lineages. In mammals, this role extends from both pluripotent stem cells to those encompassing all embryonic germ layers, as well as extra-embryonic lineages. E2F/PP-mediated regulation of stem cell decisions is highly evolutionarily conserved, and is likely a pivotal biological mechanism underlying stem cell homeostasis. This has immense implications for organismal development, tissue maintenance, and regeneration. In this article, we discuss the roles of E2F factors and PPs in stem cell populations, focusing on mammalian systems. We discuss emerging findings that position the E2F and PP families as widespread and dynamic epigenetic regulators of cell fate decisions. Additionally, we focus on the ever expanding landscape of E2F/PP target genes, and explore the possibility that E2Fs are not simply regulators of general ‘multi-purpose’ cell fate genes but can execute tissue- and cell type-specific gene regulatory programs. PMID:25972892

Changes in cell surface structure by viral transformation studied by binding of lectins differing in sugar specificity.

PubMed

Tsuda, M; Kurokawa, T; Takeuchi, M; Sugino, Y

1975-10-01

Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown in coverslips. The following 125I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetyl-galactosamine). Cells for a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure. The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

Effects of tunicamycin, mannosamine, and other inhibitors of glycoprotein processing on skeletal alkaline phosphatase in human osteoblast-like cells.

PubMed

Farley, J R; Magnusson, P

2005-01-01

Skeletal alkaline phosphatase (sALP) is a glycoprotein- approximately 20% carbohydrate by weight, with five presumptive sites for N-linked glycosylation, as well as a carboxy-terminal site for attachment of the glycolipid structure (glycosylphosphatidylinositol, GPI), which anchors sALP to the outer surface of osteoblasts. The current studies were intended to characterize the effects of inhibiting glycosylation and glycosyl-processing on the synthesis, plasma membrane attachment, cellular-extracellular distribution, and reaction kinetics of sALP in human osteosarcoma (SaOS-2) cells. sALP synthesis, glycosylation, and GPI-anchor attachment were assessed as total protein synthesis/immunospecific sALP synthesis, sialic acid content (i.e., wheat germ agglutinin precipitation), and insolubility (i.e., temperature-dependent phase-separation), respectively. sALP reaction kinetics were characterized by analysis of dose-dependent initial velocity data, with a phosphoryl substrate. The results of these studies revealed that the inhibition of either N-linked glycosylation or oligosaccharide synthesis for GPI-anchor addition could affect the synthesis and the distribution of sALP, but not the kinetics of the phosphatase reaction. Tunicamycin-which blocks N-linked glycosylation by inhibiting core oligosaccharide synthesis-decreased cell layer protein and the total amount of sALP in the cells, while increasing the relative level of sALP in the cell-conditioned culture medium (CM, i.e., the amount of sALP released). These effects were attributed to dose- and time-dependent decreases in sALP synthesis and N-linked glycosylation, and an increase in apoptotic cell death (P <0.001 for each). In contrast to the effects of tunicamycin on N-linked glycosylation, the effects of mannosamine, which inhibits GPI-anchor glycosylation/formation, included (1) an increase in cell layer protein; (2) decreases in sALP specific activity, in the cells and in the CM; and (3) increases in the percentages of both anchorless and wheat germ agglutinin (WGA)-soluble sALP in the medium, but not in the cells (P <0.005 for each). These effects of mannosamine were, presumably, a consequence of inhibiting the insertion/attachment of sALP to the outside of the plasma membrane surface. Neither mannosammine nor tunicamycin had any effect on the reaction kinetics of sALP or on the apparent affinity (the value of KM) for the phosphoryl substrate.

Isolation and purification of wheat germ agglutinin and analysis of its properties

NASA Astrophysics Data System (ADS)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Extragonadal Germ Cell Cancer (EGC)

MedlinePlus

The Testicular Cancer Resource Center Extragonadal Germ Cell Cancer (EGC) 95% of all testicular tumors are germ cell tumors. That is, the tumors originate in the sperm forming cells in the testicles ( ...

Childhood Extracranial Germ Cell Tumors Treatment (PDQ®)—Patient Version

Cancer.gov

Childhood extracranial germ cell tumors treatment options include surgery, observation, and chemotherapy. Learn more about newly diagnosed and recurrent extracranial germ cell tumors in this expert-reviewed summary.

Germ Plasm Biogenesis--An Oskar-Centric Perspective.

PubMed

Lehmann, Ruth

2016-01-01

Germ granules are the hallmark of all germ cells. These membrane-less, electron-dense structures were first observed over 100 years ago. Today, their role in regulating and processing transcripts critical for the establishment, maintenance, and protection of germ cells is well established, and pathways outlining the biochemical mechanisms and physical properties associated with their biogenesis are emerging. © 2016 Elsevier Inc. All rights reserved.

Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

PubMed

Sinha, Debottam; Kalimutho, Murugan; Bowles, Josephine; Chan, Ai-Leen; Merriner, D Jo; Bain, Amanda L; Simmons, Jacinta L; Freire, Raimundo; Lopez, J Alejandro; Hobbs, Robin M; O'Bryan, Moira K; Khanna, Kum Kum

2018-04-17

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein (CEP)-55 is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 protein (TEX14), has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic mice ( Cep55 Tg/Tg ) aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55 Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

The Mouse House: A brief history of the ORNL mouse-genetics program, 1947–2009

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, Liane B.

The large mouse genetics program at the Oak Ridge National Lab is often re-membered chiefly for the germ-cell mutation-rate data it generated and their uses in estimating the risk of heritable radiation damage. In fact, it soon became a multi-faceted research effort that, over a period of almost 60 years, generated a wealth of information in the areas of mammalian mutagenesis, basic genetics (later enriched by molecular techniques), cytogenetics, reproductive biology, biochemistry of germ cells, and teratology. Research in the area of germ-cell mutagenesis explored the important physical and biological factors that affect the frequency and nature of induced mutationsmore » and made several unexpected discoveries, such as the major importance of the perigametic interval (the zygote stage) for the origin of spontaneous mutations and for the sensitivity to induced genetic change. Of practical value was the discovery that ethylnitrosourea was a supermutagen for point mutations, making high-efficiency mutagenesis in the mouse feasible worldwide. Teratogenesis findings resulted in recommendations still generally accepted in radiological practice. Studies supporting the mutagenesis research added whole bodies of information about mammalian germ-cell development and about molecular targets in germ cells. The early decision to not merely count but propagate genetic variants of all sorts made possible further discoveries, such as the Y-Chromosome s importance in mammalian sex determination and the identification of rare X-autosome translocations, which, in turn, led to the formulation of the single-active-X hypothesis and provided tools for studies of functional mosaicism for autosomal genes, male sterility, and chromosome-pairing mechanism. Extensive genetic and then molecular analyses of large numbers of induced specific-locus mutants resulted in fine-structure physical and correlated functional mapping of significant portions of the mouse genome and constituted a valuable source of mouse models for human genetic disorders.« less

Lack of commensal flora in H. pylori-infected INS-GAS mice reduces gastritis and delays intraepithelial neoplasia

PubMed Central

Lofgren, Jennifer L.; Whary, Mark T.; Ge, Zhongming; Muthupalani, Sureshkumar; Taylor, Nancy S.; Mobley, Melissa; Potter, Amanda; Varro, Andrea; Eibach, Daniel; Suerbaum, Sebastian; Wang, Timothy C.; Fox, James G.

2010-01-01

Background & Aims Transgenic, insulin–gastrin (INS–GAS) mice have high circulating levels of gastrin. On a FVB/N background, these mice develop spontaneous atrophic gastritis and gastrointestinal intraepithelial neoplasia (GIN) with 80% prevalence 6 months after Helicobacter pylori infection. GIN is associated with gastric atrophy and achlorhydria, predisposing mice to non-helicobacter microbiota overgrowth. We determined if germ-free INS–GAS mice spontaneously develop GIN and if H. pylori accelerates GIN in gnotobiotic INS–GAS mice. Methods We compared gastric lesions and levels of mRNA, serum inflammatory mediators, antibodies, and gastrin among germ-free and H. pylori-monoinfected INS-GAS mice. Microbiota composition of specific pathogen-free (SPF) INS-GAS mice was quantified by pyro-sequencing. Results Germ-free INS-GAS mice had mild hypergastrinemia but did not develop significant gastric lesions until they were 9 months old; they did not develop GIN through 13 months. H. pylori monoassociation caused progressive gastritis, epithelial defects, oxyntic gland atrophy, marked foveolar hyperplasia and dysplasia, and strong serum and tissue proinflammatory immune responses (particularly in male mice) between 5 and 11 months post infection (P<0.05, compared with germ-free controls). Only 2 of 26 female, whereas 8 of 18 male, H. pylori-infected INS-GAS mice developed low- to high-grade GIN by 11 months post infection. Stomachs of H. pylori-infected SPF male mice had significant reductions in Bacteroidetes and significant increases in Firmicutes. Conclusions Gastric lesions take 13 months longer to develop in germ-free INS–GAS mice than male SPF INS-GAS mice. H. pylori-monoassociation accelerated gastritis and GIN but caused less-severe gastric lesions and delayed onset of GIN compared to H. pylori-infected INS-GAS mice with complex gastric microbiota. Changes of gastric microbiota composition might promote GIN in the achlorhydric stomachs of SPF mice. PMID:20950613

The Mouse House: a brief history of the ORNL mouse-genetics program, 1947-2009.

PubMed

Russell, Liane B

2013-01-01

The large mouse genetics program at the Oak Ridge National Laboratory (ORNL) is often remembered chiefly for the germ-cell mutation-rate data it generated and their uses in estimating the risk of heritable radiation damage. In fact, it soon became a multi-faceted research effort that, over a period of almost 60 years, generated a wealth of information in the areas of mammalian mutagenesis, basic genetics (later enriched by molecular techniques), cytogenetics, reproductive biology, biochemistry of germ cells, and teratology. Research in the area of germ-cell mutagenesis explored the important physical and biological factors that affect the frequency and nature of induced mutations and made several unexpected discoveries, such as the major importance of the perigametic interval (the zygote stage) for the origin of spontaneous mutations and for the sensitivity to induced genetic change. Of practical value was the discovery that ethylnitrosourea was a supermutagen for point mutations, making high-efficiency mutagenesis in the mouse feasible worldwide. Teratogenesis findings resulted in recommendations still generally accepted in radiological practice. Studies supporting the mutagenesis research added whole bodies of information about mammalian germ-cell development and about molecular targets in germ cells. The early decision to not merely count but propagate genetic variants of all sorts made possible further discoveries, such as the Y-chromosome's importance in mammalian sex determination and the identification of rare X-autosome translocations, which, in turn, led to the formulation of the single-active-X hypothesis and provided tools for studies of functional mosaicism for autosomal genes, male sterility, and chromosome-pairing mechanism. Extensive genetic and then molecular analyses of large numbers of induced specific-locus mutants resulted in fine-structure physical and correlated functional mapping of significant portions of the mouse genome and constituted a valuable source of mouse models for human genetic disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

SALL4 EXPRESSION IN GERM CELL AND NON GERM-CELL TUMORS – A SYSTEMATIC IMMUNOHISTOCHEMICAL STUDY OF 3215 CASES

PubMed Central

Miettinen, Markku; Wang, Zengfeng; Mc. Cue, Peter A.; Sarlomo-Rikala, Maarit; Rys, Janusz; Biernat, Wojciech; Lasota, Jerzy; Lee, Yi-Shan

2014-01-01

SALL4 transcription factor is associated with embryonic cell pluripotency and has been shown as a useful immunohistochemical marker for germ cell tumors. However, information of SALL4 distribution in normal human tissues and non germ-cell tumors is limited. In this study we examined normal human tissues and 3215 tumors for SALL4 expression using a monoclonal antibody 6E3 and automated immunohistochemistry. In a 10th week embryo, SALL4 was expressed in ovocytes, intestine, kidney, and some hepatocytes. In adult tissues, it was only detected in germ cells. SALL4 was consistently expressed in all germ cell tumors except some trophoblastic tumors and mature components of teratomas, where it was selectively expressed in intestinal-like and some squamous epithelia. In non germ-cell carcinomas, SALL4 was detected in 20% of cases or more of serous carcinoma of ovary, urothelial high-grade carcinoma, and gastric adenocarcinoma (especially the intestinal type). SALL4 was only rarely (≤5%) expressed in mammary, colorectal, prostatic, and squamous cell carcinomas. Many SALL4 positive carcinomas showed poorly differentiated patterns and some showed positivity in most tumor cells mimicking the expression in germ cell tumors. SALL4 was commonly expressed in rhabdoid tumors of kidney and extrarenal sites, and in Wilms tumor. Expression of SALL4 was rare in other mesenchymal and neuroendocrine tumors but was occasionally detected in melanoma, desmoplastic small round cell tumor, epithelioid sarcoma, and rhabdomyosarcoma. All hematopoietic tumors were negative. SALL4 is an excellent marker of non-teratomatous germ cell tumors, but it is also expressed in other tumors, sometimes extensively. Such expression may reflect stem-cell like differentiation and must be considered when using SALL4 as a marker for germ cell tumors. Observed lack of other pluripotency factors, OCT4 and NANOG, in SALL4-positive non-germ cell tumors can also be diagnostically helpful. PMID:24525512

Combination Chemotherapy Followed by Bone Marrow Transplantation in Treating Patients With Rare Cancer

ClinicalTrials.gov

2013-06-20

Childhood Germ Cell Tumor; Extragonadal Germ Cell Tumor; Head and Neck Cancer; Kidney Cancer; Liver Cancer; Lymphoma; Neuroblastoma; Ovarian Cancer; Retinoblastoma; Sarcoma; Testicular Germ Cell Tumor

Ovarian Germ Cell Tumors Treatment (PDQ®)—Health Professional Version

Cancer.gov

Ovarian germ cell tumors treatment options include surgery, chemotherapy, and radiation therapy. Get detailed treatment information for newly diagnosed or recurrent germ cell tumors in this summary for clinicians.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

PubMed

Linhartová, Zuzana; Saito, Taiju; Kašpar, Vojtěch; Rodina, Marek; Prášková, Eva; Hagihara, Seishi; Pšenička, Martin

2015-10-15

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Ovarian Germ Cell Tumors Treatment

MedlinePlus

... Tube, & Primary Peritoneal Cancer Screening Research Ovarian Germ Cell Tumors Treatment (PDQ®)–Patient Version Treatment Option Overview ... types of treatment for patients with ovarian germ cell tumors. Different types of treatment are available for ...

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

PubMed Central

Maatouk, Danielle M.; Mork, Lindsey; Hinson, Ashley; Kobayashi, Akio; McMahon, Andrew P.; Capel, Blanche

2012-01-01

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells. PMID:23091613

Immature germ cells in semen - correlation with total sperm count and sperm motility.

PubMed

Patil, Priya S; Humbarwadi, Rajendra S; Patil, Ashalata D; Gune, Anita R

2013-07-01

Current data regarding infertility suggests that male factor contributes up to 30% of the total cases of infertility. Semen analysis reveals the presence of spermatozoa as well as a number of non-sperm cells, presently being mentioned in routine semen report as "round cells" without further differentiating them into leucocytes or immature germ cells. The aim of this work was to study a simple, cost-effective, and convenient method for differentiating the round cells in semen into immature germ cells and leucocytes and correlating them with total sperm counts and motility. Semen samples from 120 males, who had come for investigation for infertility, were collected, semen parameters recorded, and stained smears studied for different round cells. Statistical analysis of the data was done to correlate total sperm counts and sperm motility with the occurrence of immature germ cells and leucocytes. The average shedding of immature germ cells in different groups with normal and low sperm counts was compared. The clinical significance of "round cells" in semen and their differentiation into leucocytes and immature germ cells are discussed. Round cells in semen can be differentiated into immature germ cells and leucocytes using simple staining methods. The differential counts mentioned in a semen report give valuable and clinically relevant information. In this study, we observed a negative correlation between total count and immature germ cells, as well as sperm motility and shedding of immature germ cells. The latter was statistically significant with a P value 0.000.

Germ line determinants are not localized early in sea urchin development, but do accumulate in the small micromere lineage.

PubMed

Juliano, Celina E; Voronina, Ekaterina; Stack, Christie; Aldrich, Maryanna; Cameron, Andrew R; Wessel, Gary M

2006-12-01

Two distinct modes of germ line determination are used throughout the animal kingdom: conditional-an inductive mechanism, and autonomous-an inheritance of maternal factors in early development. This study identifies homologs of germ line determinants in the sea urchin Strongylocentrotus purpuratus to examine its mechanism of germ line determination. A list of conserved germ-line associated genes from diverse organisms was assembled to search the S. purpuratus genome for homologs, and the expression patterns of these genes were examined during embryogenesis by whole mount in situ RNA hybridization and QPCR. Of the 14 genes tested, all transcripts accumulate uniformly during oogenesis and Sp-pumilio, Sp-tudor, Sp-MSY, and Sp-CPEB1 transcripts are also uniformly distributed during embryonic development. Sp-nanos2, Sp-seawi, and Sp-ovo transcripts, however, are enriched in the vegetal plate of the mesenchyme blastula stage and Sp-vasa, Sp-nanos2, Sp-seawi, and Sp-SoxE transcripts are localized in small micromere descendents at the tip of the archenteron during gastrulation and are then enriched in the left coelomic pouch of larvae. The results of this screen suggest that sea urchins conditionally specify their germ line, and support the hypothesis that this mechanism is the basal mode of germ line determination amongst deuterostomes. Furthermore, accumulation of germ line determinants selectively in small micromere descendents supports the hypothesis that these cells contribute to the germ line.

Cancer incidence in men with Klinefelter syndrome.

PubMed Central

Hasle, H.; Mellemgaard, A.; Nielsen, J.; Hansen, J.

1995-01-01

Many case reports have suggested an association between Klinefelter syndrome (KS) and cancer, but studies of the cancer incidence in larger groups of men with KS are lacking. A cohort of 696 men with KS was established from the Danish Cytogenetic Register. Information on the cancer incidence in the cohort was obtained from the Danish Cancer Registry and compared with the expected number calculated from the age, period and site specific cancer rates for Danish men. A total of 39 neoplasms were diagnosed (relative risk = 1.1). Four mediastinal tumours were observed (relative risk = 67); all four were malignant germ cell tumours. No cases of breast cancer or testis cancer were observed. One case of prostate cancer occurred within a previously irradiated field. No excess of leukaemia or lymphoma was found. An increased risk of cancer occurred in the age group 15-30 years (relative risk = 2.7). All six tumours in this group were germ cell tumours or sarcomas. The overall cancer incidence is not increased and no routine cancer screening seems to be justified. A considerably elevated risk of mediastinal germ cell tumours occurs in the period from early adolescence until the age of 30. PMID:7841064

Effect of KnockOut serum replacement on germ cell development of immature testis tissue culture.

PubMed

Liu, Feng; Cai, Chunhong; Wu, Xin; Cheng, Yanxia; Lin, Tao; Wei, Guanghui; He, Dawei

2016-01-15

To compare KnockOut serum replacement (KSR) and fetal bovine serum (FBS) for the development of germ cells. Testicular tissues from Sprague-Dawley rats were cultured for 4 weeks in culture media supplemented with FBS or KSR. Tissue area was measured at the beginning and end of the culturing period. Testicular histology, development of the germ cells, and the diameter of seminiferous tubules were analyzed by hematoxylin and eosin staining. After 4 weeks in culture, apoptosis and expression of the stage-specific spermatogenesis marker genes Kit, Sycp3, and Crisp1 were assayed. Tissues cultured in KSR-supplemented media were able to sustain growth and gradually increase seminiferous tubule diameter during the culture period. In addition, spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids were observed after 4 weeks in culture, and reverse transcription-PCR confirmed expression of the marker genes. In comparison, tissues cultured in FBS-supplemented media showed dwindling testicular organization, necrotic seminiferous tubules, and expression of Kit, but inconsistent expression of Sycp3 and Crisp1 KnockOut serum replacement outperforms FBS as a growth media supplements for culturing immature spermatogonial tissue culture. Copyright © 2016 Elsevier Inc. All rights reserved.

Scrotal insulation and sperm production in the boar.

PubMed

Parrish, John J; Willenburg, Kilby L; Gibbs, Katelynn M; Yagoda, Kylie B; Krautkramer, Megan M; Loether, Teyanna M; Melo, Fabiana C S A

2017-09-01

Seasonal infertility is a limiting factor in boar fertility, and is increasingly important as climate changes. Spermatogenesis in the boar produces 256 spermatozoa per type A 1 spermatogonium, but the process is inefficient such that only 10-30% of these potential spermatozoa are actually produced. Heat further impacts spermatogenesis by reducing the number of specific germ cells produced while increasing the fraction of abnormal sperm. Early studies used whole-animal exposure to simulate seasonal exposure to heat under production settings, but this approach is associated with many confounding factors that make assessment of the mechanisms of heat-induced damage to spermatogenesis difficult. Scrotal insulation provides a better model to investigate the mechanisms and potential mitigation strategies of heat-induce damage. For example, scrotal insulation helped identify a link between short-term heat stress and damage to meiotic germ cells. This outcome is likely due to changes in the integrity of the blood-testis barrier, which induce apoptosis, autophagy and DNA damage in the germ cells. Further understanding how heat damages spermatogenesis, and whether or not this can be repaired, are crucial to mitigating heat effects on boars in production settings. © 2017 Wiley Periodicals, Inc.

Restriction of the Xenopus DEADSouth mRNA to the primordial germ cells is ensured by multiple mechanisms.

PubMed

Yamaguchi, Takeshi; Kataoka, Kensuke; Watanabe, Kenji; Orii, Hidefumi

2014-02-01

DEADSouth mRNA encoding the RNA helicase DDX25 is a component of the germ plasm in Xenopus laevis. We investigated the mechanisms underlying its specific mRNA expression in primordial germ cells (PGCs). Based on our previous findings of several microRNA miR-427 recognition elements (MREs) in the 3' untranslated region of the mRNA, we first examined whether DEADSouth mRNA was degraded by miR-427 targeting in somatic cells. Injection of antisense miR-427 oligomer and reporter mRNA for mutated MREs revealed that DEADSouth mRNA was potentially degraded in somatic cells via miR-427 targeting, but not in PGCs after the mid-blastula transition (MBT). The expression level of miR-427 was very low in PGCs, which probably resulted in the lack of miR-427-mediated degradation. In addition, the DEADSouth gene was expressed zygotically after MBT. Thus, the predominant expression of DEADSouth mRNA in the PGCs is ensured by multiple mechanisms including zygotic expression and prohibition from miR-427-mediated degradation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.