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Sample records for preimplantation mammalian development

  1. Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development.

    PubMed

    Palmer, N; Kaldis, P

    2016-01-01

    The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages. This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events. Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family. This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development. © 2016 Elsevier Inc. All rights reserved.

  2. The influence of growth factors on the development of preimplantation mammalian embryos.

    PubMed

    Díaz-Cueto, L; Gerton, G L

    2001-01-01

    The development of the preimplantation mammalian embryo from a fertilized egg to a blastocyst capable of implanting in the uterus is a complex process. Cell division must be carefully programmed. The embryonic genome must be activated at the appropriate stage of development, and the pattern of gene expression must be carefully coordinated for the initiation of the correct program of differentiation. Cell fates must be chosen to establish specific cell types such as the inner cell mass and the trophectoderm, which give rise to the embryo proper and the placenta, respectively. This review summarizes recent findings concerning the influence of growth factors on the development of preimplantation mammalian embryos. Maternal factors secreted into the lumen of the female reproductive tract as well as substances synthesized by the developing embryo itself help to regulate this process. Studies of embryos in culture and investigations using homologous recombination to create embryos and animals null for specific genes have enabled the identification of several growth factors that appear essential for preimplantation mammalian embryo development. Some of the factors are required maternal factors; others are embryo-derived autocrine and paracrine factors. Studies using molecular biology are beginning to identify differences in the patterns of genes expressed by naturally derived embryos and those developing in culture. The knowledge gained from studies on growth factors, media, embryonic development, and gene expression should help improve culture conditions for embryos and will provide for safer outcomes from assisted reproductive procedures in human and animal clinics.

  3. Impact of maternal malnutrition during the periconceptional period on mammalian preimplantation embryo development.

    PubMed

    Velazquez, M A

    2015-04-01

    During episodes of undernutrition and overnutrition the mammalian preimplantation embryo undergoes molecular and metabolic adaptations to cope with nutrient deficits or excesses. Maternal adaptations also take place to keep a nutritional microenvironment favorable for oocyte development and embryo formation. This maternal-embryo communication takes place via several nutritional mediators. Although adaptive responses to malnutrition by both the mother and the embryo may ensure blastocyst formation, the resultant quality of the embryo can be compromised, leading to early pregnancy failure. Still, studies have shown that, although early embryonic mortality can be induced during malnutrition, the preimplantation embryo possesses an enormous plasticity that allows it to implant and achieve a full-term pregnancy under nutritional stress, even in extreme cases of malnutrition. This developmental strategy, however, may come with a price, as shown by the adverse developmental programming induced by even subtle nutritional challenges exerted exclusively during folliculogenesis and the preimplantation period, resulting in offspring with a higher risk of developing deleterious phenotypes in adulthood. Overall, current evidence indicates that malnutrition during the periconceptional period can induce cellular and molecular alterations in preimplantation embryos with repercussions for fertility and postnatal health. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Effects of simulated microgravity on mammalian fertilization and preimplantation embryonic development in vitro.

    PubMed

    Kojima, Y; Sasaki, S; Kubota, Y; Ikeuchi, T; Hayashi, Y; Kohri, K

    2000-12-01

    To study the effects of simulated microgravity on mammalian fertilization and preimplantation embryonic development in vitro with the use of a horizontal clinostat device. Controlled animal study. Research laboratory at a university medical school. B6D2F1 (C57BL/6 x DBA/2) and ICR mice between 8 and 10 weeks old. The first experiment was performed to investigate whether gravity is required for fertilization in vitro under three conditions: clinostat rotation, rotational control, and stationary control. In the second experiment, one-cell embryos were cultured under each condition and their morphology and viability were assessed at 96 hours. The fertilized numbers and embryonic numbers at the morula and blastocyst stages were recorded in each condition. In the first experiment, there were no statistically significant differences in the efficiency of achieving normal fertilization in vitro among the conditions. In the second experiment, there was a statistically significant decrease in the number of embryos reaching the morula and blastocyst stages after 96 hours in culture under clinostat rotation. These results suggest that the process of fertilization in vitro is not sensitive to the gravitational vector. However, the possibility exists that the frequency of early embryonic lethality is increased by microgravity.

  5. Cell Arrest and Cell Death in Mammalian Preimplantation Development: Lessons from the Bovine Model

    PubMed Central

    Leidenfrost, Sandra; Boelhauve, Marc; Reichenbach, Myriam; Güngör, Tuna; Reichenbach, Horst-Dieter; Sinowatz, Fred; Wolf, Eckhard; Habermann, Felix A.

    2011-01-01

    Background The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. Methods and Findings To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. Conclusions In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development. PMID

  6. Cell arrest and cell death in mammalian preimplantation development: lessons from the bovine model.

    PubMed

    Leidenfrost, Sandra; Boelhauve, Marc; Reichenbach, Myriam; Güngör, Tuna; Reichenbach, Horst-Dieter; Sinowatz, Fred; Wolf, Eckhard; Habermann, Felix A

    2011-01-01

    The causes, modes, biological role and prospective significance of cell death in preimplantation development in humans and other mammals are still poorly understood. Early bovine embryos represent a very attractive experimental model for the investigation of this fundamental and important issue. To obtain reference data on the temporal and spatial occurrence of cell death in early bovine embryogenesis, three-dimensionally preserved embryos of different ages and stages of development up to hatched blastocysts were examined in toto by confocal laser scanning microscopy. In parallel, transcript abundance profiles for selected apoptosis-related genes were analyzed by real-time reverse transcriptase-polymerase chain reaction. Our study documents that in vitro as well as in vivo, the first four cleavage cycles are prone to a high failure rate including different types of permanent cell cycle arrest and subsequent non-apoptotic blastomere death. In vitro produced and in vivo derived blastocysts showed a significant incidence of cell death in the inner cell mass (ICM), but only in part with morphological features of apoptosis. Importantly, transcripts for CASP3, CASP9, CASP8 and FAS/FASLG were not detectable or found at very low abundances. In vitro and in vivo, errors and failures of the first and the next three cleavage divisions frequently cause immediate embryo death or lead to aberrant subsequent development, and are the main source of developmental heterogeneity. A substantial occurrence of cell death in the ICM even in fast developing blastocysts strongly suggests a regular developmentally controlled elimination of cells, while the nature and mechanisms of ICM cell death are unclear. Morphological findings as well as transcript levels measured for important apoptosis-related genes are in conflict with the view that classical caspase-mediated apoptosis is the major cause of cell death in early bovine development.

  7. Signaling pathways in mammalian preimplantation development: Linking cellular phenotypes to lineage decisions.

    PubMed

    Menchero, Sergio; Rayon, Teresa; Andreu, Maria Jose; Manzanares, Miguel

    2017-04-01

    The first stages of mammalian development, before implantation of the embryo in the maternal uterus, result in the establishment of three cell populations in the blastocyst: trophectoderm, epiblast, and primitive endoderm. These events involve only a small number of cells, and are initiated by morphological differences among them related to cell adhesion and polarity. Much attention has been paid to the master transcription factors that are critical for establishing and maintaining early lineage choices. Nevertheless, a large body of work also reveals that additional molecular mechanisms are involved. Here, we provide an updated view of the role of different signaling pathways in the first stages of mouse development, and how their cross-talk and interplay determine the initial lineage decisions occurring in the blastocyst. We will also discuss how these pathways are critical for translating cellular phenotypes, the product of the morphogenetic events occurring at these stages, into transcriptional responses and expression of lineage-specifying transcription factors. Developmental Dynamics 246:245-261, 2017. © 2016 Wiley Periodicals, Inc.

  8. Towards Functional Annotation of the Preimplantation Transcriptome: An RNAi Screen in Mammalian Embryos

    PubMed Central

    Cui, Wei; Dai, Xiangpeng; Marcho, Chelsea; Han, Zhengbin; Zhang, Kun; Tremblay, Kimberly D.; Mager, Jesse

    2016-01-01

    With readily available transcriptome-wide data, understanding the role of each expressed gene is an essential next step. Although RNAi technologies allow for genome-wide screens in cell culture, these approaches cannot replace strategies for discovery in the embryo. Here we present, for the first time, a knockdown screen in mouse preimplantation embryos. Early mammalian development encompasses dynamic cellular, molecular and epigenetic events that are largely conserved from mouse to man. We assayed 712 genes for requirements during preimplantation. We identified 59 genes required for successful development or outgrowth and implantation. We have characterized each phenotype and revealed cellular, molecular, and lineage specific defects following knockdown of transcript. Induced network analyses demonstrate this as a valid approach to identify networks of genes that play important roles during preimplantation. Our approach provides a robust and efficient strategy towards identification of novel phenotypes during mouse preimplantation and facilitates functional annotation of the mammalian transcriptome. PMID:27869233

  9. Genetic redundancy of GATA factors in the extraembryonic trophoblast lineage ensures the progression of preimplantation and postimplantation mammalian development

    PubMed Central

    Home, Pratik; Kumar, Ram Parikshan; Ganguly, Avishek; Saha, Biswarup; Milano-Foster, Jessica; Bhattacharya, Bhaswati; Ray, Soma; Gunewardena, Sumedha; Paul, Arindam; Camper, Sally A.; Fields, Patrick E.

    2017-01-01

    GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure. PMID:28232602

  10. Epigenetics in fertilization and preimplantation embryo development.

    PubMed

    Rivera, Rocio Melissa; Ross, Jason Wayne

    2013-12-01

    Epigenetic reprogramming of the parental genomes upon fertilization is required for proper embryonic development. It has long been appreciated that asymmetric distribution of histone modifications as well as differences in the level of DNA methylation exist between the parental pronuclei in mammalian zygotes and during preimplantation development. The speed at which the paternal genome is demethylated after entering the oocyte and the fact that rapid demethylation occurs in the absence of DNA replication have led many to hypothesize that a DNA demethylase must exist. However, such an enzyme has not been found. That the genome of mammalian preimplantation embryos undergo a wave of global demethylation was first reported 25 years ago but only in the past three years has data surfaced that can partially explain the elusive nature of this phenomenon. In addition to the global reorganization of the methylation and histone modification patterns, oocyte development prior to germinal vesicle breakdown involves the production of numerous small RNA, including miRNA. Despite their presence, miRNA functional activity is thought to be limited in the mature mouse oocyte. Additionally, molecular signatures in the 3' untranslated region of maternally expressed transcripts may impact mRNA stability during the transcriptionally quiescent period following germinal vesicle breakdown and prior to the maternal to zygote transition. In this review, we reference some of the recent works which attempt to shed light into the importance of the dynamic epigenetic landscape observed during oocyte maturation and preimplantation embryo development in mammals.

  11. Comparative dynamics of 5-methylcytosine reprogramming and TET family expression during preimplantation mammalian development in mouse and sheep.

    PubMed

    Jafarpour, F; Hosseini, S M; Ostadhosseini, S; Abbasi, H; Dalman, A; Nasr-Esfahani, M H

    2017-02-01

    Despite previous assumption that paternal active DNA demethylation is an evolutionary conserved phenomenon in mammals, emerging studies in other species, particularly sheep, do not support this issue. Recently, ten eleven translocation (TET) enzymes have been suggested as intermediates in genome-wide DNA demethylation through the iterative conversion of five methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC)/5-formylcytosine/5-carboxylcytosine (5caC) derivatives. This study investigated whether TET enzymes and 5mC derivatives are also involved in dynamic reprogramming of early sheep embryos derived by fertilization. Mouse zygotes and developing embryos were considered as control. Obtained results reported substantial differences in dynamics of parent-of-origin-specific patterns of 5mC reprogramming and generation/dilution of 5mC derivatives (5hmC and 5caC) between mouse and sheep early zygotes. Sheep zygotes reported a gradual and insignificant decrease pattern of parental pronucleus 5mC, which was notably replication independent, coincided with gradual generation of 5hmC and 5caC. Although the expression profiles of TET family of enzymes (Tet1, Tet2, and Tet3), with the main exception being Tet2 at later developmental stages, were similar between mouse and sheep developing embryos. In addition, although the expression level of Tet3 was higher than Tet1 and Tet2 in MII oocytes and zygotes in both mouse and sheep, the expression of Tet3 in mouse was higher than sheep in both MII oocytes and zygotes. The contrasting dynamics of 5mC reprogramming between these two species may be associated with the particular evolutionary differences that exist between developmental program of rodents and ruminants, particularly during peri-implantation stages. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Epigenetics in preimplantation mammalian development.

    PubMed

    Canovas, Sebastian; Ross, Pablo Juan

    2016-07-01

    Fertilization is a very dynamic period of comprehensive chromatin remodeling, from which two specialized cells result in a totipotent zygote. The formation of a totipotent cell requires extensive epigenetic remodeling that, although independent of modifications in the DNA sequence, still entails a profound cell-fate change, supported by transcriptional profile modifications. As a result of finely tuned interactions between numerous mechanisms, the goal of fertilization is to form a full healthy new individual. To avoid the persistence of alterations in epigenetic marks, the epigenetic information contained in each gamete is reset during early embryogenesis. Covalent modification of DNA by methylation, as well as posttranslational modifications of histone proteins and noncoding RNAs, appears to be the main epigenetic mechanisms that control gene expression. These allow different cells in an organism to express different transcription profiles, despite each cell containing the same DNA sequence. In the context of replacement of spermatic protamine with histones from the oocyte, active cell division, and specification of different lineages, active and passive mechanisms of epigenetic remodeling have been revealed as critical for editing the epigenetic profile of the early embryo. Importantly, redundant factors and mechanisms are likely in place, and only a few have been reported as critical for fertilization or embryo survival by the use of knockout models. The aim of this review is to highlight the main mechanisms of epigenetic remodeling that ensue after fertilization in mammals.

  13. A growth factor phenotype map for ovine preimplantation development.

    PubMed

    Watson, A J; Watson, P H; Arcellana-Panlilio, M; Warnes, D; Walker, S K; Schultz, G A; Armstrong, D T; Seamark, R F

    1994-04-01

    The reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the patterns of expression for several growth factor ligand and receptor genes during ovine preimplantation development. Transcripts for insulin-like growth factor (IGF)-I, IGF-II, and the receptors for insulin and IGF-I were detected throughout ovine preimplantation development from the 1-cell to the blastocyst stage. Transforming growth factor alpha (TGF alpha) transcripts were also detected throughout ovine preimplantation development. The mRNAs encoding basic fibroblast growth factor (bFGF) were detected in all stages of the ovine preimplantation embryo, although the relative abundance of this transcript consistently decreased from the 1-cell to the blastocyst stage, suggesting that it may represent a maternal transcript in early sheep embryos. Transcripts encoding ovine trophoblast protein (oTP) were detected only within blastocyst-stage embryos. Primary ovine oviduct cell cultures express the transcripts for IGF-II, IGF-I, TGF alpha, bFGF, TGF beta 1, and the receptors for insulin and IGF-I, suggesting that paracrine growth factor circuits may exist between the oviduct epithelium and the early ovine embryo. Transcripts for insulin, epidermal growth factor (EGF), and nerve growth factor (NGF) were not detected in any stage of the ovine preimplantation embryo or within the oviduct cell preparations. The expression of growth factor transcripts very early in mammalian development would predict that these molecules fulfil a necessary role(s) in supporting the progression of early embryos through the preimplantation interval. Our future efforts will be directed to understanding the nature of these putative regulatory pathways.

  14. Transcriptomics analysis and human preimplantation development.

    PubMed

    Freour, Thomas; Vassena, Rita

    2016-10-17

    The study of oocyte and preimplantation embryo biology has been regarded with great curiosity throughout scientific history, but it is not until the development of robust methods for in vitro observation and manipulation of animal gametes that developmental biology has flourished as a discipline. By far the biggest technical challenge in studying transcription in oocytes and early embryo has been the necessity of developing techniques that retain a high level of accuracy when starting from small amount of material. The objective of this narrative review is to summarize the knowledge gained about the embryonic preimplantation period in the human species from transcriptomics experiments, and to discuss technical limitations and solutions to the study of transcriptomics in these samples.

  15. Detrimental effects of microgravity on mouse preimplantation development in vitro.

    PubMed

    Wakayama, Sayaka; Kawahara, Yumi; Li, Chong; Yamagata, Kazuo; Yuge, Louis; Wakayama, Teruhiko

    2009-08-25

    Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (microG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under microG. However, although we obtained 75 healthy offspring from microG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under microG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under microG environment in a mammal, but normal preimplantation embryo development might require 1G.

  16. Detrimental Effects of Microgravity on Mouse Preimplantation Development In Vitro

    PubMed Central

    Wakayama, Sayaka; Kawahara, Yumi; Li, Chong; Yamagata, Kazuo; Yuge, Louis; Wakayama, Teruhiko

    2009-01-01

    Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (µG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10–3 G using 3D rotation. Fertilization occurred normally in vitro under µG. However, although we obtained 75 healthy offspring from µG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under µG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under µG environment in a mammal, but normal preimplantation embryo development might require 1G. PMID:19707597

  17. Long-term live-cell imaging of mammalian preimplantation development and derivation process of pluripotent stem cells from the embryos.

    PubMed

    Yamagata, Kazuo; Ueda, Jun

    2013-05-01

    Mammalian fertilization is a process in which two highly specialized haploid gametes unite and endow totipotency to the resulting diploid zygote. This is followed by cell proliferation and the onset of differentiation during the brief period leading up to implantation. In these processes, a number of cellular components and structures are regulated spatially and temporally, as seen in repeated cell division, cell cycle progression, and epigenetic reprogramming. In mammals, the numbers of oocytes and embryos that can be collected are very limited. Therefore, analyses of molecular mechanisms are hampered because of difficulties in conducting biochemical analyses on such limited material. Furthermore, immunostaining methods require cell fixation and are insufficient for understanding ontogeny, because the processes observed in fertilization and early embryonic development progress in time-dependent manners and each phenomenon is connected with others by cause-and-effect relationships. Consequently, it is important to develop an experimental system that enables molecular imaging without affecting embryonic development. To achieve the above advantages, especially retrospective and prospective analyses, we have established a live-cell imaging system that enables observations under minimally invasive conditions. Using this approach, we have succeeded in visualizing and predicting the developmental potential of embryos after various perturbations. We also succeeded in imaging embryonic stem (ES) cell derivation in natural conditions. In this review, we describe a brief history of embryonic imaging and detailed protocols. We also discuss promising aspects of imaging in the fields of developmental and stem cell biology.

  18. A medium-chain fatty acid as an alternative energy source in mouse preimplantation development.

    PubMed

    Yamada, Mitsutoshi; Takanashi, Kazumi; Hamatani, Toshio; Hirayama, Akiyoshi; Akutsu, Hidenori; Fukunaga, Tomoko; Ogawa, Seiji; Sugawara, Kana; Shinoda, Kosaku; Soga, Tomoyoshi; Umezawa, Akihiro; Kuji, Naoaki; Yoshimura, Yasunori; Tomita, Masaru

    2012-01-01

    To further optimize the culturing of preimplantation embryos, we undertook metabolomic analysis of relevant culture media using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We detected 28 metabolites: 23 embryo-excreted metabolites including 16 amino acids and 5 media-derived metabolites (e.g., octanoate, a medium-chain fatty acid (MCFA)). Due to the lack of information on MCFAs in mammalian preimplantation development, this study examined octanoate as a potential alternative energy source for preimplantation embryo cultures. No embryos survived in culture media lacking FAs, pyruvate, and glucose, but supplementation of octanoate rescued the embryonic development. Immunoblotting showed significant expression of acyl-CoA dehydrogenase and hydroxyacyl-CoA dehydrogenase, important enzymes for ß-oxidation of MCFAs, in preimplantation embryo. Furthermore, CE-TOFMS traced [1-(13)C(8)] octanoate added to the culture media into intermediate metabolites of the TCA cycle via ß-oxidation in mitochondria. These results are the first demonstration that octanoate could provide an efficient alternative energy source throughout preimplantation development.

  19. Functional Analysis of Rex1 During Preimplantation Development

    PubMed Central

    Climent, María; Alonso-Martin, Sonia; Pérez-Palacios, Raquel; Guallar, Diana; Benito, Alfredo A.; Larraga, Ana; Fernández-Juan, Marta; Sanz, Marta; de Diego, Alicia; Seisdedos, María T.; Muniesa, Pedro

    2013-01-01

    Rex1/Zfp42 is a nuclear protein that is highly conserved in mammals, and widely used as an embryonic stem (ES) cell marker. Although Rex1 expression is associated with enhanced pluripotency, loss-of-function models recently described do not exhibit major phenotypes, and both preimplantation development and ES cell derivation appear normal in the absence of Rex1. To better understand the functional role of Rex1, we examined the expression and localization of Rex1 during preimplantation development. Our studies indicated that REX1 is expressed at all stages during mouse preimplantation development, with a mixed pattern of nuclear, perinuclear, and cytoplasmic localization. Chromatin association seemed to be altered in 8-cell embryos, and in the blastocyst, we found REX1 localized almost exclusively in the nucleus. A functional role for Rex1 in vivo was assessed by gain- and loss-of-function approaches. Embryos with attenuated levels of Rex1 after injection of zygotes with siRNAs did not exhibit defects in preimplantation development in vitro. In contrast, overexpression of Rex1 interfered with cleavage divisions and with proper blastocyst development, although we failed to detect alterations in the expression of lineage and pluripotency markers. Rex1 gain- and loss-of-function did alter the expression levels of Zscan4, an important regulator of preimplantation development and pluripotency. Our results suggest that Rex1 plays a role during preimplantation development. They are compatible with a role for Rex1 during acquisition of pluripotency in the blastocyst. PMID:22897771

  20. Mechanisms of epigenetic remodelling during preimplantation development.

    PubMed

    Ross, Pablo Juan; Canovas, Sebastian

    2016-01-01

    Epigenetics involves mechanisms independent of modifications in the DNA sequence that result in changes in gene expression and are maintained through cell divisions. Because all cells in the organism contain the same genetic blueprint, epigenetics allows for cells to assume different phenotypes and maintain them upon cell replication. As such, during the life cycle, there are moments in which the epigenetic information needs to be reset for the initiation of a new organism. In mammals, the resetting of epigenetic marks occurs at two different moments, which both happen to be during gestation, and include primordial germ cells (PGCs) and early preimplantation embryos. Because epigenetic information is reversible and sensitive to environmental changes, it is probably no coincidence that both these extensive periods of epigenetic remodelling happen in the female reproductive tract, under a finely controlled maternal environment. It is becoming evident that perturbations during the extensive epigenetic remodelling in PGCs and embryos can lead to permanent and inheritable changes to the epigenome that can result in long-term changes to the offspring derived from them, as indicated by the Developmental Origins of Health and Disease (DOHaD) hypothesis and recent demonstration of inter- and trans-generational epigenetic alterations. In this context, an understanding of the mechanisms of epigenetic remodelling during early embryo development is important to assess the potential for gametic epigenetic mutations to contribute to the offspring and for new epimutations to be established during embryo manipulations that could affect a large number of cells in the offspring. It is of particular interest to understand whether and how epigenetic information can be passed on from the gametes to the embryo or offspring, and whether abnormalities in this process could lead to transgenerationally inheritable phenotypes. The aim of this review is to highlight recent progress made in

  1. Myo-Inositol Safety in Pregnancy: From Preimplantation Development to Newborn Animals

    PubMed Central

    Kuşcu, Nilay

    2016-01-01

    Myo-inositol (myo-Ins) has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction. We have previously shown that mouse embryo exposure to myo-Ins through preimplantation development in vitro increases proliferation activity and blastocyst production, representing an improvement in culture conditions. We have herein investigated biochemical mechanisms elicited by myo-Ins in preimplantation embryos and evaluated myo-Ins effects on postimplantation/postnatal development. To this end naturally fertilized embryos were cultured in vitro to blastocyst in the presence or absence of myo-Ins and analyzed for activation of the PKB/Akt pathway, known to modulate proliferation/survival cellular processes. In parallel, blastocyst-stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning. Results obtained provide evidence that myo-Ins induces cellular pathways involving Akt and show that (a) exposure of preimplantation embryos to myo-Ins increases the number of blastocysts available for uterine transfer and of delivered animals and (b) the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo-Ins, up to three weeks of age, overlap. These data further identify myo-Ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology. PMID:27698667

  2. Cell identity in the preimplantation mammalian embryo: an epigenetic perspective from the mouse

    PubMed Central

    Torres-Padilla, Maria Elena

    2008-01-01

    The early preimplantation mouse embryo is a unique system where it is possible to explore the foundations of totipotency and differentiation. Following fertilization, a single cell, the zygote, will give rise to all tissues of the organism. The first signs of differentiation in the embryo are evident at the blastocyst stage with the formation of the trophectoderm, a differentiated tissue that envelopes the inner cell mass. The question of when and how the cells start to be different from each other in the embryo is central to developmental biology: as cell fate decisions are undertaken, loss of totipotency comes about. Although the blastomeres of the preimplantation embryo are totipotent, as the embryo develops some differences appear to develop between them which are, at least partially, related to the epigenetic information of each of these cells. The hypothesis of epigenetic asymmetries acting as driver for lineage allocation is presented. Although there are now some indications that epigenetic mechanisms are involved in cell fate determination, much work is needed to discover how such mechanisms are set in play upon fertilization and how they are transmitted through cell division. These considerations are further discussed in the context of preimplantation genetic diagnosis: does it matter to the embryo which cell is used for genetic diagnosis? The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future. PMID:18272526

  3. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos

    PubMed Central

    Kawamura, Kazuhiro; Kawamura, Nanami; Mulders, Sabine M.; Gelpke, Maarten D. Sollewijn; Hsueh, Aaron J. W.

    2005-01-01

    Optimal development of fertilized eggs into preimplantation embryos is essential for reproduction. Although mammalian oocytes ovulated after luteinizing hormone (LH) stimulation can be fertilized and promoted into early embryos in vitro, little is known about ovarian factors important for the conditioning of eggs for early embryo development. Because LH interacts only with ovarian somatic cells, its potential regulation of oocyte functions is presumably mediated by local paracrine factors. We performed DNA microarray analyses of ovarian transcripts and identified brain-derived neurotrophic factor (BDNF) secreted by granulosa and cumulus cells as an ovarian factor stimulated by the preovulatory LH surge. Ovarian BDNF acts on TrkB receptors expressed exclusively in oocytes to enhance first polar body extrusion of oocytes and to promote the in vitro development of zygotes into preimplantation embryos. Furthermore, in vivo treatment with a Trk receptor inhibitor suppressed first polar body extrusion and the progression of zygotes into blastocysts. Thus, ovarian BDNF is important to nuclear and cytoplasmic maturation of the oocyte, which is essential for successful oocyte development into preimplantation embryos. Treatment with BDNF could condition the cultured oocytes for optimal progression into the totipotent blastocysts. PMID:15967989

  4. Sex and the preimplantation embryo: implications of sexual dimorphism in the preimplantation period for maternal programming of embryonic development.

    PubMed

    Hansen, Peter J; Dobbs, Kyle B; Denicol, Anna C; Siqueira, Luiz G B

    2016-01-01

    The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into postnatal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on the maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.

  5. Mammalian development in space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  6. Mammalian development in space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  7. Mammalian development in space.

    PubMed

    Ronca, April E

    2003-01-01

    Life on Earth, and thus the reproductive and ontogenetic processes of all extant species and their ancestors, evolved under the constant influence of the Earth's l g gravitational field. These considerations raise important questions about the ability of mammals to reproduce and develop in space. In this chapter, I review the current state of our knowledge of spaceflight effects on developing mammals. Recent studies are revealing the first insights into how the space environment affects critical phases of mammalian reproduction and development, viz., those events surrounding fertilization, embryogenesis, pregnancy, birth, postnatal maturation and parental care. This review emphasizes fetal and early postnatal life, the developmental epochs for which the greatest amounts of mammalian spaceflight data have been amassed. The maternal-offspring system, the coordinated aggregate of mother and young comprising mammalian development, is of primary importance during these early, formative developmental phases. The existing research supports the view that biologically meaningful interactions between mothers and offspring are changed in the weightlessness of space. These changes may, in turn, cloud interpretations of spaceflight effects on developing offspring. Whereas studies of mid-pregnant rats in space have been extraordinarily successful, studies of young rat litters launched at 9 days of postnatal age or earlier, have been encumbered with problems related to the design of in-flight caging and compromised maternal-offspring interactions. Possibilities for mammalian birth in space, an event that has not yet transpired, are considered. In the aggregate, the results indicate a strong need for new studies of mammalian reproduction and development in space. Habitat development and systematic ground-based testing are important prerequisites to future research with young postnatal rodents in space. Together, the findings support the view that the environment within which young

  8. Roles of one-carbon metabolism in preimplantation period--effects on short-term development and long-term programming--.

    PubMed

    Ikeda, Shuntaro; Koyama, Hiroyuki; Sugimoto, Miki; Kume, Shinichi

    2012-01-01

    One-carbon metabolism (OCM) can be seen as integrated metabolic pathways centered on the metabolism of two nutritional substances, folate and methionine. Mammalian oocytes and preimplantation embryos express almost all enzymes that participate in OCM, suggesting that they can independently metabolize OCM nutrients. A deficiency or excess of OCM nutrients and their metabolites during in vitro culture affects preimplantation development of mammalian embryos. Recent in vivo studies have demonstrated that specific OCM dietary interventions during the periconceptional (mainly oocyte growth and preimplantation) period can cause epigenetic alterations in DNA of offspring and program the long-term consequences in their health in adulthood. The epigenetic processes are likely to be implicated in the effects of OCM nutrients; however, understanding their effects at the level of specific genes and their implications in assisted reproductive technology will require further investigations.

  9. Sex and the Preimplantation Embryo - Implications of Sexual Dimorphism in the Preimplantation Period for Maternal Programming of Embryonic Development

    PubMed Central

    Hansen, Peter J.; Dobbs, Kyle B.; Denicol, Anna C.; Siqueira, Luiz G. B.

    2015-01-01

    The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into post-natal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion, and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development. PMID:26391275

  10. Effect of sericin on preimplantation development of bovine embryos cultured individually.

    PubMed

    Isobe, T; Ikebata, Y; Onitsuka, T; Wittayarat, M; Sato, Y; Taniguchi, M; Otoi, T

    2012-09-01

    The silk protein sericin has been identified as a potent antioxidant in mammalian cells. This study was conducted to examine the effects of sericin on preimplantation development and quality of bovine embryos cultured individually. When two-cell-stage embryos were cultured individually for 7 days in CR1aa medium supplemented with 0, 0.1, 0.5, or 1% sericin, rates of total blastocyst formation and development to expanded blastocysts from embryos cultured with 0.5% sericin were higher (P < 0.05) than those from embryos cultured with 0 or 1% sericin. When embryos were cultured individually for 7 days in the CR1aa medium supplemented with 0 or 0.5% sericin under two oxidative stress conditions (50 or 100 μm H(2)O(2)), the addition of sericin significantly improved the blastocyst formation rate of embryos exposed to 100 μm H(2)O(2). However, the protective effect of sericin was not observed in development of embryos exposed to 50 μm H(2)O(2). When embryos were exposed to 100 μm H(2)O(2) during culture, the DNA fragmentation index of total blastocysts from embryos cultured with 0.5% sericin was lower than blastocysts derived from embryos cultured without sericin (4.4 vs. 6.8%; P < 0.01). In conclusion, the addition of 0.5% sericin to in vitro culture medium improved preimplantation development and quality of bovine embryos cultured individually by preventing oxidative stress.

  11. mRNAs encoding aquaporins are present during murine preimplantation development.

    PubMed

    Offenberg, H; Barcroft, L C; Caveney, A; Viuff, D; Thomsen, P D; Watson, A J

    2000-12-01

    The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

  12. Exposure to mono-n-butyl phthalate disrupts the development of preimplantation embryos.

    PubMed

    Chu, Da-Peng; Tian, Shi; Sun, Da-Guang; Hao, Chan-Juan; Xia, Hong-Fei; Ma, Xu

    2013-01-01

    Dibutyl phthalate (DBP), a widely used phthalate, is known to cause many serious diseases, especially in the reproductive system. However, little is known about the effects of its metabolite, mono-n-butyl phthalate (MBP), on preimplantation embryo development. In the present study, we found that treatment of embryos with 10⁻³ M MBP impaired developmental competency, whereas exposure to 10⁻⁴ M MBP delayed the progression of preimplantation embryos to the blastocyst stage. Furthermore, reactive oxygen species (ROS) levels in embryos were significantly increased following treatment with 10⁻³ M MBP. In addition, 10⁻³ M MBP increased apoptosis via the release of cytochrome c, whereas immunofluorescent analysis revealed that exposure of preimplantation embryos to MBP concentration-dependently (10⁻⁵, 10⁻⁴ and 10⁻³ M) decreased DNA methylation. Together, the results indicate a possible relationship between MBP exposure and developmental failure in preimplantation embryos.

  13. Preimplantational Ectogenesis

    PubMed Central

    Karp, Laurence E.; Donahue, Roger P.

    1976-01-01

    In recent years, technical advances have made preimplantational ectogenesis (in vitro maturation, fertilization and early embryonic development) more than a theoretical concept. Such procedures hold great promise in medical research. However, despite our newly-acquired skills in tissue culture and microsurgical manipulation, and contrary to many sensational articles in the lay press, it is not likely that preimplantational ectogenesis will soon attain wide clinical use in humans. Adverse societal attitudes, based largely upon moral and ethical dilemmas, will probably combine with still-unresolved technical difficulties to restrict the clinical applications. ImagesFigure 2.Figure 1. PMID:1266215

  14. Cell fate regulation in early mammalian development

    NASA Astrophysics Data System (ADS)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

    Preimplantation development in mammals encompasses a period from fertilization to implantation and results in formation of a blastocyst composed of three distinct cell lineages: epiblast, trophectoderm and primitive endoderm. The epiblast gives rise to the organism, while the trophectoderm and the primitive endoderm contribute to extraembryonic tissues that support embryo development after implantation. In many vertebrates, such as frog or fish, maternally supplied lineage determinants are partitioned within the egg. Cell cleavage that follows fertilization results in polarization of these factors between the individual blastomeres, which become restricted in their developmental fate. In contrast, the mouse oocyte and zygote lack clear polarity and, until the eight-cell stage, individual blastomeres retain the potential to form all lineages. How are cell lineages specified in the absence of a maternally supplied blueprint? This is a fundamental question in the field of developmental biology. The answer to this question lies in understanding the cell-cell interactions and gene networks involved in embryonic development prior to implantation and using this knowledge to create testable models of the developmental processes that govern cell fates. We provide an overview of classic and contemporary models of early lineage development in the mouse and discuss the emerging body of work that highlights similarities and differences between blastocyst development in the mouse and other mammalian species.

  15. Embryonic genotype and inbreeding affect preimplantation development in cattle.

    PubMed

    Lazzari, G; Colleoni, S; Duchi, R; Galli, A; Houghton, F D; Galli, C

    2011-05-01

    Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7-8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

  16. DNA methylation signature of long noncoding RNA genes during human pre-implantation embryonic development

    PubMed Central

    Shen, Xiaoli; Han, Shubiao; Ye, Hong; Huang, Guoning

    2017-01-01

    DNA methylation have crucial roles in regulating the expression of developmental genes during mammalian pre-implantation embryonic development (PED). However, the DNA methylation dynamic pattern of long noncoding RNA (lncRNA) genes, one type of epigenetic regulators, in human PED have not yet been demonstrated. Here, we performed a comprehensive analysis of lncRNA genes in human PED based on public reduced representation bisulphite sequencing (RRBS) data. We observed that both lncRNA and protein-coding genes complete the major demethylation wave at the 2-cell stage, whereas the promoters of lncRNA genes show higher methylation level than protein-coding genes during PED. Similar methylation distribution was observed across the transcription start sites (TSS) of lncRNA and protein-coding genes, contrary to previous observations in tissues. Besides, not only the gamete-specific differentially methylated regions (G-DMRs) but also the embryonic developmental-specific DMRs (D-DMRs) showed more paternal bias, especially in promoter regions in lncRNA genes. Moreover, coding-non-coding gene co-expression network analysis of genes containing D-DMRs suggested that lncRNA genes involved in PED are associated with gene expression regulation through several means, such as mRNA splicing, translational regulation and mRNA catabolic. This firstly provides study provides the methylation profiles of lncRNA genes in human PED and improves the understanding of lncRNA genes involvement in human PED. PMID:28915634

  17. Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development

    PubMed Central

    Yuan, Shuiqiao; Schuster, Andrew; Tang, Chong; Yu, Tian; Ortogero, Nicole; Bao, Jianqiang; Zheng, Huili; Yan, Wei

    2016-01-01

    Although it is believed that mammalian sperm carry small noncoding RNAs (sncRNAs) into oocytes during fertilization, it remains unknown whether these sperm-borne sncRNAs truly have any function during fertilization and preimplantation embryonic development. Germline-specific Dicer and Drosha conditional knockout (cKO) mice produce gametes (i.e. sperm and oocytes) partially deficient in miRNAs and/or endo-siRNAs, thus providing a unique opportunity for testing whether normal sperm (paternal) or oocyte (maternal) miRNA and endo-siRNA contents are required for fertilization and preimplantation development. Using the outcome of intracytoplasmic sperm injection (ICSI) as a readout, we found that sperm with altered miRNA and endo-siRNA profiles could fertilize wild-type (WT) eggs, but embryos derived from these partially sncRNA-deficient sperm displayed a significant reduction in developmental potential, which could be rescued by injecting WT sperm-derived total or small RNAs into ICSI embryos. Disrupted maternal transcript turnover and failure in early zygotic gene activation appeared to associate with the aberrant miRNA profiles in Dicer and Drosha cKO spermatozoa. Overall, our data support a crucial function of paternal miRNAs and/or endo-siRNAs in the control of the transcriptomic homeostasis in fertilized eggs, zygotes and two-cell embryos. Given that supplementation of sperm RNAs enhances both the developmental potential of preimplantation embryos and the live birth rate, it might represent a novel means to improve the success rate of assisted reproductive technologies in fertility clinics. PMID:26718009

  18. Preimplantation exposure to bisphenol A (BPA) affects embryo transport, preimplantation embryo development, and uterine receptivity in mice

    PubMed Central

    Xiao, Shuo; Diao, Honglu; Smith, Mary Alice; Song, Xiao; Ye, Xiaoqin

    2011-01-01

    To investigate the effects of bisphenol A (BPA) on embryo and uterine factors in embryo implantation, timed pregnant C57BL6 females were treated subcutaneously with 0, 0.025, 0.5, 10, 40, and 100 mg/kg/day BPA from gestation days 0.5 to 3.5. In 100 mg/kg/day BPA-treated females, no implantation sites were detected on day 4.5 but retention of embryos in the oviduct and delayed embryo development were detected on day 3.5. When untreated healthy embryos were transferred to pseudopregnant females treated with 100 mg/kg/day BPA, no implantation sites were detected on day 4.5. In 40 mg/kg/day BPA-treated females, delayed implantation and increased perinatal lethality of their offspring were observed. Implantation seemed normal in the rest BPA-treated groups or the female offspring from 40 mg/kg/day BPA-treated group. These data demonstrate the adverse effects of high doses of BPA on processes critical for embryo implantation: embryo transport, preimplantation embryo development, and establishment of uterine receptivity. PMID:21907787

  19. Development of mammalian ovary.

    PubMed

    Smith, Peter; Wilhelm, Dagmar; Rodgers, Raymond J

    2014-06-01

    Pre-natal and early post-natal ovarian development has become a field of increasing importance over recent years. The full effects of perturbations of ovarian development on adult fertility, through environmental changes or genetic anomalies, are only now being truly appreciated. Mitigation of these perturbations requires an understanding of the processes involved in the development of the ovary. Herein, we review some recent findings from mice, sheep, and cattle on the key events involved in ovarian development. We discuss the key process of germ cell migration, ovigerous cord formation, meiosis, and follicle formation and activation. We also review the key contributions of mesonephric cells to ovarian development and propose roles for these cells. Finally, we discuss polycystic ovary syndrome, premature ovarian failure, and pre-natal undernutrition; three key areas in which perturbations to ovarian development appear to have major effects on post-natal fertility.

  20. Simulated Microgravity Influences Bovine Oocyte In Vitro Fertilization and Preimplantation Embryo Development

    USDA-ARS?s Scientific Manuscript database

    The aim of this study was to investigate whether in vitro fertilization and preimplantation embryos exposed to a simulated microgravity environment in vitro would improve, or be deleterious to, their fertilization and embryonic development. A Rotating Cell Culture System™ (RCCS) bioreactor with a Hi...

  1. Adaptive Transition of Aquaporin 5 Expression and Localization during Preimplantation Embryo Development by In Vitro Culture.

    PubMed

    Park, Jae-Won; Shin, Yun Kyung; Choen, Yong-Pil

    2014-09-01

    Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second midpreimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

  2. Adaptive Transition of Aquaporin 5 Expression and Localization during Preimplantation Embryo Development by In Vitro Culture

    PubMed Central

    Park, Jae-Won; Shin, Yun Kyung; Choen, Yong-Pil

    2014-01-01

    Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second midpreimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization. PMID:25949184

  3. Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development.

    PubMed

    Wu, B J; Dong, F L; Ma, X S; Wang, X G; Lin, F; Liu, H L

    2014-08-07

    Epigenetic modifications of the genome, such as histone H2A variants, ensure appropriate gene activation or silencing during oogenesis and preimplantation embryo development. We examined global localization and expression of the histone H2A variants, including H2A.Bbd, H2A.Z and H2A.X, during mouse oogenesis and preimplantation embryo development. Immunocytochemistry with specific antibodies against various histone H2A variants showed their localization and changes during oogenesis and preimplantation development. H2A.Bbd and H2A.Z were almost absent from nuclei of growing oocytes (except 5-day oocyte), whereas H2A.X was deposited in nuclei throughout oogenesis and in preimplantation embryos. In germinal vesicle (GV) oocyte chromatin, H2A.Bbd was detected as a weak signal, whereas no fluorescent signal was detected in GV breakdown (GVBD) or metaphase II (MII) oocytes; H2A.Z showed intense signals in chromatin of GV, GVBD and MII oocytes. H2A. Bbd showed very weak signals in both pronucleus and 2-cell embryo nuclei, but intense signals were detected in nuclei from 4-cell embryo to blastula. The H2A.Z signal was absent from pronucleus to morula chromatin, whereas a fluorescent signal was detected in blastula nuclei. Our results suggest that histone H2A variants are probably involved in reprogramming of genomes during oocyte meiosis or after fertilization.

  4. Stress exposure during the preimplantation period affects blastocyst lineages and offspring development

    PubMed Central

    BURKUŠ, Ján; KAČMAROVÁ, Martina; KUBANDOVÁ, Janka; KOKOŠOVÁ, Natália; FABIANOVÁ, Kamila; FABIAN, Dušan; KOPPEL, Juraj; ČIKOŠ, Štefan

    2015-01-01

    We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life. PMID:25985793

  5. Some aspects of the early development and implantation of the mammalian egg.

    PubMed

    Bloch, S

    1976-05-15

    Some aspects of the early development and implantation of the mammalian egg in various species are reviewed. Those phases reviewed include ovulation, fertilization and tubal passage of sperm and fertilized ova, capacitation of spermatozoa, the spacing of ova along the uterus, preimplantation stages of the uterus and uterus-blastocyst interaction, decidualization, the zona pellucida, normal and delayed nidation, superfetation, and the parthogenetic activation of eggs. Explanations for problems arising from research in these areas are discussed.

  6. Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice

    PubMed Central

    Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Kobayashi, Hisato; Umezawa, Akihiro

    2016-01-01

    Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming. PMID:27495230

  7. Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development

    PubMed Central

    Vassena, Rita; Boué, Stéphanie; González-Roca, Eva; Aran, Begoña; Auer, Herbert; Veiga, Anna; Belmonte, Juan Carlos Izpisua

    2011-01-01

    The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming. PMID:21775417

  8. Effects of American Ginseng on Preimplantation Development and Pregnancy in Mice.

    PubMed

    Belanger, Danyka; Calder, Michele D; Gianetto-Berruti, Alessandra; Lui, Edmund M; Watson, Andrew J; Feyles, Valter

    2016-01-01

    In North America, a high proportion of pregnant women use herbal medications including North American ginseng. This medicinal plant contains high amounts of triterpene saponins (ginsenosides), which are the main bioactive compounds. It is important to assess ginseng's impact on all reproductive functions to ensure the safety of pregnant women and fetuses. In this study, we defined the concentration-responsive effects of North American alcoholic and aqueous ginseng extracts on preimplantation development in vitro and on pregnancy and post-partum development in the mouse. Two-cell mouse embryos were cultured with 5 different concentrations of whole ginseng root extracts, or ginsenosides Rb1, Rg1 and Re alone, a combinatorial ginsenoside solution and a crude polysaccharide fraction solution. Embryonic development and recovery from each treatment was assessed. To investigate the in vivo effects of ginseng extracts, female mice were gavaged with 50[Formula: see text]mg/kg/day, 500[Formula: see text]mg/kg/day or 2000[Formula: see text]mg/kg/day of either extract (treatment) or water (sham) for 2 weeks prior to mating and throughout gestation. Gestation period, litter size, pup growth and pup sex ratio were evaluated. Oral ginseng consumption did not significantly affect fertility or pregnancy in the mouse. High doses of ginseng (2000[Formula: see text]mg/kg/day) decreased maternal weight gain. Direct treatment of preimplantation embryos in vitro demonstrated that ALC and AQ extract treatment reduced development in a concentration responsive manner, while only ALC extract effects were largely reversible. Treatments with individual or combinatorial ginsenosides, or the polysaccharide fraction solution alone did not impair preimplantation development, in vitro. In conclusion, maternal oral consumption of ginseng has little negative impact on pregnancy in the mouse, however, direct exposure to ginseng extract during mouse preimplantation development in vitro is detrimental.

  9. Removal of O-GlcNAcylation is important for pig preimplantation development

    PubMed Central

    SHIBUTANI, Mihiro; MORI, Takeshi; MIYANO, Takashi; MIYAKE, Masashi

    2015-01-01

    Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error. PMID:26004176

  10. Removal of O-GlcNAcylation is important for pig preimplantation development.

    PubMed

    Shibutani, Mihiro; Mori, Takeshi; Miyano, Takashi; Miyake, Masashi

    2015-01-01

    Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error.

  11. Preimplantation development regulatory pathway construction through a text-mining approach

    PubMed Central

    2011-01-01

    Background The integration of sequencing and gene interaction data and subsequent generation of pathways and networks contained in databases such as KEGG Pathway is essential for the comprehension of complex biological processes. We noticed the absence of a chart or pathway describing the well-studied preimplantation development stages; furthermore, not all genes involved in the process have entries in KEGG Orthology, important information for knowledge application with relation to other organisms. Results In this work we sought to develop the regulatory pathway for the preimplantation development stage using text-mining tools such as Medline Ranker and PESCADOR to reveal biointeractions among the genes involved in this process. The genes present in the resulting pathway were also used as seeds for software developed by our group called SeedServer to create clusters of homologous genes. These homologues allowed the determination of the last common ancestor for each gene and revealed that the preimplantation development pathway consists of a conserved ancient core of genes with the addition of modern elements. Conclusions The generation of regulatory pathways through text-mining tools allows the integration of data generated by several studies for a more complete visualization of complex biological processes. Using the genes in this pathway as “seeds” for the generation of clusters of homologues, the pathway can be visualized for other organisms. The clustering of homologous genes together with determination of the ancestry leads to a better understanding of the evolution of such process. PMID:22369103

  12. Preimplantation development regulatory pathway construction through a text-mining approach.

    PubMed

    Donnard, Elisa; Barbosa-Silva, Adriano; Guedes, Rafael L M; Fernandes, Gabriel R; Velloso, Henrique; Kohn, Matthew J; Andrade-Navarro, Miguel A; Ortega, J Miguel

    2011-12-22

    The integration of sequencing and gene interaction data and subsequent generation of pathways and networks contained in databases such as KEGG Pathway is essential for the comprehension of complex biological processes. We noticed the absence of a chart or pathway describing the well-studied preimplantation development stages; furthermore, not all genes involved in the process have entries in KEGG Orthology, important information for knowledge application with relation to other organisms. In this work we sought to develop the regulatory pathway for the preimplantation development stage using text-mining tools such as Medline Ranker and PESCADOR to reveal biointeractions among the genes involved in this process. The genes present in the resulting pathway were also used as seeds for software developed by our group called SeedServer to create clusters of homologous genes. These homologues allowed the determination of the last common ancestor for each gene and revealed that the preimplantation development pathway consists of a conserved ancient core of genes with the addition of modern elements. The generation of regulatory pathways through text-mining tools allows the integration of data generated by several studies for a more complete visualization of complex biological processes. Using the genes in this pathway as "seeds" for the generation of clusters of homologues, the pathway can be visualized for other organisms. The clustering of homologous genes together with determination of the ancestry leads to a better understanding of the evolution of such process.

  13. Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing.

    PubMed

    Moreira de Mello, Joana C; Fernandes, Gustavo R; Vibranovski, Maria D; Pereira, Lygia V

    2017-09-07

    In female mammals, one X chromosome is transcriptionally inactivated (XCI), leading to dosage compensation between sexes, fundamental for embryo viability. A previous study using single-cell RNA-sequencing (scRNA-seq) data proposed that female human preimplantation embryos achieve dosage compensation by downregulating both Xs, a phenomenon named dampening of X expression. Using a novel pipeline on those data, we identified a decrease in the proportion of biallelically expressed X-linked genes during development, consistent with XCI. Moreover, we show that while the expression sum of biallelically expressed X-linked genes decreases with embryonic development, their median expression remains constant, rejecting the hypothesis of X dampening. In addition, analyses of a different dataset of scRNA-seq suggest the appearance of X-linked monoallelic expression by the late blastocyst stage in females, another hallmark of initiation of XCI. Finally, we addressed the issue of dosage compensation between the single active X and autosomes in males and females for the first time during human preimplantation development, showing emergence of X to autosome dosage compensation by the upregulation of the active X chromosome in both male and female embryonic stem cells. Our results show compelling evidence of an early process of X chromosome inactivation during human preimplantation development.

  14. Chemical exposure of embryos during the preimplantation stages of pregnancy: mortality rate and intrauterine development.

    PubMed

    Fabro, S; McLachlan, J A; Dames, N M

    1984-04-01

    Exposure of CD-1 mouse embryos at the eight- to 16-cell stage for 1 hour to methylmethanesulfonate (MMS; 0.25, 0.5, and 1.0 mM) produced DNA breakage and interfered with embryonic development in a dose-related manner. MMS-exposed blastocysts were transferred to oviducts of untreated recipient female mice, and the conceptuses were allowed to develop to term. MMS exposure resulted in an increased intrauterine death rate, although the number of implantation sites was not decreased. Surviving MMS-treated offspring showed intrauterine growth retardation, but there was no increase in the incidence of gross abnormalities. Intrauterine growth retardation, without an increase in gross abnormalities, was also observed in the offspring of pregnant New Zealand White rabbits dosed during the preimplantation stages of pregnancy with an "environmental cocktail" composed of ethanol, nicotine, caffeine, sodium salicylate, and dichloro-diphenyl-trichloro-ethane (DDT). When the compounds were tested individually, nicotine and DDT were the only two that produced intrauterine growth retardation. DDT-treated 8-day rabbit conceptuses were smaller than controls and showed abnormal persistence of preimplantation proteins in the yolk sac fluid. These results suggest that exposure to chemicals during the preimplantation stages of pregnancy may result in a cessation of growth and development before implantation or during later intrauterine development. Damage can be repaired but it may result in offspring that show intrauterine growth retardation without gross abnormalities.

  15. ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development.

    PubMed

    Zhou, Lin; Wang, Pei; Zhang, Juanjuan; Heng, Boon Chin; Tong, Guo Qing

    2016-02-01

    ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.

  16. Manifestations of diabetes mellitus on mouse preimplantation development: effect of elevated concentration of metabolic intermediates.

    PubMed

    Moley, K H; Vaughn, W K; Diamond, M P

    1994-01-01

    The metabolic derangements of pregnancies complicated by diabetes mellitus, specifically hyperglycaemia and hyperketonaemia, are known to be teratogenic during the period of organogenesis in animals. We have shown previously that poorly controlled diabetes mellitus impairs in-vivo and in-vitro mouse preimplantation embryo growth, and that culturing embryos in elevated glucose concentrations only partially recreates this developmental delay. To extend this observation we examined the effect on mouse preimplantation embryo growth of elevated concentrations of other metabolic intermediates, which may be deranged in diabetes mellitus, namely lipids, lactate, glycerol, amino acids, and ketones. Two-cell embryos from ovulation-induced B6C3F1 mice were cultured for 72 h in the presence of added lipids (250 mg/dl), lactate (5 mM), glycerol (160 microM) or mixed amino acids (8.5% travasol, 7 mM) and showed no significant difference in growth over 72 h versus their control groups. However, growth of preimplantation embryos in acetoacetate (10 mM) or in the racemic mixture of DL-beta-hydroxybutyrate (16 and 32 mM) revealed marked retardation versus controls when assessed either by distribution of developmental stages over time (24, 48, 72 h, P < 0.001) or by the difference in the average rank of sums indicating a delay in maturation (P < 0.0001). We conclude that elevated ketone concentrations adversely affect preimplantation embryo development. These findings extend previous studies which correlate uncontrolled diabetes mellitus as well as hyperglycaemia with abnormal organogenesis, and demonstrate that exposure to metabolic derangements may also hinder reproductive performance at even earlier stages in gestation.

  17. The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming

    PubMed Central

    Durruthy-Durruthy, Jens; Sebastiano, Vittorio; Wossidlo, Mark; Cepeda, Diana; Cui, Jun; Grow, Edward J; Davila, Jonathan; Mall, Moritz; Wong, Wing H; Wysocka, Joanna; Au, Kin Fai; Pera, Renee A Reijo

    2016-01-01

    Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate. PMID:26595768

  18. Environmental and epigenetic effects upon preimplantation embryo metabolism and development

    PubMed Central

    Chason, Rebecca J; Csokmay, John; Segars, James H.; DeCherney, Alan H.; Armant, D. Randall

    2011-01-01

    In vitro fertilization has provided a unique window into the metabolic processes that drive embryonic growth and development from a fertilized ovum to a competent blastocyst. Post-fertilization development is dependent upon a dramatic reshuffling of the parental genomes during meiosis, as well as epigenetic changes that provide a new and autonomous set of instructions to guide cellular differentiation both in the embryo and beyond. While early literature focused simply on the substrates and culture conditions required for progress through embryonic development, more recent insights lead us to suggest that the surrounding environment can alter the epigenome, which can, in turn, impact embryonic metabolism and developmental competence. PMID:21741268

  19. Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos.

    PubMed

    Takahashi, Kazuki; Sakurai, Nobuyuki; Emura, Natsuko; Hashizume, Tsutomu; Sawai, Ken

    2015-01-01

    Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.

  20. Effects of downregulating GLIS1 transcript on preimplantation development and gene expression of bovine embryos

    PubMed Central

    TAKAHASHI, Kazuki; SAKURAI, Nobuyuki; EMURA, Natsuko; HASHIZUME, Tsutomu; SAWAI, Ken

    2015-01-01

    Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos. PMID:26074126

  1. The effect of molybdenum on the in vitro development of mouse preimplantation embryos.

    PubMed

    Bi, Cong-Ming; Zhang, Yu-Ling; Liu, Feng-Jun; Zhou, Tie-Zhong; Yang, Zi-Jun; Gao, Shen-Yang; Wang, Shu-De; Chen, Xiao-Li; Zhai, Xiao-Wei; Ma, Xue-Gang; Jin, Li-Jun; Wang, Shen

    2013-04-01

    The object of this study was to investigate the effect of molybdenum on the development of mouse preimplantation embryos cultured in vitro. Zygotes were flushed from one outbred mouse strain (Kunming), and then were cultured in potassium simplex optimized medium (KSOM) containing 0, 5, 10, 20, 40, 80, 120, and 160 µg/ml of molybdenum for 5 days until the mid-blastocyst stage. The addition of ≤ 20 µg/ml molybdenum did not affect the blastocyst and birth rates. Molybdenum at doses of 40 µg/ml and higher significantly decreased the cleavage, blastocyst and birth rates, the average cell number, and significantly increased the proportion of degenerative blastocysts. At 120 µg/ml molybdenum inhibited the blastocysts development to birth. At 160 µg/ml molybdenum caused overall developmental arrest (up to 16-cells) of embryos and their massive degeneration. In conclusion, molybdenum negatively affected the development of embryos in a dose-dependent manner. With lower doses (≤ 20 µg/ml), mouse embryos were not apparently damaged. With very high doses (≥ 40 µg/ml), embryo quality significantly decreased. This assessment of the effect of molybdenum on the preimplantation embryo is an initial survey of toxicological risk.

  2. Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development.

    PubMed

    Park, Mi-Ryung; Lee, Ah-Reum; Bui, Hong-Thuy; Park, Chankyu; Park, Keun-Kyu; Cho, Ssang-Goo; Song, Hyuk; Kim, Jae-Hwan; Nguyen, Van Thuan; Kim, Jin-Hoi

    2011-07-01

    Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two-cell embryos were fused and cultured in vitro in Chatot-Ziomek-Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8-10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12-14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm.

  3. Building up the nucleus: nuclear organization in the establishment of totipotency and pluripotency during mammalian development

    PubMed Central

    Borsos, Máté; Torres-Padilla, Maria-Elena

    2016-01-01

    In mammals, epigenetic reprogramming, the acquisition and loss of totipotency, and the first cell fate decision all occur within a 3-d window after fertilization from the one-cell zygote to the formation of the blastocyst. These processes are poorly understood in molecular detail, yet this is an essential prerequisite to uncover principles of stem cells, chromatin biology, and thus regenerative medicine. A unique feature of preimplantation development is the drastic genome-wide changes occurring to nuclear architecture. From studying somatic and in vitro cultured embryonic stem cells (ESCs) it is becoming increasingly established that the three-dimensional (3D) positions of genomic loci relative to each other and to specific compartments of the nucleus can act on the regulation of gene expression, potentially driving cell fate. However, the functionality, mechanisms, and molecular characteristics of the changes in nuclear organization during preimplantation development are only now beginning to be unraveled. Here, we discuss the peculiarities of nuclear compartments and chromatin organization during mammalian preimplantation development in the context of the transition from totipotency to pluripotency. PMID:26980186

  4. Nitric oxide affects preimplantation embryonic development in a rotating wall vessel bioreactor simulating microgravity.

    PubMed

    Cao, Yu-jing; Fan, Xun-jun; Shen, Zheng; Ma, Bao-hua; Duan, En-kui

    2007-01-01

    Microgravity was simulated with a rotating wall vessel bioreactor (RWVB) in order to study its effect on pre-implantation embryonic development in mice. Three experimental groups were used: stationary control, rotational control and clinostat rotation. Three experiments were performed as follows. The first experiment showed that compared with the other two (control) groups, embryonic development was significantly retarded after 72 h in the clinostat rotation group. The second experiment showed that more nitric oxide (NO) was produced in the culture medium in the clinostat rotation group after 72 h (P<0.05), and the nitric oxide synthase (NOS) activity in this group was significantly higher than in the controls (P<0.01). In the third experiment, we studied apoptosis in the pre-implantation mouse embryos after 72 h in culture and found that Annexin-V staining was negative in the normal (stationary and rotational control) embryos, but the developmentally retarded (clinostat rotation) embryos showed a strong green fluorescence. These results indicate that microgravity induced developmental retardation and cell apoptosis in the mouse embryos. We presume that these effects are related to the higher concentration of NO in the embryos under microgravity, which have cause cytotoxic consequences.

  5. Distinct roles of ROCK1 and ROCK2 during development of porcine preimplantation embryos.

    PubMed

    Zhang, Jin Yu; Dong, Huan Sheng; Oqani, Reza K; Lin, Tao; Kang, Jung Won; Jin, Dong Il

    2014-07-01

    Cell-to-cell contact mediated by cell adhesion is fundamental to the compaction process that ensures blastocyst quality during embryonic development. In this study, we first showed that Rho-associated coiled-coil protein kinases (ROCK1 and ROCK2) were expressed both in porcine oocytes and IVF preimplantation embryos, playing different roles in oocytes maturation and embryo development. The amount of mRNA encoding ROCK1 and the protein concentration clearly increased between the eight-cell and morula stages, but decreased significantly when blastocysts were formed. Conversely, ROCK2 was more abundant in the blastocyst compared with other embryonic stages. Moreover, immunostaining showed that ROCK1 protein distribution changed as the embryo progressed through cleavage and compaction to the morula stage. Initially, the protein was predominantly associated with the plasma membrane but later became cytoplasmic. By contrast, ROCK2 protein was localized in both the cytoplasm and the spindle rotation region during oocyte meiosis, but in the cytoplasm and nucleus as the embryo developed. In addition, ROCK2 was present in the trophectoderm cells of the blastocyst. Treatment with 15 μM Y27632, a specific inhibitor of ROCKs, completely blocked further development of early four-cell stage embryos. Moreover, we did not detect the expression of ROCK1 but did detect ROCK2 expression in blastocysts. Moreover, lysophosphatidic acid an activator of ROCKs significantly improved the rates of blastocyst formation. These data demonstrate that ROCKs are required for embryo development to the blastocyst stage. Together, our results indicate that ROCK1 and ROCK2 may exert different biological functions during the regulation of compaction and in ensuring development of porcine preimplantation embryos to the blastocyst stage. © 2014 Society for Reproduction and Fertility.

  6. Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications

    PubMed Central

    Popken, Jens; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Schmid, Volker J.; Strauss, Axel; Guengoer, Tuna; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

    2015-01-01

    The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA. PMID:25932910

  7. Aberrant DNA methylation reprogramming in bovine SCNT preimplantation embryos

    PubMed Central

    Zhang, Sheng; Chen, Xin; Wang, Fang; An, Xinglan; Tang, Bo; Zhang, Xueming; Sun, Liguang; Li, Ziyi

    2016-01-01

    DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner. PMID:27456302

  8. Effects of alcohols on murine preimplantation development: relationship to relative membrane disordering potency.

    PubMed

    Kowalczyk, C L; Stachecki, J J; Schultz, J F; Leach, R E; Armant, D R

    1996-05-01

    During in vitro culture of murine preimplantation embryos, we have observed that exposure to 0.1% ethanol induces an immediate increase in intracellular calcium levels and subsequently accelerates embryogenesis. If the observed effects of ethanol on developing embryos is mediated by its membrane disordering potency, we hypothesized that the relative membrane disordering potencies of related alcohols would correspondingly effect embryonic intracellular calcium levels and developmental rates. Two-cell embryos were exposed to 0.1% ethanol or 0.05 to 1.0% (w/v) n-butanol, n-propanol, isopropanol, 1,2-propanediol, glycerol, or methanol for 24 hr at 37 degrees C, and development to the blastocyst stage was monitored after 5 days. n-Butanol, n-propanol, isopropanol, and methanol treatment caused a dose-dependent inhibition (p < 0.01) of development to the blastocyst stage, whereas 1,2-propanediol or glycerol neither accelerated nor inhibited development. In a second experiment, 8-cell morulae were treated with 1,2-propanediol or glycerol, and cavitation rates were examined. There was no significant difference from control embryos in the onset of cavitation or the blastocoel expansion rate of 1,2-propanediol- or glycerol-exposed embryos, whereas exposure to 0.1% ethanol accelerate cavitation (p > 0.05). In a third experiment, morulae were exposed to 0.1% or 1.0% of each alcohol and were monitored for changes in intracellular calcium levels using the fluorescent indicator, fluo-3-acetoxymethyl ester. There was an immediate increase in intracellular calcium levels when morulae were treated with 1.0% ethanol or n-butanol, but only ethanol induced an increase (p < 0.05) in the level of intracellular calcium at 0.1%. These data suggest that ethanol is unique in its ability to accelerate embryogenesis and that the membrane disordering potency of ethanol does not directly underlie its effects on intracellular calcium release and the acceleration of preimplantation development.

  9. Paternal Low Protein Diet Programs Preimplantation Embryo Gene Expression, Fetal Growth and Skeletal Development in Mice.

    PubMed

    Watkins, Adam J; Sirovica, Slobodan; Stokes, Ben; Isaacs, Mark; Addison, Owen; Martin, Richard A

    2017-02-08

    Defining the mechanisms underlying the programming of early life growth is fundamental for improving adult health and wellbeing. While the association between maternal diet, offspring growth and adult disease risk is well-established, the effect of father's diet on offspring development are largely unknown. Therefore, we fed male mice an imbalanced low protein diet (LPD) to determine the impact on post-fertilisation development and fetal growth. We observed that in preimplantation embryos derived from LPD fed males, expression of multiple genes within the central metabolic AMPK pathway was reduced. In late gestation, paternal LPD programmed increased fetal weight, however, placental weight was reduced, resulting in an elevated fetal:placental weight ratio. Analysis of gene expression patterns revealed increased levels of transporters for calcium, amino acids and glucose within LPD placentas. Furthermore, placental expression of the epigenetic regulators Dnmt1 and Dnmt3L were increased also, coinciding with altered patterns of maternal and paternal imprinted genes. More strikingly, we observed fetal skeletal development was perturbed in response to paternal LPD. Here, while offspring of LPD fed males possessed larger skeletons, their bones comprised lower volumes of high mineral density in combination with reduced maturity of bone apatite. These data offer new insight in the underlying programming mechanisms linking poor paternal diet at the time of conception with the development and growth of his offspring.

  10. Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

    PubMed

    Kim, Jeesun; Zhao, Hongbo; Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

    2016-04-01

    Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.

  11. Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse

    PubMed Central

    Dan, Jiameng; Kim, Soojin; Hardikar, Swanand; Hollowell, Debra; Lin, Kevin; Lu, Yue; Takata, Yoko; Shen, Jianjun; Chen, Taiping

    2016-01-01

    Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression. PMID:27070551

  12. Studies on lysophosphatidic acid action during in vitro preimplantation embryo development.

    PubMed

    Boruszewska, D; Sinderewicz, E; Kowalczyk-Zieba, I; Grycmacher, K; Woclawek-Potocka, I

    2016-01-01

    Assisted reproductive technologies, including in vitro embryo production (IVP), have been successfully used in animal reproduction to optimize breeding strategies for improved production and health in animal husbandry. Despite the progress in IVP techniques over the years, further improvements in in vitro embryo culture systems are required for the enhancement of oocyte and embryo developmental competence. One of the most important issues associated with IVP procedures is the optimization of the in vitro culture of oocytes and embryos. Studies in different species of animals and in humans have identified important roles for receptor-mediated lysophosphatidic acid (LPA) signaling in multiple aspects of human and animal reproductive tract function. The data on LPA signaling in the ovary and uterus suggest that LPA can directly contribute to embryo-maternal interactions via its influence on early embryo development beginning from the influence of the ovarian environment on the oocyte to the influence of the uterine environment on the preimplantation embryo. This review discusses the current status of LPA as a potential supplement in oocyte maturation, fertilization, and embryo culture media and current views on the potential involvement of the LPA signaling pathway in early embryo development. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro.

    PubMed

    Pei, Yijin

    2010-01-01

    This study aimed to investigate the effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro. Zygotes were collected from superovulated New Zealand rabbits 19 h after injection of hCG and immediately mating and cultured in TCM-199 plus fatty-acid free BSA with different concentrations of NGF. Zygotes not treated with NGF served as control. At 24 h, 48 h, 72 h and 96 h of the culture, the numbers of the early cleavage stage, morulae, blastocysts and hatching blastocysts were determined. The intrazonal diameter of the blastocyst and the total cell numbers per blastocyst were measured after 96 h of culture. The results showed: (1) NGF at 100 ng/mL and 1000 ng/mL could improve the numbers of the hatching blastocysts which developed compared to the control treatment (p < 0.05); (2) All concentrations of NGF increased the total cell numbers in the blastocysts compared to the control treatment (p < 0.05); (3) NGF had no significant effect on the blastocyst intrazonal diameter of the blastocysts at 96 h of culture (p = 0.493); (4) The proportion in the early cleavage stage at 24 h of culture (p = 0.635), of morulae at 48 h of culture (p = 0.812) and of blastocysts at 72 h of culture (p = 0.812) in all treatments were not significantly different.

  14. Effect of ICSI on gene expression and development of mouse preimplantation embryos

    PubMed Central

    Giritharan, G.; Li, M.W.; De Sebastiano, F.; Esteban, F.J.; Horcajadas, J.A.; Lloyd, K.C.K.; Donjacour, A.; Maltepe, E.; Rinaudo, P.F.

    2010-01-01

    BACKGROUND In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. METHODS We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). CONCLUSIONS Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used. PMID:20889529

  15. Genetics on stage: public engagement in health policy development on preimplantation genetic diagnosis.

    PubMed

    Cox, Susan M; Kazubowski-Houston, Magdalena; Nisker, Jeff

    2009-04-01

    Arts-based approaches to public engagement offer unique advantages over traditional methods of consultation. Here we describe and assess our use of theatre as a method of public engagement in the development of health policy on preimplantation genetic diagnosis, a controversial method for selecting the genetic characteristics of embryos created through in vitro fertilization. Funding from the Canadian Institutes for Health Research and Health Canada supported 16 performances of the play Orchids in Vancouver, Toronto, and Montréal and post-performance discussion in English and French (with Hubert Doucet) in 2005. A total of 741 individuals attended. The methods used to assess audience engagement and elicit policy-relevant dialogue included in-theatre observation of audience responses, moderated post-performance large audience discussion and focus groups, audience feedback forms and researcher fieldnotes. Emphasizing process and context over emerging outcomes, we reflect on the distinctive contributions of theatre in stimulating public engagement and the need to utilize multiple methods to adequately assess these contributions. We suggest continued dialogue about the possible uses of theatre in health policy development and conclude that greater clarity is needed with regard to citizens' (as well as specific stakeholders, policy makers' and sponsors') desired outcomes if there is to be a suitably nuanced and reflexive basis for assessing the effectiveness of various strategies for public engagement.

  16. ECAT1 is essential for human oocyte maturation and pre-implantation development of the resulting embryos

    PubMed Central

    Liu, Changyu; Li, Min; Li, Tianjie; Zhao, Hongcui; Huang, Jin; Wang, Yun; Gao, Qian; Yu, Yang; Shi, Qinghua

    2016-01-01

    ECAT1 is a subunit of the subcortical maternal complex that is required for cell cycle progression during pre-implantation embryonic development; however, its exact function remains to be elucidated. Here we investigated the expression of ECAT1 in human ovarian tissue, oocytes and pre-implantation embryos and assessed its function by using RNA interference (RNAi) in oocytes. ECAT1 mRNA was highly expressed in human oocytes and zygotes, as well as in two-cell, four-cell and eight-cell embryos, but declined significantly in morulae and blastocysts. ECAT1 was expressed in the cytoplasm of oocytes and pre-implantation embryos and was localized more specifically in the cortical region than in the inner cytoplasm. RNAi experiments demonstrated that down-regulation of ECAT1 expression not only impaired spindle assembly and reduced maturation and fertilization rates of human oocytes but also decreased the cleavage rate of the resulting zygotes. In conclusion, our study indicates that ECAT1 may play a role in meiotic progression by maintaining the accuracy of spindle assembly in human oocytes, thus promoting oocyte maturation and subsequent development of the embryo. PMID:27917907

  17. Gene activation-associated long noncoding RNAs function in mouse preimplantation development

    PubMed Central

    Hamazaki, Nobuhiko; Uesaka, Masahiro; Nakashima, Kinichi; Agata, Kiyokazu; Imamura, Takuya

    2015-01-01

    In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development. PMID:25633350

  18. The early embryonic expression of TFII-I during mouse preimplantation development.

    PubMed

    Enkhmandakh, Badam; Bitchevaia, Natalia; Ruddle, Frank; Bayarsaihan, Dashzeveg

    2004-01-01

    We studied the developmentally regulated expression of mouse TFII-I, a founding member of a family of transcription factors characterized by the presence of multiple helix-loop-helix repeat domains. TFII-I and BEN, a second member of this family, are involved in histone modification and SUMOylation. The genes, GTF2I and GTF2IRD1, encoding these proteins in human are located at chromosomal band 7q11.23, within the Williams syndrome critical region. Our immunohistochemical analysis revealed extensive expression of TFII-I at early stages of embryogenesis. Like BEN, TFII-I is detected in the cytoplasm and nuclei of postfertilization through first cleavage stage embryos. However, in E4.5 blastocysts, at the time of implantation, TFII-I is localized in the nucleus and cytoplasm of the inner cell mass (ICM) and trophectoderm. BEN, at this stage, is expressed only in the cytoplasm of trophoblast cells, but not in the ICM [Gene Expr. Patterns, 2003; 3, 577-587]. Using RT-PCR, we detected Gtf2i and Gtf2ird1 mRNA transcripts in unfertilized oocytes, which indicates the maternal expression of these genes. Thus, the early embryonic expression of TFII-I implicates this family of transcription factors in preimplantation development.

  19. Essential role of maternal UCHL1 and UCHL3 in fertilization and preimplantation embryo development

    PubMed Central

    Mtango, Namdori R.; Sutovsky, Miriam; Susor, Andrej; Zhong, Zhisheng; Latham, Keith E.; Sutovsky, Peter

    2015-01-01

    Posttranslational protein modification by ubiquitination, a signal for lysosomal or proteasomal proteolysis, can be regulated and reversed by deubiquitinating enzymes (DUBs). This study examined the roles of UCHL1 and UCHL3, two members of ubiquitin C-terminal hydrolase (UCH) family of DUBs, in murine fertilization and preimplantation development. Before fertilization, these proteins were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Intracytoplasmic injection of the general UCH-family inhibitor ubiquitin-aldehyde (UBAL) or antibodies against UCHL3 into mature metaphase II oocytes blocked fertilization by reducing sperm penetration of the zona pellucida and incorporation into the ooplasm, suggesting a role for cortical UCHL1 in sperm incorporation. Both UBAL and antibodies against UCHL1 injected at the onset of oocyte maturation (germinal vesicle stage) reduced the fertilizing ability of oocytes. The subfertile Uchl1gad−/− mutant mice showed an intriguing pattern of switched UCH localization, with UCHL3 replacing UCHL1 in the oocyte cortex. While fertilization defects were not observed, the embryos from homozygous Uchl1gad−/− mutant females failed to undergo morula compaction and did not form blastocysts in vivo, indicating a maternal effect related to UCHL1 deficiency. We conclude that the activity of oocyte UCHs contributes to fertilization and embryogenesis by regulating the physiology of the oocyte and blastomere cortex. PMID:21678411

  20. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    SciTech Connect

    Yoneda, Akihiro; Watanabe, Tomomasa

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  1. Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development

    PubMed Central

    Canovas, Sebastian; Cibelli, Jose B.; Ross, Pablo J.

    2012-01-01

    Understanding the mechanisms of epigenetic remodeling that follow fertilization is a fundamental step toward understanding the bases of early embryonic development and pluripotency. Extensive and dynamic chromatin remodeling is observed after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate embryonic genome activation. In particular, trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcription repression. Global levels of this epigenetic mark are high in oocyte chromatin and decrease to minimal levels at the time of embryonic genome activation. We provide evidence that the decrease in H3K27me3 observed during early development is cell-cycle independent, suggesting an active mechanism for removal of this epigenetic mark. Among H3K27me3-specific demethylases, Jumonji domain-containing protein 3 (JMJD3), but not ubiquitously transcribed tetratricopeptide repeat X (UTX), present high transcript levels in oocytes. Soon after fertilization JMJD3 protein levels increase, concurrent with a decrease in mRNA levels. This pattern of expression suggests maternal inheritance of JMJD3. Knockdown of JMJD3 by siRNA injection in parthenogenetically activated metaphase II oocytes resulted in inhibition of the H3K27me3 decrease normally observed in preimplantation embryos. Moreover, knockdown of JMJD3 in oocytes reduced the rate of blastocyst development. Overall, these results indicate that JMJD3 is involved in active demethylation of H3K27me3 during early embryo development and that this mark plays an important role during the progression of embryos to blastocysts. PMID:22308433

  2. Dynamic imaging of preimplantation embryos in the murine oviduct

    NASA Astrophysics Data System (ADS)

    Burton, Jason C.; Wang, Shang; Larina, Irina V.

    2015-03-01

    Studying the dynamic events involved in early preimplantation embryo development during their transport from the ovary to the uterus is of great significance to improve the understanding of infertility, and eventually to help reduce the infertility rate. The mouse is a widely used mammalian model in reproductive biology, however, dynamic imaging studies of mouse preimplantation embryos have been very limited due to the lack of proper imaging tools for such analysis. Here, we introduce an innovative approach, which can potentially be used for three-dimensional imaging and tracking of murine oocytes with optical coherence tomography (OCT) as they exit the ovary and migrate through the oviduct to the uterus. The imaging is performed with spectral-domain OCT system operating at 70 kHz A-scan rate. The preimplantation embryos and surrounding cumulus cells can be clearly visualized. Results from our experiments indicate that OCT has great potential for dynamic imaging of the oviduct and oocyte tracking, which provides the foundation for future investigations aimed at understanding dynamic events during preimplantation stages in normal development as well as in mouse models of infertility.

  3. Suppression of the transcription factor MSX1 gene delays bovine preimplantation embryo development in vitro.

    PubMed

    Tesfaye, D; Regassa, A; Rings, F; Ghanem, N; Phatsara, C; Tholen, E; Herwig, R; Un, C; Schellander, K; Hoelker, M

    2010-05-01

    This study was conducted to investigate the effect of suppressing transcription factor gene MSX1 on the development of in vitro produced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reduced MSX1 mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reduced MSX1 mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression of MSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression of MSX1. In conclusion, suppression of MSX1 affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.

  4. Genes of the transforming growth factor-beta signalling pathway are associated with pre-implantation embryonic development in cattle.

    PubMed

    Li, Geng; Khateeb, Karam; Schaeffer, Erin; Zhang, Bao; Khatib, Hasan

    2012-08-01

    One of the main factors affecting cattle fertility is pre-implantation development of the bovine embryo, which is a complex process regulated by various signal-transduction pathways. The transforming growth factor-β (TGF-β) signalling system, which is responsible for many biological processes including cell proliferation, differentiation and apoptosis, also is involved in embryo development. We hypothesized that altered expression of TGF-β genes in pre-implantation bovine embryos is associated with morphological abnormalities of these embryos. To test this hypothesis, we produced embryos in vitro and classified them at the blastocyst stage as either normally developed blastocysts or degenerates (growth-arrested embryos). The expression patterns of 25 genes from the TGF-β pathway were assessed using quantitative real time PCR. Ten genes showed differential expression between the two embryo groups, four genes displayed similar expressional profiles, and 11 genes had no detectable expression. An altered expression profile was statistically significant for 10 of the 14 expressed genes, and all were up-regulated in degenerate embryos vs. blastocysts. Furthermore, genomic association analysis of the cows from which embryos were produced revealed a significant association of ID3 and BMP4 polymorphisms--two of the most significant differentially expressed genes--with fertilization rate and blastocyst rate, respectively. Taken together, we conclude that TGF-β pathway genes, especially BMP4 and ID3 play a vital function in the regulation of pre-implantation embryo development at both embryo and maternal levels. Hence, these genes may be suitable as genetic markers for embryo development and fertility in cattle.

  5. Caffeine supplementation during IVM improves frequencies of nuclear maturation and preimplantation development of dromedary camel oocytes following IVF.

    PubMed

    Fathi, Mohamed; Seida, Adel A; Sobhy, Refaat R; Darwish, Gamal M; Badr, Magdy R; Moawad, Adel R

    2014-06-01

    Caffeine supplementation during oocyte IVM has been reported to improve preimplantation embryo development and the quality of in vitro-produced blastocysts in a range of species; but no studies have been done in camels. The present study investigated the effect of caffeine supplementation during dromedary camel oocyte IVM on nuclear maturation rates, IVF events, and subsequent preimplantation development. Cumulus-oocyte complexes obtained at slaughter were matured in vitro in caffeine supplemented medium either for 30 hours (caffeine 30 hours) or in the medium without caffeine supplement for 24 hours and then transferred to freshly prepared IVM medium supplemented with 10 mM caffeine for another 6 hours (caffeine 6 hours). Cumulus-oocyte complexes matured for 30 hours in the medium without caffeine supplement were used as a control. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels collected from a local slaughterhouse. Eighteen hours after insemination, presumptive zygotes were cultured in modified KSOMaa medium for 7 days. Maturation and fertilization rates were significantly higher in the caffeine 6-hour group compared with the control group (P < 0.05), whereas IVM of oocytes in caffeine-supplemented medium for 30 hours did not affect these parameters (P > 0.05). Interestingly, IVM of oocytes in caffeine supplemented medium for 6 hours significantly (P < 0.05) increased the frequencies of blastocyst development by more than two-fold when compared with control (27.78% vs. 11.76%). In conclusion, culturing dromedary camel oocytes in maturation medium without caffeine for 24 hours and then in the medium supplemented with 10 mM caffeine for 6 hours during 30-hour IVM can significantly improve frequencies of nuclear maturation, fertilization rate, and subsequent preimplantation development.

  6. Lack of Maternal Glutamate Cysteine Ligase Modifier Subunit (Gclm) Decreases Oocyte Glutathione Concentrations and Disrupts Preimplantation Development in Mice

    PubMed Central

    Nakamura, Brooke N.; Fielder, Thomas J.; Hoang, Yvonne D.; Lim, Jinhwan; McConnachie, Lisa A.; Kavanagh, Terrance J.

    2011-01-01

    Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm−/− mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm−/− females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm−/− females. Prepubertal Gclm−/− and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm−/− females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/− females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm−/− females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm−/− females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development. PMID:21558310

  7. Preimplantation development of cloned canine embryos recovered by hysterectomy or surgical uterine flushing and subsequent pregnancy outcomes.

    PubMed

    Jeong, Yeon Woo; Kim, Joung Joo; Kim, Hyun Duk; Hwang, Kyu Chan; Hyun, Sang Hwan; Kim, Nam-Hyung; Jeung, Eui-Bae; Hwang, Woo Suk

    2016-11-01

    Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos. Two hundred cloned embryos were created and transferred into 11 surrogates. The preimplantation stage cloned embryos were then collected on Days 7, 8, and 9 using an ovariohysterectomy or the Foley balloon catheter method. The recovery rate of reconstructed embryos was 63.6% and 60.6% for the ovariohysterectomy and Foley balloon catheter methods, respectively. Although significant differences were observed in the early developmental stages (one-cell and 16-cell stages), no significant difference was observed in the blastocyst stage. Significantly higher blastocyst rate was observed when the embryos were collected on Day 8 (11.4%) than on Day 7 (0.0%; P < 0.05). At the proximal uterine horn on Day 7, no embryos at any stage were found, whereas on Days 8 and 9, blastocysts were found. We have observed a 63% initial pregnancy rate at 25 to 30 days after embryo transfer and a 50% full-term pregnancy rate, whereas 6.3% of the puppies were born, and 5.5% were born live among the total transferred embryos. Our results suggest that cloned embryos can develop to blastocysts by Day 8, and full-term pregnancy can be achieved after embryo transfer in canine. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Preimplantation paternal effects of acrylamide treatment on development and micronuclei formation in mice

    SciTech Connect

    Titenko-Holland, N.; Shang, N.; Smith, M.T.

    1997-10-01

    Treatment of male mice with acrylamide (AA, 50 mg/kg, 5 days) at postmeiotic stages of spermatogenesis was used to assess paternally mediated developmental and genetic toxicity in preimplantation embryos. There was a significant increase in the proportion of morphologically abnormal embryos (88.7 v 14.8%). The proliferation of morphologically normal embryos was significantly delayed, size of nuclei and number of cells were decreased, and fragmented nuclei were found in the majority of abnormal embryos (P<0.001). Cytogenetic damage was determined by the presence of micronuclei (MN) stained with DAPI or FISH with a pancentromeric probe. AA caused 10- and 20-fold increases in cells with MN in morphologically normal and abnormal embryos, respectively (41 and 93 MN per 1000 cells). There were no detectable effects of AA on size or ratio of centromere-positive MN. The presence of MN was not associated with the number or size of cells in the embryos. These findings suggest that multiple mechanisms are involved in male-mediated developmental and genetic toxicity of AA on preimplantation stages of embryogenesis. Future studies are needed to establish how preimplantation defects contribute to postimplantation and postnatal abnormalities.

  9. Mammalian Kidney Development: Principles, Progress, and Projections

    PubMed Central

    Little, Melissa H.; McMahon, Andrew P.

    2012-01-01

    The mammalian kidney is a vital organ with considerable cellular complexity and functional diversity. Kidney development is notable for requiring distinct but coincident tubulogenic processes involving reciprocal inductive signals between mesenchymal and epithelial progenitor compartments. Key molecular pathways mediating these interactions have been identified. Further, advances in the analysis of gene expression and gene activity, coupled with a detailed knowledge of cell origins, are enhancing our understanding of kidney morphogenesis and unraveling the normal processes of postnatal repair and identifying disease-causing mechanisms. This article focuses on recent insights into central regulatory processes governing organ assembly and renal disease, and predicts future directions for the field. PMID:22550230

  10. FOXO1, FOXO3, AND FOXO4 are differently expressed during mouse oocyte maturation and preimplantation embryo development.

    PubMed

    Kuscu, Nilay; Celik-Ozenci, Ciler

    2015-01-01

    Preimplantation embryo development is affected by its environment. FoxO transcription factors are regulated by PI3K/Akt signaling pathway that essentially supports growth and development. FoxO transcription factors are at the interface of crucial cellular processes, orchestrating programs of gene expression that regulate apoptosis, cell-cycle arrest, oxidative stress resistance, DNA repair, glucose metabolism, and differentiation. In the presence of growth factors, FoxO transcription factors are localized in the cytoplasm, whereas under stress conditions they move to the nucleus and trigger transcriptional activities of their target genes. The aim of the present study is to investigate whether FoxO transcription factors are present during in vivo oocyte maturation and preimplantation embryo development. Presence and localizations of FoxO1, FoxO3 and FoxO4 proteins have been determined with immunofluorescence staining. Our results have confirmed that FoxO1, FoxO3 and FoxO4 proteins are differentially expressed in prophase I, metaphase I, metaphase II oocytes, as well as in fertilized oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst. FoxOs translocate to nucleus in embryos with developmental delay. Our findings indicate that FoxO transcription factors are present during both oocyte and embryo in vivo maturation and provide fundamental knowledge that FoxOs may regulate in vitro embryo development under stress conditions.

  11. Combined parental obesity negatively impacts preimplantation mouse embryo development, kinetics, morphology and metabolism.

    PubMed

    Finger, Bethany J; Harvey, Alexandra J; Green, Mark P; Gardner, David K

    2015-09-01

    Does combined parental obesity, both an obese mother and father, have a greater effect on mouse preimplantation embryo development and quality than single-parent obesity? Combined parental obesity causes a greater reduction in the blastocyst rate and a greater delay to the timing of key embryonic developmental events than single-parental obesity, as well as altering embryonic characteristics, such as zona pellucida width. Maternal or paternal obesity alone are known to have significant and detrimental impacts on preimplantation embryo development. Furthermore, these early embryonic perturbations can have long-term impacts on both offspring health and further generations. This is one of the first studies to examine the effects of having both an obese mother and an obese father. A cross-sectional control versus treatment mouse study of diet-induced obesity was employed, in which 300 embryos per group were generated and studied from reciprocal matings: (i) control female and control male (Lean Parented Embryos); (ii) control female and obese male (Paternal Obese Parented Embryos); (iii) obese female and control male (Maternal Obese Parented Embryos) and (iv) obese female and obese male (Combined Obese Parented embryos). Assessments of the embryonic development rate, timing of development, morphological characteristics, metabolic gene expression, metabolism and cell lineage allocation were made at selected time points and analysed in relation to parental obesity status. Three-week-old C57BL6 male and female mice were fed control (7% total fat) or high fat (21% total fat) diets for a minimum of 8 weeks. Females were superovulated, mated, fertilized zygotes recovered and standard mouse in vitro embryo culture performed. Time-lapse monitoring was undertaken to compare developmental timings and morphological characteristics (embryonic area and zona pellucida width) for embryos from all four reciprocal matings. Differential staining identified cell lineage allocation. Real

  12. Genome-wide expression profiling reveals distinct clusters of transcriptional regulation during bovine preimplantation development in vivo.

    PubMed

    Kues, W A; Sudheer, S; Herrmann, D; Carnwath, J W; Havlicek, V; Besenfelder, U; Lehrach, H; Adjaye, J; Niemann, H

    2008-12-16

    Bovine embryos can be generated by in vitro fertilization or somatic nuclear transfer; however, these differ from their in vivo counterparts in many aspects and exhibit a higher proportion of developmental abnormalities. Here, we determined for the first time the transcriptomes of bovine metaphase II oocytes and all stages of preimplantation embryos developing in vivo up to the blastocyst using the Affymetrix GeneChip Bovine Genome Array which examines approximately 23,000 transcripts. The data show that bovine oocytes and embryos transcribed a significantly higher number of genes than somatic cells. Several hundred genes were transcribed well before the 8-cell stage, at which the major activation of the bovine genome expression occurs. Importantly, stage-specific expression patterns in 2-cell, 4-cell, and 8-cell stages, and in morulae and blastocysts, were detected, indicating dynamic changes in the embryonic transcriptome and in groups of transiently active genes. Pathway analysis revealed >120 biochemical pathways that are operative in early preimplantation bovine development. Significant differences were observed between the mRNA expression profiles of in vivo and in vitro matured oocytes, highlighting the need to include in vivo derived oocytes/embryos in studies evaluating assisted reproductive techniques. This study provides the first comprehensive analysis of gene expression and transcriptome dynamics of in vivo developing bovine embryos and will serve as a basis for improving assisted reproductive technology.

  13. Lack of effect of 2. 45-GHz microwave radiation on the development of preimplantation embryos of mice

    SciTech Connect

    Inouye, M.; Matsumoto, N.; Galvin, M.J.; McRee, D.I.

    1982-01-01

    The development of preimplantation embryos after exposure to microwave radiation was studied. Female CD-1 mice were induced to superovulate, mated, and exposed to 2.45-GHz microwave or sham radiation for 3 h at power densities of 9 mW/cm2 and 19 mW/cm2 on either day 2 or 3 of pregnancy (plug day was considered day 1). Another group of mice was exposed to heat stress by placing the dams in an environmental room at an ambient temperature of 38 degrees C and relative humidity at 62% for 3 h on day 2 of pregnancy. All groups were euthanized on day 4 of pregnancy and embryos were recovered by flushing excised uterine horns. Embryos were examined for abnormalities and classified by the developmental stages. They were then treated with hypotonic solution and dissociated for counting blastomeres. Heat stress caused stunted development of embryos, but no remarkable effect of microwave radiation could be found on the development of preimplantation embryos.

  14. [Current options of preimplantion genetic screening and preimplantation genetic diagnostics].

    PubMed

    Šimečková, V

    The aim of this work is to summarize the current knowledge about preimplantation genetic screening and diagnostics. A review article. Department of Gynecology and Obstetrics, District Hospital Šternberk, IVF Clinic, Olomouc. Preimplantation genetic testing is a complex of genetic and molecular cytogenetic examinations, which can help to detect abnormalities in embryos before transfer into the uterus of the mother. These specialized examinations are based on the latest findings in genetics and assisted reproduction. The preimplantation genetic testing is necessarily associated with a method of in vitro fertilization. It is performed on isolated blastomeres on the third day of embryo cultivation. Nowadays, it is preferred trophectoderm examination of cells from the five-day blastocysts. Generally speaking, after preimplantation genetic testing, we can select only embryos without genetic load to transfer into uterus. Preimplantation genetic testing is an important part of treatment of infertility. Complex diagnostics and treatment of infertile couples are increasingly influenced by the development and use of advanced genomic technologies. Further development and application of these modern methods require close cooperation between the field of assisted reproduction and clinical genetics.

  15. Molecular analysis of radiation-induced albino (c)-locus mutations that cause death at preimplantation stages of development

    SciTech Connect

    Rinchik, E.M. ); Toenjes, R.R.; Paul, D. ); Potter, M.D. )

    1993-12-01

    Deletion mutations at the albino (c) locus have been useful for continuing the development of fine-structure physical and functional maps of the Fes-Hbb region of mouse chromosome 7. This report describes the molecular analysis of a number of radiation-induced c deletions that, when homozygous, cause death of the embryo during preimplantation stages. The distal extent of these deletions defines a locus, pid, (preimplantation development) genetically associated with this phenotype. The proximal breakpoints of eight of these deletions were mapped with respect to the Tyr (tyrosinase; albino) gene as well as to anonymous loci within the Fah-Tyr region that are defined by the Pmv-31 viral integration site and by chromosome-microdissection clones. Rearrangements corresponding to the proximal breakpoints of two of these deletions were detected by Southern blot analysis, and a size-altered restriction fragment carrying the breakpoint of one of them was cloned. A probe derived from this deletion fusion fragment defines a locus, D7Rn6, which maps within (or distal to) the pid region, and which discriminates among the distal extents of deletions eliciting the pid phenotype. Extension of physical maps from D7Rn6 should provide access both to the pid region and to loci mapping distal to pid that are defined by N-ethyl-N-nitrosourea-induced lethal mutations. 36 refs., 10 figs.

  16. 61 REVERSIBLE INHIBITION OF BOVINE MINOR EMBRYONIC GENOME ACTIVATION IMPAIRS PRE-IMPLANTATION DEVELOPMENT.

    PubMed

    Nociti, R P; Sampaio, R V; de Lima, V F M H; Schultz, R M; Ross, P J

    2016-01-01

    Bovine pre-implantation embryos can develop in the absence of gene expression up to the 8/16-cell stage, the time when the major embryonic genome activation (EGA) occurs. Some embryonic genes, however, are transcribed before EGA (minor EGA). This study used a reversible inhibitor of RNA Polymerase II (5,6 dichlorobenzimidazole 1-β-D-ribofuranoside; DRB) to assess the importance of minor EGA for development to the blastocyst stage. Oocytes were matured and inseminated in vitro, and the fertilized eggs were cultured in supplemented KSOMaa and allocated to different treatments 16h post-insemination (hpi). Development was recorded at 44 and 72 hpi, and the incidence of blastocyst formation on Day 7 (IVF=Day 0) was recorded. Data were analysed by ANOVA followed by Duncan test. First, we tested different DRB concentrations [50μM (D50), 75μM (D75), 100μM (D100), and dimethyl sulfoxide vehicle control (CTRL)] to block development to blastocyst when continuously present. Only embryos in CTRL produced blastocysts (45.0±5.8%; 4 replicates with a total of 391 oocytes examined). No difference in development was observed at 44h (57.9±16.5, 53.3±10.5, 60.5±19.0, and 52.3±5.8% for D50, D75, D100, and CTRL, respectively) and 72h (78.9±8.8, 66.1±11.7, 71.5±16.5, and 70.8±5.6% for D50, D75, D100, and CTRL, respectively). Next, in 7 replicates (751 oocytes) we determined the effect of blocking transcription (50μM DRB) spanning 2 embryo stages (periods of 28h), initiated at 16hpi (1&2C), 30hpi (2&4C), and 44hpi (4&8C). Controls included DRB treatment from 16 to 72hpi (1-8C) and CTRL. There was no difference in development at 44 and 72h. The incidence of blastocyst formation, however, was significantly decreased in all treatment groups compared with CTRL (27.7±4.7; 15.1±3.5; 23.3±3.1; 20.5±1.9; and 42.1±3.2% for 1&2C, 2&4C, 4&8C, 1-8C, and CTRL, respectively). Finally, in 12 replicates (1499 oocytes), the effect of blocking transcription for 14-h periods, spanning

  17. Global Epigenomic Reconfiguration During Mammalian Brain Development

    PubMed Central

    Nery, Joseph R.; Urich, Mark; Puddifoot, Clare A.; Johnson, Nicholas D.; Lucero, Jacinta; Huang, Yun; Dwork, Andrew J.; Schultz, Matthew D.; Yu, Miao; Tonti-Filippini, Julian; Heyn, Holger; Hu, Shijun; Wu, Joseph C.; Rao, Anjana; Esteller, Manel; He, Chuan; Haghighi, Fatemeh G.; Sejnowski, Terrence J.; Behrens, M. Margarita; Ecker, Joseph R.

    2013-01-01

    DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity. PMID:23828890

  18. Involvement of free cholesterol and high-density lipoprotein in development and resistance of the preimplantation bovine embryo to heat shock.

    PubMed

    Moss, J I; Garrett, T J; Hansen, P J

    2012-11-01

    Development of the mammalian preimplantation embryo is susceptible to disruption by elevated temperature. The molecular and biochemical bases for developmental, genetic, and other differences in embryonic resistance to heat shock are largely not known. Here we tested the hypothesis that increasing free cholesterol content could improve embryonic resistance to heat shock. Culture of bovine embryos at 41.0°C for 15 h beginning at 30 h after insemination (1- to 2-cell stage) reduced development to the blastocyst stage. Reduction in embryonic cholesterol content by culture with methyl-β-cyclodextrin (MBCD) reduced development. This effect of MBCD could be abrogated in 1 of 2 experiments if the molecule was loaded with cholesterol before addition to culture medium. Even though culture with cholesterol-loaded MBCD increased free cholesterol content, it did not increase resistance of embryos to heat shock. Treatment of embryos with cholesterol-loaded high density lipoprotein (HDL) increased embryonic resistance to heat shock even though it slightly reduced embryo cholesterol content. It is likely that other actions of HDL (e.g., protection from free radicals) were responsible for the thermoprotective properties of this molecule. A final experiment was performed to determine whether the increased resistance of embryos at d 5 of development to heat shock as compared with the 2-cell embryo was due to changes in free cholesterol content. However, there was no significant difference in cholesterol content between 2-cell embryos and d 5 embryos that were > 16 cells in development. In conclusion, raising cholesterol content does not improve embryonic survival in response to heat shock. Depletion of cholesterol, in contrast, reduces competence of embryos to develop to the blastocyst stage. High density lipoprotein is thermoprotective to embryos and probably acts through a mechanism independent of its actions on embryonic content of free cholesterol.

  19. Protective Effect of Quercetin on the Development of Preimplantation Mouse Embryos against Hydrogen Peroxide-Induced Oxidative Injury

    PubMed Central

    Zhang, Qin-hua; Yan, Zhi-guang; Liang, Hong-xing; Chai, Wei-ran; Yan, Zheng; Kuang, Yan-ping; Qi, Cong

    2014-01-01

    Quercetin, a plant-derived flavonoid in Chinese herbs, fruits and wine, displays antioxidant properties in many pathological processes associated with oxidative stress. However, the effect of quercetin on the development of preimplantation embryos under oxidative stress is unclear. The present study sought to determine the protective effect and underlying mechanism of action of quercetin against hydrogen peroxide (H2O2)-induced oxidative injury in mouse zygotes. H2O2 treatment impaired the development of mouse zygotes in vitro, decreasing the rates of blastocyst formation and hatched, and increasing the fragmentation, apoptosis and retardation in blastocysts. Quercetin strongly protected zygotes from H2O2-induced oxidative injury by decreasing the reactive oxygen species level, maintaining mitochondrial function and modulating total antioxidant capability, the activity of the enzymatic antioxidants, including glutathione peroxidase and catalase activity to keep the cellular redox environment. Additionally, quercetin had no effect on the level of glutathione, the main non-enzymatic antioxidant in embryos. PMID:24586844

  20. The Anti-Apoptotic Role of Berberine in Preimplantation Embryo In Vitro Development through Regulation of miRNA-21.

    PubMed

    Zhang, Chao; Shi, Ya-Ran; Liu, Xiao-Ran; Cao, Yong-Chun; Zhen, Di; Jia, Zi-Ye; Jiang, Jin-Qi; Tian, Jian-Hui; Gao, Jian-Ming

    2015-01-01

    Traditional Chinese medicinal herbs containing berberine have been historically used to prevent miscarriage. Here, we investigated whether the anti-apoptotic effects of berberine on pre-implantation embryonic development are regulated by miRNA-21. Mouse pronuclear embryos were cultured in medium with or without berberine, and some were then microinjected with a miRNA-21 inhibitor. The in vitro developmental rates of 2- and 4-cell embryos and blastocysts, blastocyst cell numbers, apoptotic rates, and apoptotic cell numbers were measured in each group. Furthermore, we examined the transcription levels of miRNA-21 and its target genes (caspase-3, PTEN, and Bcl-2) and their translation levels. Comparisons were made with in vivo-developed and untreated embryos. We found that berberine significantly increased the developmental rates and cell numbers of mouse blastocysts and decreased apoptotic cell rates in vitro. Berberine also significantly increased miRNA-21 and Bcl-2 transcription levels and significantly decreased caspase-3 and PTEN transcription levels. In embryos treated with a miRNA-21 inhibitor, the results followed the opposite trend; PTEN and caspase-3 transcription levels increased significantly, while the transcription level of Bcl-2 decreased significantly. Additionally, berberine treatment significantly increased the Bcl-2 protein level and significantly decreased the caspase-3 and PTEN protein levels in blastocysts, but there were no significant differences observed in the levels of these proteins in 2- and 4-cell embryos. This study revealed that miRNA-21 is important for pre-implantation embryonic development, especially blastocyst development in vitro. Berberine elevates miRNA-21 expression, decreases PTEN and caspase-3 levels, increases Bcl-2 levels, and exerts anti-apoptotic and pro-growth effects.

  1. Ontogenetic development of the mammalian circadian system.

    PubMed

    Weinert, Dietmar

    2005-01-01

    This review summarizes the current knowledge about the ontogenetic development of the circadian system in mammals. The developmental changes of overt rhythms are discussed, although the main focus of the review is the underlying neuronal and molecular mechanisms. In addition, the review describes ontogenetic development, not only as a process of morpho-functional maturation. The need of repeated adaptations and readaptations due to changing developmental stage and environmental conditions is also considered. The review analyzes mainly rodent data, obtained from the literature and from the author's own studies. Results from other species, including humans, are presented to demonstrate common features and species-dependent differences. The review first describes the development of the suprachiasmatic nuclei as the central pacemaker system and shows that intrinsic circadian rhythms are already generated in the mammalian fetus. As in adult organisms, the period length is different from 24 h and needs continuous correction by environmental periodicities, or zeitgebers. The investigation of the ontogenetic development of the mechanisms of entrainment reveals that, at prenatal and early postnatal stages, non-photic cues deriving from the mother are effective. Light-dark entrainment develops later. At a certain age, both photic and non-photic zeitgebers may act in parallel, even though the respective time information is 12 h out of phase. That leads to a temporary internal desynchronization. Because rhythmic information needs to be transferred to effector organs, the corresponding neural and humoral signalling pathways are also briefly described. Finally, to be able to transform a rhythmic signal into an overt rhythm, the corresponding effector organs must be functionally mature. As many of these organs are able to generate their own intrinsic rhythms, another aspect of the review is dedicated to the development of peripheral oscillators and mechanisms of their entrainment

  2. Programmed cell senescence during mammalian embryonic development.

    PubMed

    Muñoz-Espín, Daniel; Cañamero, Marta; Maraver, Antonio; Gómez-López, Gonzalo; Contreras, Julio; Murillo-Cuesta, Silvia; Rodríguez-Baeza, Alfonso; Varela-Nieto, Isabel; Ruberte, Jesús; Collado, Manuel; Serrano, Manuel

    2013-11-21

    Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

    PubMed Central

    McCoy, Rajiv C.; Demko, Zachary P.; Ryan, Allison; Banjevic, Milena; Hill, Matthew; Sigurjonsson, Styrmir; Rabinowitz, Matthew; Petrov, Dmitri A.

    2015-01-01

    Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight

  4. Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

    PubMed Central

    Miao, Yi-Liang; Williams, Carmen J.

    2012-01-01

    Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

  5. HUWE1 plays important role in mouse preimplantation embryo development and the dysregulation is associated with poor embryo development in humans

    PubMed Central

    Chen, L. J.; Xu, W. M.; Yang, M.; Wang, K.; Chen, Y.; Huang, X. J.; Ma, Q. H.

    2016-01-01

    HUWE1 is a HECT domain containing ubiquitin ligase implicated in neurogenesis, spermatogenesis and cancer development. The purpose of the current study is to investigate the role of HUWE1 in early embryo development. Here we demonstrate that Huwe1 is expressed in both nucleus and cytoplasm of preimplantation mouse embryos as well as gametes. Hypoxia (5% O2) treatment could significantly increase Huwe1 expression during mouse embryo development process. HUWE1 knockdown inhibited normal embryonic development and reduced blastocyst formation, and increased apoptotic cell numbers were observed in the embryos of HUWE1 knockdown group. Human embryo staining result showed that reduced HUWE1 staining was observed in the poor-quality embryos. Furthermore, Western blot result showed that significantly reduced expression of HUWE1 was observed in the villi of miscarriage embryos compared with the normal control, indicating that reduced expression of HUWE1 is related to poor embryo development. Oxidative reagent, H2O2 inhibited HUWE1 expression in human sperm, indicating that HUWE1 expression in sperm is regulated by oxidative stress. In conclusion, these results suggest that HUWE1 protein could contribute to preimplantation embryo development and dysregulated expression of HUWE1 could be related to poor embryo development and miscarriage in IVF clinic. PMID:27901130

  6. Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment.

    PubMed

    Swain, J E; Smith, G D

    2011-01-01

    The majority of research aimed at improving embryo development in vitro has focused on manipulation of the chemical environment, examining details such as energy substrate composition and impact of various growth factors or other supplements. In comparison, relatively little work has been done examining the physical requirements of preimplantation embryos and the role culture platforms or devices can play in influencing embryo development. Electronic searches were performed using keywords centered on embryo culture techniques using PUBMED through June 2010 and references were searched for additional research articles. Various approaches to in vitro embryo culture that involve manipulations of the physical culture environment are emerging. Novel culture platforms being developed examine issues such as media volume and embryo spacing. Furthermore, methods to permit dynamic embryo culture with fluid flow and embryo movement are now available, and novel culture surfaces are being tested. Although several factors remain to be studied to optimize efficiency, manipulations of the embryo culture microenvironment through novel culture devices may offer a means to improve embryo development in vitro. Reduced volume systems that reduce embryo spacing, such as the well-of-the-well approach, appear beneficial, although more work is needed to verify the source of their true benefit in human embryos. Emerging microfluidic technology appears to be a promising approach. However, along with the work on specialized culture surfaces, more information is required to determine the impact on human embryo development.

  7. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages.

  8. Clinical applications of preimplantation genetic testing.

    PubMed

    Brezina, Paul R; Kutteh, William H

    2015-02-19

    Genetic diagnostic technologies are rapidly changing the way medicine is practiced. Preimplantation genetic testing is a well established application of genetic testing within the context of in vitro fertilization cycles. It involves obtaining a cell(s) from a developing embryo in culture, which is then subjected to genetic diagnostic analysis; the resulting information is used to guide which embryos are transferred into the uterus. The potential applications and use of this technology have increased in recent years. Experts agree that preimplantation genetic diagnosis is clinically appropriate for many known genetic disorders. However, some applications of such testing, such as preimplantation genetic screening for aneuploidy, remain controversial. Clinical data suggest that preimplantation genetic screening may be useful, but further studies are needed to quantify the size of the effect and who would benefit most. © BMJ Publishing Group Ltd 2015.

  9. Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development

    PubMed Central

    Ruzov, Alexey; Tsenkina, Yanina; Serio, Andrea; Dudnakova, Tatiana; Fletcher, Judy; Bai, Yu; Chebotareva, Tatiana; Pells, Steve; Hannoun, Zara; Sullivan, Gareth; Chandran, Siddharthan; Hay, David C; Bradley, Mark; Wilmut, Ian; De Sousa, Paul

    2011-01-01

    Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are different from those of 5-methylcytosine (5-mC); in particular 5-hmC is enriched in embryonic contexts compared to adult tissues. A detectable 5-hmC signal appears in pre-implantation development starting at the zygote stage, where the paternal genome is subjected to a genome-wide hydroxylation of 5-mC, which precisely coincides with the loss of the 5-mC signal in the paternal pronucleus. Levels of 5-hmC are high in cells of the inner cell mass in blastocysts, and the modification colocalises with nestin-expressing cell populations in mouse post-implantation embryos. Compared to other adult mammalian organs, 5-hmC is strongly enriched in bone marrow and brain, wherein high 5-hmC content is a feature of both neuronal progenitors and post-mitotic neurons. We show that high levels of 5-hmC are not only present in mouse and human embryonic stem cells (ESCs) and lost during differentiation, as has been reported previously, but also reappear during the generation of induced pluripotent stem cells; thus 5-hmC enrichment correlates with a pluripotent cell state. Our findings suggest that apart from the cells of neuronal lineages, high levels of genomic 5-hmC are an epigenetic feature of embryonic cell populations and cellular pluri- and multi-lineage potency. To our knowledge, 5-hmC represents the first epigenetic modification of DNA discovered whose enrichment is so cell-type specific. PMID:21747414

  10. The impact of transposable elements on mammalian development.

    PubMed

    Garcia-Perez, Jose L; Widmann, Thomas J; Adams, Ian R

    2016-11-15

    Despite often being classified as selfish or junk DNA, transposable elements (TEs) are a group of abundant genetic sequences that have a significant impact on mammalian development and genome regulation. In recent years, our understanding of how pre-existing TEs affect genome architecture, gene regulatory networks and protein function during mammalian embryogenesis has dramatically expanded. In addition, the mobilization of active TEs in selected cell types has been shown to generate genetic variation during development and in fully differentiated tissues. Importantly, the ongoing domestication and evolution of TEs appears to provide a rich source of regulatory elements, functional modules and genetic variation that fuels the evolution of mammalian developmental processes. Here, we review the functional impact that TEs exert on mammalian developmental processes and discuss how the somatic activity of TEs can influence gene regulatory networks. © 2016. Published by The Company of Biologists Ltd.

  11. Identification and functional analysis of long intergenic noncoding RNA genes in porcine pre-implantation embryonic development

    PubMed Central

    Li, Jingyu; Gao, Zhengling; Wang, Xingyu; Liu, Hongbo; Zhang, Yan; Liu, Zhonghua

    2016-01-01

    Genome-wide transcriptome studies have identified thousands of long intergenic noncoding RNAs (lincRNAs), some of which play important roles in pre-implantation embryonic development (PED). Pig is an ideal model for reproduction, however, porcine lincRNAs are still poorly characterized and it is unknown if they are associated with porcine PED. Here we reconstructed 195,531 transcripts in 122,007 loci, and identified 7,618 novel lincRNAs from 4,776 loci based on published RNA-seq data. These lincRNAs show low exon number, short length, low expression level, tissue-specific expression and cis-acting, which is consistent with previous reports in other species. By weighted co-expression network analysis, we identified 5 developmental stages specific co-expression modules. Gene ontology enrichment analysis of these specific co-expression modules suggested that many lincRNAs are associated with cell cycle regulation, transcription and metabolism to regulate the process of zygotic genome activation. Futhermore, we identified hub lincRNAs in each co-expression modules, and found two lincRNAs TCONS_00166370 and TCONS_00020255 may play a vital role in porcine PED. This study systematically analyze lincRNAs in pig and provides the first catalog of lincRNAs that might function as gene regulatory factors of porcine PED. PMID:27922056

  12. Exposure of mouse embryos to ethanol during preimplantation development: effect on DNA methylation in the h19 imprinting control region.

    PubMed

    Haycock, Philip C; Ramsay, Michéle

    2009-10-01

    In the present study, it was hypothesized that disruption of imprinting control in the H19/Igf2 domain may be a mechanism of ethanol-induced growth retardation-a key clinical feature of the fetal alcohol spectrum disorders (FASD). To test this prediction, genomic bisulphite sequencing was carried out on 473 bp of the H19 imprinting control region in DNA obtained from midgestation F(1) hybrid mouse embryos (C57BL/6 x Mus musculus castaneus) exposed to ethanol during preimplantation development. Although ethanol-exposed placentae and embryos were severely growth retarded in comparison with saline-treated controls, DNA methylation at paternal and maternal alleles was unaffected in embryos. However, paternal alleles were significantly less methylated in ethanol-treated placentae in comparison with saline-treated controls. Partial correlations suggested that the relationship between ethanol and placental weight partly depended on DNA methylation at a CCCTC-binding factor site on the paternal allele in placentae, suggesting a novel mechanism of ethanol-induced growth retardation. In contrast, partial correlations suggested that embryo growth retardation was independent of placental growth retardation. Relaxation of allele-specific DNA methylation in control placentae in comparison with control embryos was also observed, consistent with a model of imprinting in which 1) regulation of allele-specific DNA methylation in the placenta depends on a stochastic interplay between silencer and enhancer chromatin assembly factors and 2) imprinting control mechanisms in the embryo are more robust to environmental perturbations.

  13. The Role of Peroxisome Proliferator-Activated Receptors in the Development and Physiology of Gametes and Preimplantation Embryos

    PubMed Central

    Huang, Jaou-Chen

    2008-01-01

    In several species, a family of nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) composed of three isotypes, is expressed in somatic cells and germ cells of the ovary as well as the testis. Invalidation of these receptors in mice or stimulation of these receptors in vivo or in vitro showed that each receptor has physiological roles in the gamete maturation or the embryo development. In addition, synthetic PPAR γ ligands are recently used to induce ovulation in women with polycystic ovary disease. These results reveal the positive actions of PPAR in reproduction. On the other hand, xenobiotics molecules (in herbicides, plasticizers, or components of personal care products), capable of activating PPAR, may disrupt normal PPAR functions in the ovary or the testis and have consequences on the quality of the gametes and the embryos. Despite the recent data obtained on the biological actions of PPARs in reproduction, relatively little is known about PPARs in gametes and embryos. This review summarizes the current knowledge on the expression and the function of PPARs as well as their partners, retinoid X receptors (RXRs), in germ cells and preimplantation embryos. The effects of natural and synthetic PPAR ligands will also be discussed from the perspectives of reproductive toxicology and assisted reproductive technology. PMID:18354728

  14. Preimplantation factor (PIF) analog prevents type I diabetes mellitus (TIDM) development by preserving pancreatic function in NOD mice.

    PubMed

    Weiss, Lola; Bernstein, Steve; Jones, Richard; Amunugama, Ravi; Krizman, David; Jebailey, Lellean; Almogi-Hazan, Osnat; Hazan, Osnat; Yekhtin, Zhanna; Yachtin, Janna; Shiner, Reut; Reibstein, Israel; Triche, Elizabeth; Slavin, Shimon; Or, Reuven; Barnea, Eytan R

    2011-08-01

    Preimplantation factor (PIF) is a novel embryo-secreted immunomodulatory peptide. Its synthetic analog (sPIF) modulates maternal immunity without suppression. There is an urgent need to develop agents that could prevent the development of type 1 diabetes mellitus (TIDM). Herein, we examine sPIF's preventive effect on TIDM development by using acute adoptive-transfer (ATDM) and spontaneously developing (SDM) in non-obese diabetic (NOD) murine models. Diabetes was evaluated by urinary and plasma glucose, intraperitoneal glucose tolerance test (IPGTT), pancreatic islets insulin staining by immunohistochemistry and by pancreatic proteome evaluation using mass spectrometry, followed by signal pathway analysis. Continuous administration of sPIF for 4-weeks prevents diabetes development in ATDM model in >90% of recipients demonstrated by normal IPGTT, preserved islets architecture, number, and insulin staining. (P < 0.01). sPIF effect was specific; its protective effects are not replicated by scrambled PIF (χ(2) = 0.009) control. sPIF led also to increased circulating Th2 and Th1 cytokines. In SDM model, 4-week continuous sPIF administration prevented onset of diabetes for 21 weeks post-therapy (P < 0.01). Low-dose sPIF administration for 16 weeks prevented diabetes development up to 14 weeks post-therapy, evidenced by preserved islets architecture and insulin staining. In SDM model, pancreatic proteome pathway analysis demonstrated that sPIF regulates protein traffic, prevents protein misfolding and aggregation, and reduces oxidative stress and islets apoptosis, leading to preserved insulin staining. sPIF further increased insulin receptor expression and reduced actin and tubulin proteins, thereby blocking neutrophil invasion and inflammation. Exocrine pancreatic function was also preserved. sPIF administration results in marked prevention of spontaneous and induced adoptive-transfer diabetes suggesting its potential effectiveness in treating early-stage TIDM.

  15. Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis

    PubMed Central

    Steffann, J; Frydman, N; Gigarel, N; Burlet, P; Ray, P F; Fanchin, R; Feyereisen, E; Kerbrat, V; Tachdjian, G; Bonnefont, J‐P; Frydman, R; Munnich, A

    2006-01-01

    Background Diseases arising from mitochondrial DNA (mtDNA) mutations are usually serious pleiotropic disorders with maternal inheritance. Owing to the high recurrence risk in the progeny of carrier females, “at‐risk” couples often ask for prenatal diagnosis. However, reliability of such practices remains under debate. Preimplantation diagnosis (PGD), a theoretical alternative to conventional prenatal diagnosis, requires that the mutant load measured in a single cell from an eight cell embryo accurately reflects the overall heteroplasmy of the whole embryo, but this is not known to be the case. Objective To investigate the segregation of an mtDNA length polymorphism in blastomeres of 15 control embryos from four unrelated couples, the NARP mutation in blastomeres of three embryos from a carrier of this mutation. Results Variability of the mtDNA polymorphism heteroplasmy among blastomeres from each embryo was limited, ranging from zero to 19%, with a mean of 7%. PGD for the neurogenic ataxia retinitis pigmentosa (NARP) mtDNA mutation (8993T→G) was therefore carried out in the carrier mother of an affected child. One of three embryos was shown to carry 100% of mutant mtDNA species while the remaining two were mutation‐free. These two embryos were transferred, resulting in a singleton pregnancy with delivery of a healthy child. Conclusions This PGD, the first reported for a mtDNA mutation, illustrates the skewed meiotic segregation of the NARP mtDNA mutation in early human development. However, discrepancies between the segregation patterns of the NARP mutation and the HV2 polymorphism indicate that a particular mtDNA nucleotide variant might differentially influenced the mtDNA segregation, precluding any assumption on feasibility of PGD for other mtDNA mutations. PMID:16155197

  16. Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo.

    PubMed Central

    Zwingman, T; Erickson, R P; Boyer, T; Ao, A

    1993-01-01

    We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation. Images PMID:8430091

  17. Transcription of the sex-determining region genes Sry and Zfy in the mouse preimplantation embryo.

    PubMed

    Zwingman, T; Erickson, R P; Boyer, T; Ao, A

    1993-02-01

    We have confirmed the faster growth of male preimplantation mouse embryos. We have also studied the transcription of Y chromosomal genes postulated to have a role in sex determination, using the highly sensitive technique of reverse-transcription polymerase chain reaction at these early stages. We find that two sex-determining region genes, Sry and Zfy, are transcribed during mouse preimplantation development, while the Zfy homologs Zfx and Zfa and a sex-determining region gene originally called A1s9 (now called Ube1y-1) are not. We also show that the anti-Müllerian hormone gene, which contains a Sry consensus binding element in its 5' promoter region, is not transcribed at this time. Developmental curves show that Sry and Zfy are expressed commencing at the two-cell stage. These results suggest that mammalian sex determination starts prior to gonad differentiation.

  18. Innovative developments for long-term mammalian pest control.

    PubMed

    Blackie, Helen M; MacKay, Jamie W B; Allen, Will J; Smith, Des H V; Barrett, Brent; Whyte, Belinda I; Murphy, Elaine C; Ross, James; Shapiro, Lee; Ogilvie, Shaun; Sam, Shona; MacMorran, Duncan; Inder, Shane; Eason, Charles T

    2014-03-01

    Invasive mammalian pests have inflicted substantial environmental and economic damage on a worldwide scale. Over the last 30 years there has been minimal innovation in the development of new control tools. The development of new vertebrate pesticides, for example, has been largely restricted due to the costly and time-consuming processes associated with testing and registration. In this article we discuss recent progress and trends in a number of areas of research aimed to achieve long-term population suppression or eradication of mammalian pest species. The examples discussed here are emerging from research being conducted in New Zealand, where invasive mammalian pests are one of the greatest threats facing the national environment and economy. © 2013 Society of Chemical Industry.

  19. Erythroid development in the mammalian embryo.

    PubMed

    Baron, Margaret H; Vacaru, Andrei; Nieves, Johnathan

    2013-12-01

    Erythropoiesis is the process by which progenitors for red blood cells are produced and terminally differentiate. In all vertebrates, two morphologically distinct erythroid lineages (primitive, embryonic, and definitive, fetal/adult) form successively within the yolk sac, fetal liver, and marrow and are essential for normal development. Red blood cells have evolved highly specialized functions in oxygen transport, defense against oxidation, and vascular remodeling. Here we review key features of the ontogeny of red blood cell development in mammals, highlight similarities and differences revealed by genetic and gene expression profiling studies, and discuss methods for identifying erythroid cells at different stages of development and differentiation.

  20. Preimplantation genetic screening.

    PubMed

    Harper, Joyce C

    2017-01-01

    Preimplantation genetic diagnosis was first successfully performed in 1989 as an alternative to prenatal diagnosis for couples at risk of transmitting a genetic or chromosomal abnormality, such as cystic fibrosis, to their child. From embryos generated in vitro, biopsied cells are genetically tested. From the mid-1990s, this technology has been employed as an embryo selection tool for patients undergoing in vitro fertilisation, screening as many chromosomes as possible, in the hope that selecting chromosomally normal embryos will lead to higher implantation and decreased miscarriage rates. This procedure, preimplantation genetic screening, was initially performed using fluorescent in situ hybridisation, but 11 randomised controlled trials of screening using this technique showed no improvement in in vitro fertilisation delivery rates. Progress in genetic testing has led to the introduction of array comparative genomic hybridisation, quantitative polymerase chain reaction, and next generation sequencing for preimplantation genetic screening, and three small randomised controlled trials of preimplantation genetic screening using these new techniques indicate a modest benefit. Other trials are still in progress but, regardless of their results, preimplantation genetic screening is now being offered globally. In the near future, it is likely that sequencing will be used to screen the full genetic code of the embryo.

  1. Where hearing starts: The development of the mammalian cochlea

    PubMed Central

    Basch, Martin L.; Brown, Rogers M.; Jen, Hsin-I; Groves, Andrew K.

    2016-01-01

    The mammalian cochlea is a remarkable sensory organ, capable of perceiving sound over a range of 1012 in pressure and discriminating both infrasonic and ultrasonic frequencies in different species. The sensory hair cells of the mammalian cochlea are exquisitely sensitive, responding to atomic-level deflections at speeds on the order of tens of microseconds. The number and placement of hair cells are precisely determined during inner ear development, and a large number of developmental processes sculpt the shape, size and morphology of these cells along the length of the cochlear duct to make them optimally responsive to different sound frequencies. In this review, we briefly discuss the evolutionary origins of the mammalian cochlea, and then describe the successive developmental processes that lead to its induction, cell cycle exit, cellular patterning and the establishment of topologically distinct frequency responses along its length. PMID:26052920

  2. Mammalian oocyte development: checkpoints for competence.

    PubMed

    Fair, Trudee

    2010-01-01

    During the lifespan of the female, biochemical changes occur in the ovarian environment. These changes are brought about by numerous endogenous and exogenous factors, including husbandry practices, production demands and disease, and can have a profound effect on ovarian oocyte quality and subsequent embryo development. Despite many investigations, there is no consensus regarding the time or period of follicular oocyte development that is particularly sensitive to insult. Here, the key molecular and morphological events that occur during oocyte and follicle growth are reviewed, with a specific focus on identifying critical checkpoints in oocyte development. The secondary follicle stage appears to be a key phase in follicular oocyte development because major events such as activation of the oocyte transcriptome, sequestration of the zona pellucida, establishment of bidirectional communication between the granulosa cells and the oocyte and cortical granule synthesis occur during this period of development. Several months later, the periovulatory period is also characterised by the occurrence of critical events, including appropriate degradation or polyadenylation of mRNA transcripts, resumption of meiosis, spindle formation, chromosome alignment and segregation, and so should also be considered as a potential checkpoint of oocyte development.

  3. Lung development of monotremes: evidence for the mammalian morphotype.

    PubMed

    Ferner, Kirsten; Zeller, Ulrich; Renfree, Marilyn B

    2009-02-01

    The reproductive strategies and the extent of development of neonates differ markedly between the three extant mammalian groups: the Monotremata, Marsupialia, and Eutheria. Monotremes and marsupials produce highly altricial offspring whereas the neonates of eutherian mammals range from altricial to precocial. The ability of the newborn mammal to leave the environment in which it developed depends highly on the degree of maturation of the cardio-respiratory system at the time of birth. The lung structure is thus a reflection of the metabolic capacity of neonates. The lung development in monotremes (Ornithorhynchus anatinus, Tachyglossus aculeatus), in one marsupial (Monodelphis domestica), and one altricial eutherian (Suncus murinus) species was examined. The results and additional data from the literature were integrated into a morphotype reconstruction of the lung structure of the mammalian neonate. The lung parenchyma of monotremes and marsupials was at the early terminal air sac stage at birth, with large terminal air sacs. The lung developed slowly. In contrast, altricial eutherian neonates had more advanced lungs at the late terminal air sac stage and postnatally, lung maturation proceeded rapidly. The mammalian lung is highly conserved in many respects between monotreme, marsupial, and eutherian species and the structural differences in the neonatal lungs can be explained mainly by different developmental rates. The lung structure of newborn marsupials and monotremes thus resembles the ancestral condition of the mammalian lung at birth, whereas the eutherian newborns have a more mature lung structure.

  4. Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media.

    PubMed

    Summers, Michael C; Bird, Sophie; Mirzai, Fatima Mirzazadeh; Thornhill, Alan; Biggers, John D

    2013-12-01

    A comparison was made of the development of human zygotes in either a one-step (Global® medium) or two-step culture system (Quinn's Advantage®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global® medium and Quinn's Advantage® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global® medium and Quinn's Advantage® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.

  5. The evolution and development of mammalian flight.

    PubMed

    Cooper, Lisa Noelle; Cretekos, Chris J; Sears, Karen E

    2012-01-01

    Mammals have evolved a stunning diversity of limb morphologies (e.g., wings, flippers, hands, and paws) that allowed access to a wide range of habitats. Over 50 million years ago, bats (Order Chiroptera) evolved a wing (composed of a thin membrane encasing long digits) and thereby achieved powered flight. Unfortunately, the fossil record currently lacks any transitional fossils between a rodent-like ancestor and a winged bat. To reconstruct how this important evolutionary transition occurred, researchers have begun to employ an evolutionary developmental approach. This approach has revealed some of the embryological and molecular changes that have contributed to the evolution of the bat wing. For example, bat and mouse forelimb morphologies are similar during earliest limb development. Despite this, some key signaling centers for limb development are already divergent in bat and mouse at these early stages. Bat and mouse limb development continues to diverge, such that at later stages many differences are apparent. For example, at these later stages bats redeploy expression of toolkit genes (i.e., Fgf, Shh, Bmp, Grem) in a novel expression domain to inhibit apoptosis of the interdigital tissues. When results are taken together, a broad picture of the developmental changes that drove the transition from a hand to a wing over 50 million years ago is beginning to take shape. Moreover, studies seem to suggest that small changes in gene regulation during organogenesis can generate large evolutionary changes in phenotype.

  6. Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects

    PubMed Central

    Mehaisen, Gamal M. K.; Saeed, Ayman M.; Gad, Ahmed; Abass, Ahmed O.; Arafa, Mahmoud; El-Sayed, Ashraf

    2015-01-01

    Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10−3 M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the

  7. Glycine increases preimplantation development of mouse oocytes following vitrification at the germinal vesicle stage

    PubMed Central

    Cao, Xin-Yan; Rose, Jack; Wang, Shi-Yong; Liu, Yong; Zhao, Meng; Xing, Ming-Jie; Chang, Tong; Xu, Baozeng

    2016-01-01

    Ice-free cryopreservation, referred to as vitrification, is receiving increased attention in the human and animal assisted reproduction. However, it introduces the detrimental osmotic stress by adding and removing high contents of cryoprotectants. In this study, we evaluated the effects of normalizing cell volume regulation by adding glycine, an organic osmolyte, during vitrification of mouse germinal vesicle stage oocyte and/or subsequent maturation on its development. The data showed that glycine supplementation in either vitrification/thawing or maturation medium significantly improved the cytoplasmic maturation of MII oocytes manifested by spindle assembly, chromosomal alignment, mitochondrial distribution, euploidy rate, and blastocyst development following fertilization in vitro, compared to the control without glycine treatment. Furthermore, glycine addition during both vitrification/thawing and maturation further enhanced the oocyte quality demonstrated by various markers, including ATP contents and embryo development. Lastly, the effect of anti-apoptosis was also observed when glycine was added during vitrification. Our result suggests that reducing osmotic stress induced by vitrification could improve the development of vitrified mouse oocyte. PMID:27845423

  8. Phylogenetic memory of developing mammalian dentition.

    PubMed

    Peterkova, Renata; Lesot, Hervé; Peterka, Miroslav

    2006-05-15

    Structures suppressed during evolution can be retraced due to atavisms and vestiges. Atavism is an exceptional emergence of an ancestral form in a living individual. In contrast, ancestral vestige regularly occurs in all members of an actual species. We surveyed data about the vestigial and atavistic teeth in mammals, updated them by recent findings in mouse and human embryos, and discussed their ontogenetic and evolutionary implications. In the mouse incisor and diastema regions, dental placodes are transiently distinct being morphologically similar to the early tooth primordia in reptiles. Two large vestigial buds emerge in front of the prospective first molar and presumably correspond to the premolars eliminated during mouse evolution. The incorporation of the posterior premolar vestige into the lower first molar illustrates the putative mechanism of evolutionary disappearance of the last premolar in the mice. In mutant mice, devious development of the ancestral tooth primordia might lead to their revivification and origin of atavistic supernumerary teeth. Similarity in the developmental schedule between three molars in mice and the respective third and fourth deciduous premolar and the first molar in humans raises a question about putative homology of these teeth. The complex patterning of the vestibular and dental epithelium in human embryos is reminiscent of the pattern of "Zahnreihen" in lower vertebrates. A hypothesis was presented about the developmental relationship between the structures at the external aspect of the dentition in mammals (oral vestibule, pre-lacteal teeth, paramolar cusps/teeth), the tooth glands in reptiles, and the earliest teeth in lower vertebrates.

  9. Analysis of transcription factor Stk40 expression and function during mouse pre-implantation embryonic development.

    PubMed

    Zhang, Junqiang; Zhang, Juanjuan; Zhao, Chun; Shen, Rong; Guo, Xirong; Li, Chaojun; Ling, Xiufeng; Liu, Chang

    2014-02-01

    Determining the molecular mechanisms in the regulation of early embryonic development is crucial for assisted reproductive technology clinical applications. Serine/threonine protein kinase 40 (Stk40) is a member of the serine/threonine kinase family. It is essential in diverse signaling pathways associated with a wide range of cellular activities, including proliferation, differentiation, survival and apoptosis. However, its involvement and molecular mechanisms in pre‑implantation embryonic development have not been well‑defined. In the present study, it was demonstrated that Stk40 was involved in the development of mouse pre‑implantation embryos. Immunofluorescence and confocal microscopy analyses showed that Stk40 was equally expressed in the nuclei and cytoplasm during all stages of pre‑implantation mouse embryos of imprinting control region mice. Reverse transcription‑polymerase chain reaction showed a significantly higher transcription rate of Stk40 mRNA in the two‑cell stage. The results demonstrated that Stk40 downregulation by microinjection of small interfering RNA into the mouse zygote markedly decreased the blastulation compared with that in the control (Stk40i‑1 vs. control: 65.2% and 77.0%, P<0.05 and Stk40i‑2 vs. control: 49.8% and 70.1%, respectively, P<0.05). In addition, silencing of Stk40 significantly increased the transcription rate of reticulocalbin‑2, whereas that of the homeobox protein, Cdx2, was decreased. In conclusion, the results suggested that Stk40 may be critical in the development of pre‑implantation embryos.

  10. DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development.

    PubMed

    O'Doherty, Alan M; Magee, David A; O'Shea, Lynee C; Forde, Niamh; Beltman, Marijke E; Mamo, Solomon; Fair, Trudee

    2015-03-10

    In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation. To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization. The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.

  11. Microdrop preparation factors influence culture-media osmolality, which can impair mouse embryo preimplantation development.

    PubMed

    Swain, J E; Cabrera, L; Xu, X; Smith, G D

    2012-02-01

    Because media osmolality can impact embryo development, the effect of conditions during microdrop preparation on osmolality was examined. Various sizes of microdrops were prepared under different laboratory conditions. Drops were pipetted directly onto a dish and covered by oil (standard method) or pipetted on the dish, overlaid with oil before removing the underlying media and replaced with fresh media (wash-drop method). Drops were made at 23°C or on a heated stage (37°C) and with or without airflow. Osmolality was assessed at 5 min and 24h. The biological impact of osmolality change was demonstrated by culturing 1-cell mouse embryos in media with varying osmolality. Reduced drop volume, increased temperature and standard method were associated with a significant increase in osmolality at both 5 min and 24h (P-values <0.001, <0.0001 and <0.0001, respectively). There was a significant interaction between airflow, decreased volume, increased temperature and standard method that caused a significant increase in osmolality (40mOsm/kg) compared with controls (P<0.04). There was no significant change in osmolality over time. Mouse embryo development was significantly reduced in media with elevated osmolality (>310mOsm/kg; P<0.05). Procedures in the IVF laboratory can alter osmolality and impact embryo development.

  12. Label-free subcellular 3D live imaging of preimplantation mouse embryos with full-field optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Zheng, Jing-gao; Lu, Danyu; Chen, Tianyuan; Wang, Chengming; Tian, Ning; Zhao, Fengying; Huo, Tiancheng; Zhang, Ning; Chen, Dieyan; Ma, Wanyun; Sun, Jia-lin; Xue, Ping

    2012-07-01

    Early patterning and polarity is of fundamental interest in preimplantation embryonic development. Label-free subcellular 3D live imaging is very helpful to its related studies. We have developed a novel system of full-field optical coherence tomography (FF-OCT) for noninvasive 3D subcellular live imaging of preimplantation mouse embryos with no need of dye labeling. 3D digitized embryos can be obtained by image processing. Label-free 3D live imaging is demonstrated for the mouse embryos at various typical preimplantation stages with a spatial resolution of 0.7 μm and imaging rate of 24 fps. Factors that relate to early patterning and polarity, such as pronuclei in zygote, shapes of zona pellucida, location of second polar body, cleavage planes, and the blastocyst axis, can be quantitatively measured. The angle between the two second cleavage planes is accurately measured to be 87 deg. It is shown that FF-OCT provides a potential breakthrough for early patterning, polarity formation, and many other preimplantation-related studies in mammalian developmental biology.

  13. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    PubMed

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  14. Factors associated with preimplantation genetic diagnosis acceptance among women concerned about hereditary breast and ovarian cancer.

    PubMed

    Vadaparampil, Susan T; Quinn, Gwendolyn P; Knapp, Caprice; Malo, Teri L; Friedman, Susan

    2009-10-01

    To assess sociodemographic, clinical, awareness, and attitudinal factors associated with acceptance of preimplantation genetic diagnosis among women concerned about hereditary breast and ovarian cancer. Participants (n = 962) were members of a national advocacy organization dedicated to empowering women at high risk for developing breast or ovarian cancer. Participants completed a web-based survey assessing factors associated with preimplantation genetic diagnosis acceptance. Factors significantly associated with acceptance in the bivariate analyses were used to build a logistic regression model. Among the 962 respondents, 318 (33.1%) selected the option that they would consider preimplantation genetic diagnosis, 367 (38.2%) would not consider preimplantation genetic diagnosis, and 277 (28.8%) selected "don't know." Significant predictors of preimplantation genetic diagnosis acceptance were the desire to have more children, having had a prenatal genetic test, preimplantation genetic diagnosis awareness, belief that preimplantation genetic diagnosis is acceptable for individuals at risk for hereditary breast and ovarian cancer, belief that preimplantation genetic diagnosis information should be given to individuals at risk for hereditary breast and ovarian cancer, concerns about preimplantation genetic diagnosis, perceived benefits of preimplantation genetic diagnosis, and how preimplantation genetic diagnosis is considered. Women at increased risk for hereditary cancer may consider preimplantation genetic diagnosis as part of their reproductive decision making. Therefore, it is important to understand existing levels of awareness and attitudes toward this technology to provide optimal counseling and support.

  15. Near-infrared laser irradiation improves the development of mouse pre-implantation embryos.

    PubMed

    Yokoo, Masaki; Mori, Miho

    2017-05-27

    The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P < 0.05). When 195 irradiated blastocysts were transferred to 18 pseudopregnant mice, all became pregnant and 92 (47.2%) normal-looking pups were born alive. When 182 control blastocysts were transferred to 17 pseudopregnant mice, 14 (82.4%) became pregnant and 54 (29.7%) normal-looking pups were born alive. The growth trajectories (up to 5 weeks) of offspring from irradiated blastocysts were similar to those from control blastocysts. Second generation offspring from transplanted animals were all fertile. These results indicate that near-infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights

  16. Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development.

    PubMed

    Lin, Chih-Jen; Conti, Marco; Ramalho-Santos, Miguel

    2013-09-01

    Histone variants can replace canonical histones in the nucleosome and modify chromatin structure and gene expression. The histone variant H3.3 preferentially associates with active chromatin and has been implicated in the regulation of a diverse range of developmental processes. However, the mechanisms by which H3.3 may regulate gene activity are unclear and gene duplication has hampered an analysis of H3.3 function in mouse. Here, we report that the specific knockdown of H3.3 in fertilized mouse zygotes leads to developmental arrest at the morula stage. This phenotype can be rescued by exogenous H3.3 but not by canonical H3.1 mRNA. Loss of H3.3 leads to over-condensation and mis-segregation of chromosomes as early as the two-cell stage, with corresponding high levels of aneuploidy, but does not appear to affect zygotic gene activation at the two-cell stage or lineage gene transcription at the morula stage. H3.3-deficient embryos have significantly reduced levels of markers of open chromatin, such as H3K36me2 and H4K16Ac. Importantly, a mutation in H3.3K36 that disrupts H3K36 methylation (H3.3K36R) does not rescue the H3.3 knockdown (KD) phenotype. In addition, H3.3 KD embryos have increased incorporation of linker H1. Knockdown of Mof (Kat8), an acetyltransferase specific for H4K16, similarly leads to excessive H1 incorporation. Remarkably, pan-H1 RNA interference (RNAi) partially rescues the chromosome condensation of H3.3 KD embryos and allows development to the blastocyst stage. These results reveal that H3.3 mediates a balance between open and condensed chromatin that is crucial for the fidelity of chromosome segregation during early mouse development.

  17. Timing of human preimplantation embryonic development is confounded by embryo origin

    PubMed Central

    Kirkegaard, K.; Sundvall, L.; Erlandsen, M.; Hindkjær, J.J.; Knudsen, U.B.; Ingerslev, H.J.

    2016-01-01

    STUDY QUESTION To what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers? SUMMARY ANSWER Up to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single factor elicits a systematic influence. WHAT IS KNOWN ALREADY Several studies report that culture conditions, patient characteristics and treatment influence timing of embryo development, which have promoted the perception that each clinic must develop individual models. Most of the studies have, however, treated embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating the statistical significance of potential correlations. STUDY DESIGN, SIZE, DURATION Infertile patients were prospectively recruited to a cohort study at a hospital fertility clinic from February 2011 to May 2013. Patients aged <38 years without endometriosis were eligible if ≥8 oocytes were retrieved. Patients were included only once. All embryos were monitored for 6 days in a time-lapse incubator. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 1507 embryos from 243 patients were included. The influence of fertilization method, BMI, maternal age, FSH dose and number of previous cycles on timing of t2-t5, duration of the 2- and 3-cell stage, and development of a blastocoel (tEB) and full blastocoel (tFB) was tested in multivariate, multilevel linear regression analysis. Predictive parameters for live birth were tested in a logistic regression analysis for 223 single transferred blastocysts, where time-lapse parameters were investigated along with patient and embryo characteristics. MAIN RESULTS AND THE ROLE OF CHANCE Moderate intra-class correlation coefficients

  18. Technical Update: Preimplantation Genetic Diagnosis and Screening.

    PubMed

    Dahdouh, Elias M; Balayla, Jacques; Audibert, François; Wilson, R Douglas; Audibert, François; Brock, Jo-Ann; Campagnolo, Carla; Carroll, June; Chong, Karen; Gagnon, Alain; Johnson, Jo-Ann; MacDonald, William; Okun, Nanette; Pastuck, Melanie; Vallée-Pouliot, Karine

    2015-05-01

    technologies. Preimplantation genetic diagnosis is an alternative to prenatal diagnosis for the detection of genetic disorders in couples at risk of transmitting a genetic condition to their offspring. Preimplantation genetic screening is being proposed to improve the effectiveness of in vitro fertilization by screening for embryonic aneuploidy. Though FISH-based PGS showed adverse effects on IVF success, emerging evidence from new studies using comprehensive chromosome screening technology appears promising. Recommendations 1. Before preimplantation genetic diagnosis is performed, genetic counselling must be provided by a certified genetic counsellor to ensure that patients fully understand the risk of having an affected child, the impact of the disease on an affected child, and the benefits and limitations of all available options for preimplantation and prenatal diagnosis. (III-A) 2. Couples should be informed that preimplantation genetic diagnosis can reduce the risk of conceiving a child with a genetic abnormality carried by one or both parents if that abnormality can be identified with tests performed on a single cell or on multiple trophectoderm cells. (II-2B) 3. Invasive prenatal or postnatal testing to confirm the results of preimplantation genetic diagnosis is encouraged because the methods used for preimplantation genetic diagnosis have technical limitations that include the possibility of a false result. (II-2B) 4. Trophectoderm biopsy has no measurable impact on embryo development, as opposed to blastomere biopsy. Therefore, whenever possible, trophectoderm biopsy should be the method of choice in embryo biopsy and should be performed by experienced hands. (I-B) 5. Preimplantation genetic diagnosis of single-gene disorders should ideally be performed with multiplex polymerase chain reaction coupled with trophectoderm biopsy whenever available. (II-2B) 6. The use of comprehensive chromosome screening technology coupled with trophectoderm biopsy in preimplantation

  19. Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

    PubMed

    Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

    2008-01-01

    In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

  20. 50 SURVIVAL OF SEXED IVF-DERIVED BOVINE EMBRYOS FROZEN AT DIFFERENT PREIMPLANTATION STAGES OF DEVELOPMENT.

    PubMed

    Ferré, L; Fresno, C; Kjelland, M; Ross, P

    2016-01-01

    The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400µL of M199 medium supplemented with ALA-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µgmL(-1)), EGF (50ngmL(-1)), ovine FSH (50ngmL(-1)), bLH (3µgmL(-1)), cysteamine (0.1mM), and 10% fetal bovine serum (FBS) for 22 to 24h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1×10(6) sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90µgmL(-1)), penicillamine (3µgmL(-1)), hypotaurine (11µgmL(-1)), and heparin (20µgmL(-1)). After 18h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5-6, blastocysts at Day 6-6.5, and expanded blastocysts at Day 6.5-7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45min. The slow freezing protocol consisted of 1.5M ethylene glycol (EG)+20% FBS+0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing

  1. Methods in preimplantation genetic diagnosis.

    PubMed

    Lizcano Gil, Luis Arturo; Lucena, Carolina; Lucena, Elkin

    2001-01-01

    Preimplantation genetic diagnosis (PGD) is a new strategy, orientated toward primary prevention of congenital anomalies in couples with reproductive risk, such as advanced maternal age, carriers of chromosomal abnormalities, and carriers of monogenic conditions. For these patients, PGD is an acceptable alternative to prenatal diagnosis, mainly in those countries where pregnancy interruption is forbidden by law. PGD effectively avoids the implications linked to traditional prenatal diagnosis. Centres that provide medical servicies on reproductive biomedicine are responsible for the development and improvement of this new prevention strategy. Thanks to advances in micromanipulation techniques, associated with recent progress in molecular genetics, PGD may be employed for any genetic condition in the future.

  2. New perspectives on preimplantation genetic diagnosis and preimplantation genetic screening.

    PubMed

    Chen, Chun-Kai; Yu, Hsing-Tse; Soong, Yung-Kuei; Lee, Chyi-Long

    2014-06-01

    Preimplantation genetic diagnosis is a procedure that involves the removal of one or more nuclei from oocytes (a polar body) or embryos (blastomeres or trophectoderm cells) in order to test for problems in genome sequence or chromosomes of the embryo prior to implantation. It provides new hope of having unaffected children, as well as avoiding the necessity of terminating an affected pregnancy for genetic parents who carry an affected gene or have balanced chromosomal status. Polymerase chain reaction-based molecular techniques are the methods used to detect gene defects with a known sequence and X-linked diseases. The indication for using this approach has expanded for couples who are prevented from having babies because they carry a serious genetic disorder to couples with conditions that are not immediately life threatening, such as cancer predisposition genes and Huntington disease. In addition, fluorescent in situ hybridization (FISH) has been widely applied for the detection of chromosome abnormalities. FISH allows the evaluation of many chromosomes at the same time, up to 15 chromosome pairs in a single cell. Preimplantation genetic screening, defined as a test that screens for aneuploidy, has been most commonly used in situations of advanced maternal age, a history of recurrent miscarriage, a history of repeated implantation failure, or a severe male factor. Unfortunately, randomized controlled trials have as yet shown no benefit with respect to preimplantation genetic screening using cleavage stage biopsy, which is probably attributable to the high levels of mosaicism at early cleavage stages and the limitations of FISH. Recently, two main types of array-based technology combined with whole genome amplification have been developed for use in preimplantation genetic diagnosis; these are comparative genomic hybridization and single nucleotide polymorphism-based arrays. Both allow the analysis of all chromosomes, and the latter also allows the haplotype of

  3. Studies toward birth and early mammalian development in space.

    PubMed

    Ronca, April E

    2003-01-01

    Sustaining life beyond Earth on either space stations or other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction and development. Pregnancy, parturition (birth) and the early development of offspring are complex processes essential for successful reproduction and the proliferation of mammalian species. While no mammal has yet undergone birth within the space environment, studies spanning the gravity continuum from 0- to 2-g are revealing startling insights into how reproduction and development may proceed under gravitational conditions deviating from those typically experienced on Earth. In this report, I review studies of pregnant Norway rats and their offspring flown in microgravity onboard the NASA Space Shuttle throughout the period corresponding to mid- to late gestation, and analogous studies of pregnant rats exposed to hypergravity (hg) onboard the NASA Ames Research Center 24-ft centrifuge. Studies of postnatal rats flown in space or exposed to centrifugation are reviewed. Although many important questions remain unanswered, the available data suggest that numerous aspects of pregnancy, birth and early mammalian development can proceed under altered gravity conditions.

  4. Studies toward birth and early mammalian development in space

    NASA Astrophysics Data System (ADS)

    Ronca, April E.

    2003-10-01

    Sustaining life beyond Earth on either space stations or other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction and development. Pregnancy, parturition (birth) and the early development of offspring are complex processes essential for successful reproduction and the proliferation of mammalian species. While no mammal has yet undergone birth within the space environment, studies spanning the gravity continuum from 0- to 2-g are revealing startling insights into how reproduction and development may proceed under gravitational conditions deviating from those typically experienced on Earth. In this report, I review studies of pregnant Norway rats and their offspring flown in microgravity (μg) onboard the NASA Space Shuttle throughout the period corresponding to mid- to late gestation, and analogous studies of pregnant rats exposed to hypergravity ( ht) onboard the NASA Ames Research Center 24-ft centrifuge. Studies of postnatal rats flown in space or exposed to centrifugation are reviewed. Although many important questions remain unanswered, the available data suggest that numerous aspects of pregnancy, birth and early mammalian development can proceed under altered gravity conditions. Published by Elsevier Ltd on behalf of COSPAR.

  5. L-carnitine supplementation during vitrification of mouse oocytes at the germinal vesicle stage improves preimplantation development following maturation and fertilization in vitro.

    PubMed

    Moawad, Adel R; Tan, Seang Lin; Xu, Baozeng; Chen, Hai Ying; Taketo, Teruko

    2013-04-01

    Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

  6. Allelic reprogramming of 3D chromatin architecture during early mammalian development.

    PubMed

    Du, Zhenhai; Zheng, Hui; Huang, Bo; Ma, Rui; Wu, Jingyi; Zhang, Xianglin; He, Jing; Xiang, Yunlong; Wang, Qiujun; Li, Yuanyuan; Ma, Jing; Zhang, Xu; Zhang, Ke; Wang, Yang; Zhang, Michael Q; Gao, Juntao; Dixon, Jesse R; Wang, Xiaowo; Zeng, Jianyang; Xie, Wei

    2017-07-12

    In mammals, chromatin organization undergoes drastic reprogramming after fertilization. However, the three-dimensional structure of chromatin and its reprogramming in preimplantation development remain poorly understood. Here, by developing a low-input Hi-C (genome-wide chromosome conformation capture) approach, we examined the reprogramming of chromatin organization during early development in mice. We found that oocytes in metaphase II show homogeneous chromatin folding that lacks detectable topologically associating domains (TADs) and chromatin compartments. Strikingly, chromatin shows greatly diminished higher-order structure after fertilization. Unexpectedly, the subsequent establishment of chromatin organization is a prolonged process that extends through preimplantation development, as characterized by slow consolidation of TADs and segregation of chromatin compartments. The two sets of parental chromosomes are spatially separated from each other and display distinct compartmentalization in zygotes. Such allele separation and allelic compartmentalization can be found as late as the 8-cell stage. Finally, we show that chromatin compaction in preimplantation embryos can partially proceed in the absence of zygotic transcription and is a multi-level hierarchical process. Taken together, our data suggest that chromatin may exist in a markedly relaxed state after fertilization, followed by progressive maturation of higher-order chromatin architecture during early development.

  7. Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization.

    PubMed

    Kwak, Seong-Sung; Cheong, Seung-A; Yoon, Junchul David; Jeon, Yubyeol; Hyun, Sang-Hwan

    2012-10-15

    We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during day 4 to 7 (22.1% and 32.4) or day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol

  8. Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development

    PubMed Central

    Vogt, Edgar J.; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

    2012-01-01

    The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development. PMID:23172912

  9. Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

    PubMed

    Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

    2012-12-01

    The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

  10. Mammalian cell line developments in speed and efficiency.

    PubMed

    Estes, Scott; Melville, Mark

    2014-01-01

    Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development.

  11. Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos

    SciTech Connect

    Zhang, Ping; Wang, Ningling; Lin, Xianhua; Jin, Li; Xu, Hong; Li, Rong; Huang, Hefeng

    2016-02-26

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

  12. Fibroblast growth factor signaling in mammalian tooth development.

    PubMed

    Li, Chun-Ying; Prochazka, Jan; Goodwin, Alice F; Klein, Ophir D

    2014-01-01

    In this review, we discuss the central role of fibroblast growth factor (FGF) signaling in mammalian tooth development. The FGF family consists of 22 members, most of which bind to four different receptor tyrosine kinases, which in turn signal through a cascade of intracellular proteins. This signaling regulates a number of cellular processes, including proliferation, differentiation, cell adhesion and cell mobility. FGF signaling first becomes important in the presumptive dental epithelium at the initiation stage of tooth development, and subsequently, it controls the invagination of the dental epithelium into the underlying mesenchyme. Later, FGFs are critical in tooth shape formation and differentiation of ameloblasts and odontoblasts, as well as in the development and homeostasis of the stem cell niche that fuels the continuously growing mouse incisor. In addition, FGF signaling is critical in human teeth, as mutations in genes encoding FGF ligands or receptors result in several congenital syndromes characterized by alterations in tooth number, morphology or enamel structure. The parallel roles of FGF signaling in mouse and human tooth development demonstrate the conserved importance of FGF signaling in mammalian odontogenesis.

  13. Evolution and development of mammalian limb integumentary structures.

    PubMed

    Hamrick, Mark W

    2003-08-15

    The adaptive radiation of mammalian clades has involved marked changes in limb morphology that have affected not only the skeleton but also the integumentary structures. For example, didelphid marsupials show distinct differences in nail and claw morphology that are functionally related to the evolution of arboreal, terrestrial, and aquatic foraging behaviors. Vespertilionoid bats have evolved different volar pad structures such as adhesive discs, scales, and skin folds, whereas didelphid marsupials have apical pads covered either with scales, ridges, or small cones. Comparative analysis of pad and claw development reveals subtle differences in mesenchymal and ectodermal patterning underlying interspecific variation in morphology. Analysis of gene expression during pad and claw development reveals that signaling molecules such as Msx1 and Hoxc13 play important roles in the morphogenesis of these integumentary structures. These findings suggest that evolutionary change in the expression of these molecules, and in the response of mesenchymal and ectodermal cells to these signaling factors, may underlie interspecific differences in nail, claw, and volar pad morphology. Evidence from comparative morphology, development, and functional genomics therefore sheds new light on both the patterns and mechanisms of evolutionary change in mammalian limb integumentary structures. Copyright 2003 Wiley-Liss, Inc.

  14. Development of a monoclonal antibody specific to cooked mammalian meats.

    PubMed

    Hsieh, Y H; Sheu, S C; Bridgman, R C

    1998-04-01

    Detection of species adulteration in ground meat products is important for consumer protection and food-labeling law enforcement. This study was conducted to develop monoclonal antibodies (MAbs) that can be used in an enzyme-linked immunosorbent assay (ELISA) for rapid detection of any cooked mammalian meats in cooked poultry products. Soluble muscle proteins extracted from cooked pork (heated at 100 degrees C for 15 min) were used as the antigen to immunized mice for developing the MAb. One that was developed, MAb 2F8 (IgG2b class), strongly reacted with cooked meat of five mammalian species (beef cattle, hogs, sheep, horse, and deer) but did not react with any cooked poultry (chicken, turkey, and duck) or raw meats. At least 0.5% by weight of pork, beef, lamb, and horse meats in a chicken-based mixture could not detect using the indirect ELISA with MAb 2F8. The MAb 2F8 is useful in a single initial screening test to detect the presence of five nonpoultry meat adulterants in cooked poultry products.

  15. Development and validation of concurrent preimplantation genetic diagnosis for single gene disorders and comprehensive chromosomal aneuploidy screening without whole genome amplification.

    PubMed

    Zimmerman, Rebekah S; Jalas, Chaim; Tao, Xin; Fedick, Anastasia M; Kim, Julia G; Pepe, Russell J; Northrop, Lesley E; Scott, Richard T; Treff, Nathan R

    2016-02-01

    To develop a novel and robust protocol for multifactorial preimplantation genetic testing of trophectoderm biopsies using quantitative polymerase chain reaction (qPCR). Prospective and blinded. Not applicable. Couples indicated for preimplantation genetic diagnosis (PGD). None. Allele dropout (ADO) and failed amplification rate, genotyping consistency, chromosome screening success rate, and clinical outcomes of qPCR-based screening. The ADO frequency on a single cell from a fibroblast cell line was 1.64% (18/1,096). When two or more cells were tested, the ADO frequency dropped to 0.02% (1/4,426). The rate of amplification failure was 1.38% (55/4,000) overall, with 2.5% (20/800) for single cells and 1.09% (35/3,200) for samples that had two or more cells. Among 152 embryos tested in 17 cases by qPCR-based PGD and CCS, 100% were successfully given a diagnosis, with 0% ADO or amplification failure. Genotyping consistency with reference laboratory results was >99%. Another 304 embryos from 43 cases were included in the clinical application of qPCR-based PGD and CCS, for which 99.7% (303/304) of the embryos were given a definitive diagnosis, with only 0.3% (1/304) having an inconclusive result owing to recombination. In patients receiving a transfer with follow-up, the pregnancy rate was 82% (27/33). This study demonstrates that the use of qPCR for PGD testing delivers consistent and more reliable results than existing methods and that single gene disorder PGD can be run concurrently with CCS without the need for additional embryo biopsy or whole genome amplification. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Stem cells and lineage development in the mammalian blastocyst.

    PubMed

    Rossant, Janet

    2007-01-01

    The mammalian blastocyst is the source of the most pluripotent stem cells known: embryonic stem (ES) cells. However, ES cells are not totipotent; in mouse chimeras, they do not contribute to extra-embryonic cell types of the trophectoderm (TE) and primitive endoderm (PrE) lineages. Understanding the genetic pathways that control pluripotency v. extra-embryonic lineage restriction is key to understanding not only normal embryonic development, but also how to reprogramme adult cells to pluripotency. The trophectoderm and primitive endoderm lineages also provide the first signals that drive patterned differentiation of the pluripotent epiblast cells of the embryo. My laboratory has produced permanent mouse cell lines from both the TE and the PrE, termed trophoblast stem (TS) and eXtra-embryonic ENdoderm (XEN) cells. We have used these cells to explore the genetic and molecular hierarchy of lineage restriction and identify the key factors that distinguish the ES cell v. the TS or XEN cell fate. The major molecular pathways of lineage commitment defined in mouse embryos and stem cells are probably conserved across mammalian species, but more comparative studies of lineage development in embryos of non-rodent mammals will likely yield interesting differences in terms of timing and details.

  17. The development of the mammalian outer and middle ear.

    PubMed

    Anthwal, Neal; Thompson, Hannah

    2016-02-01

    The mammalian ear is a complex structure divided into three main parts: the outer; middle; and inner ear. These parts are formed from all three germ layers and neural crest cells, which have to integrate successfully in order to form a fully functioning organ of hearing. Any defect in development of the outer and middle ear leads to conductive hearing loss, while defects in the inner ear can lead to sensorineural hearing loss. This review focuses on the development of the parts of the ear involved with sound transduction into the inner ear, and the parts largely ignored in the world of hearing research: the outer and middle ear. The published data on the embryonic origin, signalling, genetic control, development and timing of the mammalian middle and outer ear are reviewed here along with new data showing the Eustachian tube cartilage is of dual embryonic origin. The embryonic origin of some of these structures has only recently been uncovered (Science, 339, 2013, 1453; Development, 140, 2013, 4386), while the molecular mechanisms controlling the growth, structure and integration of many outer and middle ear components are hardly known. The genetic analysis of outer and middle ear development is rather limited, with a small number of genes often affecting either more than one part of the ear or having only very small effects on development. This review therefore highlights the necessity for further research into the development of outer and middle ear structures, which will be important for the understanding and treatment of conductive hearing loss. © 2015 Anatomical Society.

  18. Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

    PubMed Central

    D’Souza, Fiona; Pudakalakatti, Shivanand M.; Uppangala, Shubhashree; Honguntikar, Sachin; Salian, Sujith Raj; Kalthur, Guruprasad; Pasricha, Renu; Appajigowda, Divya; Atreya, Hanudatta S.; Adiga, Satish Kumar

    2016-01-01

    Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs. PMID:27853269

  19. Retinoic Acid Regulates Embryonic Development of Mammalian Submandibular Salivary Glands

    PubMed Central

    Wright, Diana M.; Buenger, Deanna E.; Abashev, Timur M.; Lindeman, Robert P.; Ding, Jixiang; Sandell, Lisa L.

    2015-01-01

    Organogenesis is orchestrated by cell and tissue interactions mediated by molecular signals. Identification of relevant signals, and the tissues that generate and receive them, are important goals of developmental research. Here, we demonstrate that Retinoic Acid (RA) is a critical signaling molecule important for morphogenesis of mammalian submandibular salivary glands (SMG). By examining late stage RA deficient embryos of Rdh10 mutant mice we show that SMG development requires RA in a dose-dependent manner. Additionally, we find that active RA signaling occurs in SMG tissues, arising earlier than any other known marker of SMG development and persisting throughout gland morphogenesis. At the initial bud stage of development, we find RA production occurs in SMG mesenchyme, while RA signaling occurs in epithelium. We also demonstrate active RA signaling occurs in glands cultured ex vivo, and treatment with an inhibitor of RA signaling blocks growth and branching. Together these data identify RA signaling as a direct regulator of SMG organogenesis. PMID:26278034

  20. Studies Toward Birth and Early Mammalian Development in Space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Successful reproduction is the hallmark of a species' ability to adapt to its environment and must be realized to sustain life beyond Earth. Before taking this immense step, we need to understand the effects of altered gravity on critical phases of mammalian reproduction, viz., those events surrounding pregnancy, birth and the early development of offspring. No mammal has yet undergone birth in space. however studies spanning the gravity continuum from 0 to 2-g are revealing insights into how birth and early postnatal development will proceed in space. In this presentation, I will report the results of behavioral studies of rat mothers and offspring exposed from mid- to late pregnancy to either hypogravity (0-g) or hypergravity (1.5 or 2-g).

  1. Studies Toward Birth and Early Mammalian Development in Space

    NASA Technical Reports Server (NTRS)

    Ronca, April E.; Dalton, Bonnie (Technical Monitor)

    2002-01-01

    Successful reproduction is the hallmark of a species' ability to adapt to its environment and must be realized to sustain life beyond Earth. Before taking this immense step, we need to understand the effects of altered gravity on critical phases of mammalian reproduction, viz., those events surrounding pregnancy, birth and the early development of offspring. No mammal has yet undergone birth in space. however studies spanning the gravity continuum from 0 to 2-g are revealing insights into how birth and early postnatal development will proceed in space. In this presentation, I will report the results of behavioral studies of rat mothers and offspring exposed from mid- to late pregnancy to either hypogravity (0-g) or hypergravity (1.5 or 2-g).

  2. Different temporal gene expression patterns for ovine pre-implantation embryos produced by parthenogenesis or in vitro fertilization.

    PubMed

    Bebbere, Daniela; Bogliolo, Luisa; Ariu, Federica; Fois, Stefano; Leoni, Giovanni Giuseppe; Succu, Sara; Berlinguer, Fiammetta; Ledda, Sergio

    2010-09-15

    Parthenogenetic activation of the mammalian oocyte constitutes an essential step to a number of oocyte- or embryo-related technologies. Mammalian parthenotes are useful tools for studying the roles of paternal and maternal genomes in early mammalian development and are considered potential candidates for an ethical source of embryonic stem cells. We investigated the in vitro developmental competence of pre-implantation ovine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) together with the expression of a panel of fourteen genes at different times of development. IVF and PA embryos showed similar developmental competence. No differences in gene expression were observed between PA and IVF two cell-stage embryos, while PA morulae showed a significantly higher expression of IGF2. At the blastocyst stage, parthenotes exhibited up-regulation of TP-1, CDC2, and IGF2 transcripts and significantly lower levels of AQP3, ATP1A1, H2A.Z, hsp90beta, and OCT4, while NANOG, BAX, CCNB1, CDH1, GAPDH, and IGF2R displayed similar expression patterns in the two groups. Our study indicates that oocyte parthenogenetic activation does not impair in vitro pre-implantation development to the blastocyst stage, but affects the gene expression status of the embryo after the activation of its own genome.

  3. Mammalian development does not recapitulate suspected key transformations in the evolutionary detachment of the mammalian middle ear

    PubMed Central

    Ramírez-Chaves, Héctor E.; Wroe, Stephen W.; Selwood, Lynne; Hinds, Lyn A.; Leigh, Chris; Koyabu, Daisuke; Kardjilov, Nikolay; Weisbecker, Vera

    2016-01-01

    The ectotympanic, malleus and incus of the developing mammalian middle ear (ME) are initially attached to the dentary via Meckel's cartilage, betraying their origins from the primary jaw joint of land vertebrates. This recapitulation has prompted mostly unquantified suggestions that several suspected—but similarly unquantified—key evolutionary transformations leading to the mammalian ME are recapitulated in development, through negative allometry and posterior/medial displacement of ME bones relative to the jaw joint. Here we show, using µCT reconstructions, that neither allometric nor topological change is quantifiable in the pre-detachment ME development of six marsupials and two monotremes. Also, differential ME positioning in the two monotreme species is not recapitulated. This challenges the developmental prerequisites of widely cited evolutionary scenarios of definitive mammalian middle ear (DMME) evolution, highlighting the requirement for further fossil evidence to test these hypotheses. Possible association between rear molar eruption, full ME ossification and ME detachment in marsupials suggests functional divergence between dentary and ME as a trigger for developmental, and possibly also evolutionary, ME detachment. The stable positioning of the dentary and ME supports suggestions that a ‘partial mammalian middle ear’ as found in many mammaliaforms—probably with a cartilaginous Meckel's cartilage—represents the only developmentally plausible evolutionary DMME precursor. PMID:26763693

  4. Mammalian development does not recapitulate suspected key transformations in the evolutionary detachment of the mammalian middle ear.

    PubMed

    Ramírez-Chaves, Héctor E; Wroe, Stephen W; Selwood, Lynne; Hinds, Lyn A; Leigh, Chris; Koyabu, Daisuke; Kardjilov, Nikolay; Weisbecker, Vera

    2016-01-13

    The ectotympanic, malleus and incus of the developing mammalian middle ear (ME) are initially attached to the dentary via Meckel's cartilage, betraying their origins from the primary jaw joint of land vertebrates. This recapitulation has prompted mostly unquantified suggestions that several suspected--but similarly unquantified--key evolutionary transformations leading to the mammalian ME are recapitulated in development, through negative allometry and posterior/medial displacement of ME bones relative to the jaw joint. Here we show, using µCT reconstructions, that neither allometric nor topological change is quantifiable in the pre-detachment ME development of six marsupials and two monotremes. Also, differential ME positioning in the two monotreme species is not recapitulated. This challenges the developmental prerequisites of widely cited evolutionary scenarios of definitive mammalian middle ear (DMME) evolution, highlighting the requirement for further fossil evidence to test these hypotheses. Possible association between rear molar eruption, full ME ossification and ME detachment in marsupials suggests functional divergence between dentary and ME as a trigger for developmental, and possibly also evolutionary, ME detachment. The stable positioning of the dentary and ME supports suggestions that a 'partial mammalian middle ear' as found in many mammaliaforms--probably with a cartilaginous Meckel's cartilage--represents the only developmentally plausible evolutionary DMME precursor.

  5. Osteogenic protein-1 is required for mammalian eye development.

    PubMed

    Solursh, M; Langille, R M; Wood, J; Sampath, T K

    1996-01-17

    Osteogenic Protein-1 (OP-1/BMP-7) is a bone morphogenetic protein in the transforming growth factor-beta superfamily and has been shown to be expressed temporally and spatially during epithelial-mesenchymal interactions mediating tissue morphogenesis in early embryogenesis. In order to identify the primary role(s) for OP-1 in development, we carried out whole rat embryo cultures, over a 72-h period from primitive streak stages to early limb bud stages, in rat sera containing either OP-1 blocking antibodies (10 micrograms/ml) or nonreactive IgG. Rat embryos cultured with control antibodies developed normally, while those cultured with anti-OP-1 antibodies consistently exhibited over-all reduced size and absence of eyes. Histological sections revealed a greater reduction in neural retina development in the embryos treated with anti-OP-1 blocking antibodies. In situ hybridization and immunolocalization analyses indicate that OP-1 is expressed in the neuroepithelium of the optic vesicle at E11.5, is limited to the presumptive neural retina and developing lens placode, and is subsequently expressed in the neural retina, lens and developing cornea at E12.5-E13.5. Our results indicate that OP-1 mediates the inductive signals involved in mammalian eye development.

  6. The Development of Kisspeptin Circuits in the Mammalian Brain

    PubMed Central

    Semaan, Sheila J.; Tolson, Kristen P.

    2015-01-01

    The neuropeptide kisspeptin, encoded by the Kiss1 gene, is required for mammalian puberty and fertility. Examining the development of the kisspeptin system contributes to our understanding of pubertal progression and adult reproduction and sheds light on possible mechanisms underlying the development of reproductive disorders, such as precocious puberty or hypogonadotropic hypogonadism. Recent work, primarily in rodent models, has begun to study the development of kisspeptin neurons and their regulation by sex steroids and other factors at early life stages. In the brain, kisspeptin is predominantly expressed in two areas of the hypothalamus, the anteroventral periventricular nucleus and neighboring periventricular nucleus (pre-optic area in some species) and the arcuate nucleus. Kisspeptin neurons in these two hypothalamic regions are differentially regulated by testosterone and estradiol, both in development and in adulthood, and also display differences in their degree of sexual dimorphism. In this chapter, we discuss what is currently known and not known about the ontogeny, maturation, and sexual differentiation of kisspeptin neurons, as well as their regulation by sex steroids and other factors during development. PMID:23550009

  7. Retinoic acid regulates embryonic development of mammalian submandibular salivary glands.

    PubMed

    Wright, Diana M; Buenger, Deanna E; Abashev, Timur M; Lindeman, Robert P; Ding, Jixiang; Sandell, Lisa L

    2015-11-01

    Organogenesis is orchestrated by cell and tissue interactions mediated by molecular signals. Identification of relevant signals, and the tissues that generate and receive them, are important goals of developmental research. Here, we demonstrate that Retinoic Acid (RA) is a critical signaling molecule important for morphogenesis of mammalian submandibular salivary glands (SMG). By examining late stage RA deficient embryos of Rdh10 mutant mice we show that SMG development requires RA in a dose-dependent manner. Additionally, we find that active RA signaling occurs in SMG tissues, arising earlier than any other known marker of SMG development and persisting throughout gland morphogenesis. At the initial bud stage of development, we find RA production occurs in SMG mesenchyme, while RA signaling occurs in epithelium. We also demonstrate active RA signaling occurs in glands cultured ex vivo, and treatment with an inhibitor of RA signaling blocks growth and branching. Together these data identify RA signaling as a direct regulator of SMG organogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Monotreme ossification sequences and the riddle of mammalian skeletal development.

    PubMed

    Weisbecker, Vera

    2011-05-01

    The developmental differences between marsupials, placentals, and monotremes are thought to be reflected in differing patterns of postcranial development and diversity. However, developmental polarities remain obscured by the rarity of monotreme data. Here, I present the first postcranial ossification sequences of the monotreme echidna and platypus, and compare these with published data from other mammals and amniotes. Strikingly, monotreme stylopodia (humerus, femur) ossify after the more distal zeugopodia (radius/ulna, tibia/fibula), resembling only the European mole among all amniotes assessed. European moles also share extreme humeral adaptations to rotation digging and/or swimming with monotremes, suggesting a causal relationship between adaptation and ossification heterochrony. Late femoral ossification with respect to tibia/fibula in monotremes and moles points toward developmental integration of the serially homologous fore- and hindlimb bones. Monotreme cervical ribs and coracoids ossify later than in most amniotes but are similarly timed as homologous ossifications in therians, where they are lost as independent bones. This loss may have been facilitated by a developmental delay of coracoids and cervical ribs at the base of mammals. The monotreme sequence, although highly derived, resembles placentals more than marsupials. Thus, marsupial postcranial development, and potentially related diversity constraints, may not represent the ancestral mammalian condition.

  9. NHE1 is the sodium-hydrogen exchanger active in acute intracellular pH regulation in preimplantation mouse embryos.

    PubMed

    Siyanov, Violetta; Baltz, Jay M

    2013-06-01

    Sodium-hydrogen exchangers (NHE) of the Slc9 gene family are the major regulators of intracellular pH against acidosis in mammalian cells. Of five plasma membrane NHE isoforms, mouse oocytes and preimplantation embryos express mRNAs encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in oocytes through one-cell stage embryos and lower levels after the two-cell stage. NHE2 (SLC9A2) and NHE5 (SLC9A5) are not expressed. Measurements of intracellular pH during recovery from induced acidosis indicated that recovery occurred via NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). Recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over cariporide alone, indicating that NHE3 did not play a role. There was no indication of NHE4 activity. Another regulator of intracellular pH against acidosis, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. Thus, NHE1 appears to be the only significant regulator of intracellular pH in preimplantation mouse embryos. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development. Unexpectedly, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not affect embryo development to the blastocyst stage more substantially under conditions of chronic acidosis than at normal pH. Preimplantation mouse embryos thus appear to have limited capacity to resist chronic acidosis using intracellular pH regulatory mechanisms.

  10. The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development

    PubMed Central

    Courtois, Aurélien; Schuh, Melina; Ellenberg, Jan

    2012-01-01

    The transition from meiosis to mitosis, classically defined by fertilization, is a fundamental process in development. However, its mechanism remains largely unexplored. In this paper, we report a surprising gradual transition from meiosis to mitosis over the first eight divisions of the mouse embryo. The first cleavages still largely share the mechanism of spindle formation with meiosis, during which the spindle is self-assembled from randomly distributed microtubule-organizing centers (MTOCs) without centrioles, because of the concerted activity of dynein and kinesin-5. During preimplantation development, the number of cellular MTOCs progressively decreased, the spindle pole gradually became more focused, and spindle length progressively scaled down with cell size. The typical mitotic spindle with centrin-, odf2-, kinesin-12–, and CP110-positive centrosomes was established only in the blastocyst. Overall, the transition from meiosis to mitosis progresses gradually throughout the preimplantation stage in the mouse embryo, thus providing a unique system to study the mechanism of centrosome biogenesis in vivo. PMID:22851319

  11. Reprogenetics: Preimplantational genetics diagnosis

    PubMed Central

    Coco, Roberto

    2014-01-01

    Preimplantational Genetics Diagnosis (PGD) is requested by geneticists and reproductive specialists. Usually geneticists ask for PGD because one or both members of the couple have an increased genetic risk for having an affected offspring. On the other hand, reproductive specialists ask for embryo aneuploidy screening (PGS) to assures an euploid embryo transfer, with the purpose to achieve an ongoing pregnancy, although the couple have normal karyotypes. As embryonic aneuploidies are responsible for pre and post implantation abortions, it is logical to considerer that the screening of the embryonic aneuploidies prior to embryo transfer could improve the efficiency of the in vitro fertilization procedures. Nevertheless, it is still premature to affirm this until well-designed clinical trials were done, especially in women of advanced age where the rate of embryos with aneuploidies is much greater. Although the indications of PGD are similar to conventional prenatal diagnosis (PND), PGD has less ethical objections than the PND. As with the PGD/PGS results only unaffected embryos are transferred, both methods can avoid the decision to interrupt the pregnancy due to a genetic problem; this makes an important difference when compared to conventional prenatal diagnosis. PMID:24764761

  12. Reprogenetics: Preimplantational genetics diagnosis.

    PubMed

    Coco, Roberto

    2014-03-01

    Preimplantational Genetics Diagnosis (PGD) is requested by geneticists and reproductive specialists. Usually geneticists ask for PGD because one or both members of the couple have an increased genetic risk for having an affected offspring. On the other hand, reproductive specialists ask for embryo aneuploidy screening (PGS) to assures an euploid embryo transfer, with the purpose to achieve an ongoing pregnancy, although the couple have normal karyotypes. As embryonic aneuploidies are responsible for pre and post implantation abortions, it is logical to considerer that the screening of the embryonic aneuploidies prior to embryo transfer could improve the efficiency of the in vitro fertilization procedures. Nevertheless, it is still premature to affirm this until well-designed clinical trials were done, especially in women of advanced age where the rate of embryos with aneuploidies is much greater. Although the indications of PGD are similar to conventional prenatal diagnosis (PND), PGD has less ethical objections than the PND. As with the PGD/PGS results only unaffected embryos are transferred, both methods can avoid the decision to interrupt the pregnancy due to a genetic problem; this makes an important difference when compared to conventional prenatal diagnosis.

  13. Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig.

    PubMed

    Binh, N T; Van Thuan, N; Miyake, M

    2009-06-01

    The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 degrees C for up to 48h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Czeta (PLCzeta) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCzeta in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18h at 24 degrees C (78%, 62%, and 35%, respectively) and for 48h at 14 degrees C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCzeta in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.

  14. Contrasting changes in transport of glycine vs proline at fertilization and during preimplantation development of mouse embryos

    SciTech Connect

    Haghighat, N.; Van Winkle, L.J.

    1987-05-01

    Na/sup +/ dependent glycine transport decreased steadily during cleavage of mouse embryos and then increased dramatically upon formation of early blastocysts (approx. 80 h post coitus), while proline uptake increased several-fold upon fertilization of eggs and then decreased through the blastocyst stage. V/sub max/ and K/sub m/ values for Gly transport in unfertilized eggs, 8-cell embryos and blastocysts were 9.5, 4.0 and 20 fmol. (egg or embryo)/sup -1/ min/sup -1/ and 93, 94 and 30 ..mu..M, respectively. Gly transport in 2-cell embryos was Cl-dependent and sigmoidally related to the (Na/sup +/), whereas Cl/sup -/-dependent Gly uptake was linearly related to (Na/sup +/) in blastocysts. Uptake of 1.0 ..mu..M (/sup 3/H)Gly in cleavage stages was inhibited by 10 mM sarcosine but not by Glu, Ser, or Lys and only weakly by MeAIB, BCO and pipecolate, whereas BCO, Ser, Lys, Pipecolate, Ala and Leu strongly inhibited transport in blastocysts; and Lys inhibition was unequivocally competitive (K/sub i/ approx. 70 ..mu..M). Na/sup +/-dependent uptake of 0.9 ..mu..M L-(/sup 3/H)Pro was inhibited strongly by only pipecolate in unfertilized eggs, but MeAIB and BCO were also strong inhibitors in zygotes. Fertilization was also accompanied by an increase in the V/sub max/ (0.9 vs 6.7 fmol. cell/sup -1/ min/sup -1/) and K/sub m/ (66 vs 230 ..mu..m) values for proline transport. This appears to be the first report of a change in amino acid transport upon fertilization of mammalian eggs, although transport of several amino acids increases dramatically in sea urchin zygotes.

  15. p38 (Mapk14/11) occupies a regulatory node governing entry into primitive endoderm differentiation during preimplantation mouse embryo development

    PubMed Central

    Thamodaran, Vasanth

    2016-01-01

    During mouse preimplantation embryo development, the classically described second cell-fate decision involves the specification and segregation, in blastocyst inner cell mass (ICM), of primitive endoderm (PrE) from pluripotent epiblast (EPI). The active role of fibroblast growth factor (Fgf) signalling during PrE differentiation, particularly in the context of Erk1/2 pathway activation, is well described. However, we report that p38 family mitogen-activated protein kinases (namely p38α/Mapk14 and p38β/Mapk11; referred to as p38-Mapk14/11) also participate in PrE formation. Specifically, functional p38-Mapk14/11 are required, during early-blastocyst maturation, to assist uncommitted ICM cells, expressing both EPI and earlier PrE markers, to fully commit to PrE differentiation. Moreover, functional activation of p38-Mapk14/11 is, as reported for Erk1/2, under the control of Fgf-receptor signalling, plus active Tak1 kinase (involved in non-canonical bone morphogenetic protein (Bmp)-receptor-mediated PrE differentiation). However, we demonstrate that the critical window of p38-Mapk14/11 activation precedes the E3.75 timepoint (defined by the initiation of the classical ‘salt and pepper’ expression pattern of mutually exclusive EPI and PrE markers), whereas appropriate lineage maturation is still achievable when Erk1/2 activity (via Mek1/2 inhibition) is limited to a period after E3.75. We propose that active p38-Mapk14/11 act as enablers, and Erk1/2 as drivers, of PrE differentiation during ICM lineage specification and segregation. PMID:27605380

  16. Development of a novel mammalian cell surface antibody display platform.

    PubMed

    Zhou, Chen; Jacobsen, Frederick W; Cai, Ling; Chen, Qing; Shen, Weyen David

    2010-01-01

    Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500 fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.

  17. Genes and Conditions Controlling Mammalian Pre- and Post-implantation Embryo Development

    PubMed Central

    Anifandis, G.; Messini, C.I.; Dafopoulos, K.; Messinis, I.E.

    2015-01-01

    Embryo quality during the in vitro developmental period is of great clinical importance. Experimental genetic studies during this period have demonstrated the association between specific gene expression profiles and the production of healthy blastocysts. Although the quality of the oocyte may play a major role in embryo development, it has been well established that the post – fertilization period also has an important and crucial role in the determination of blastocyst quality. A variety of genes (such as OCT, SOX2, NANOG) and their related signaling pathways as well as transcription molecules (such as TGF-β, BMP) have been implicated in the pre- and post-implantation period. Furthermore, DNA methylation has been lately characterized as an epigenetic mark since it is one of the most important processes involved in the maintenance of genome stability. Physiological embryo development appears to depend upon the correct DNA methylation pattern. Due to the fact that soon after fertilization the zygote undergoes several morphogenetic and developmental events including activation of embryonic genome through the transition of the maternal genome, a diverse gene expression pattern may lead to clinically important conditions, such as apoptosis or the production of a chromosomically abnormal embryo. The present review focused on genes and their role during pre-implantation embryo development, giving emphasis on the various parameters that may alter gene expression or DNA methylation patterns. The pre-implantation embryos derived from in vitro culture systems (in vitro fertilization) and the possible effects on gene expression after the prolonged culture conditions are also discussed. PMID:25937812

  18. Effects of simulated weightlessness on mammalian development. Part 1: Development of clinostat for mammalian tissue culture and use in studies on meiotic maturation of mouse oocytes

    NASA Technical Reports Server (NTRS)

    Wolegemuth, D. J.; Grills, G. S.

    1984-01-01

    The effects of weightlessness on three aspects of mammalian reproduction: oocyte development, fertilization, and early embryogenesis was studied. Zero-gravity conditions within the laboratory by construction of a clinostat designed to support in vitro tissue culture were simulated and the effects of simulated weightlessness on meiotic maturation in mammalian oocytes using mouse as the model system were studied. The timing and frequency of germinal vesicule breakdown and polar body extrusion, and the structural and numerical properties of meiotic chromosomes at Metaphase and Metaphase of meiosis are assessed.

  19. Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

    PubMed

    Ding, Li-Jun; Tian, Hai-Bin; Wang, Jing-Jun; Chen, Juan; Sha, Hong-Ying; Chen, Jian-Quan; Cheng, Guo-Xiang

    2008-12-01

    The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used

  20. Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage

    PubMed Central

    2011-01-01

    Background Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known. Results Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically. Conclusions These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin. PMID:21489308

  1. The temporal relationship between oocyte maturation and early fertilisation events in relation to the pre-ovulatory LH peak and preimplantation embryo development in red deer (Cervus elaphus).

    PubMed

    Berg, D K; Thompson, J G; Peterson, A J; Asher, G W

    2008-05-01

    The temporal relationships among oocyte maturation, gamete transport and fertilisation following the pre-ovulatory luteinsing hormone surge in red deer were established; and secondly, early preimplantation development to the blastocyst stage in relation to the onset of oestrus was determined for red deer. In the first series of observations, oestrus was synchronised in April (N=22), for the fixed time recovery of gametes from 0 to 36 h after the estimated pre-ovulatory LH peak. Matings were observed and the time of the LH peak was determined from the retrospective analysis of blood plasma collected at 3h intervals. Gametes were recovered surgically and the meiotic status of follicular and ovulated oocytes assessed. Spermatozoa were recovered from the oviduct and their motility analysed by videomicroscopy. Nineteen of 22 hinds exhibited a pre-ovulatory LH surge and were observed to mate. Oocyte metaphase I occurred between 11 and 18 h, and metaphase II was completed within the follicle between 20 and 25 h following the pre-ovulatory LH peak. Fertilised ova were recovered from 30 to 36 h in both the ampulla and isthmic portions of the oviduct. Motile spermatozoa were first recovered from the isthmus and the ampulla at 13 and 21 h, respectively, after the LH peak. Hyperactive spermatozoa were observed in both the isthmus and the ampulla flushings but only from the eight hinds that had ovulated. In the second series of observations, 16 mature hinds were synchronised and allocated to groups for embryo collection on days 3, 5 and 7 after oestrus. Eight embryos were recovered; an 8-cell at 90 h, 3 morulae at 137, 138 and 186 h, and 4 blastocysts at 180, 182 and 190 h post-mating. Blastocysts were only recovered from the uterine horns and the mean+/-S.E.M. number of nuclei per blastocyst was 93.5+/-10.0 with a range of 66-114 cells. The results of this study will improve the application of assisted reproductive technologies to red deer as they indicate that oocyte

  2. Effect of chronic HTO. beta. or /sup 60/Co. gamma. radiation on preimplantation mouse development in vitro

    SciTech Connect

    Yamada, T.; Yukawa, O.; Asami, K.; Nakazawa, T.

    1982-11-01

    Response of pronuclear, early 2-cell, and late 2-cell mouse embryos to chronic HTO ..beta.. and /sup 60/Co ..gamma.. irradiation was investigated. The pronuclear embryos fertilized in vitro and 2-cell stage embryos of BC3F/sub 1/ (C3H/C57BL) mice were grown in vitro in chemically defined medically defied media containing tritium oxide. Activity levels ranged from 100 to 2000 ..mu..Ci/ml. With development to blastocyst as the end point, the LD/sub 50/ was determined to be 118, 230, and 426 ..mu..Ci/ml for pronuclear, early 2-cell, and late 2-cell embryos, respectively. Similar embryos were exposed in vitro to chronic ..gamma.. radiation from /sup 60/Co during the same period of development, and RBE values of HTO ..beta.. radiation relative to /sup 60/Co ..gamma.. rays were calculated to be within the range of 1.0 to 1.7.

  3. Preimplantation genetic diagnosis of inherited disease.

    PubMed

    Ao, A

    1996-12-01

    Research on diagnosis of inherited disease in human embryo before implantation was initiated to help those couples who would prefer to select embryos at this stage rather than during pregnancy. Following in vitro fertilization (IVF), one to two cells were removed from 3 day cleavage stage embryo and cells were analysed for genetic defects. Embryos diagnosed as unaffected were returned to the uterus and thus the resulting pregnancies were assured to be normal. First babies born after the preimplantation diagnosis were using DNA amplification of Y-linked sequences by PCR to avoid X-linked disease. Several pregnancies were obtained by identifying sex of embryos using dual fluorescent in situ hybridization (FISH) with fluorochrome labelled DNA sequences specific for X- and Y-chromosomes to interphase nuclei. Development of single cell PCR for single gene defects led to diagnose several genetic disorders. Preimplantation diagnosis was successfully achieved for predominant delta 508 deletion causing cystic fibrosis, and pregnancies were also diagnosed for Lesch-Nyhan syndrome, Tay-Sachs and Duchenne muscular dystrophy.

  4. Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos.

    PubMed

    Fan, Xiaoying; Zhang, Xiannian; Wu, Xinglong; Guo, Hongshan; Hu, Yuqiong; Tang, Fuchou; Huang, Yanyi

    2015-07-23

    Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.

  5. Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

    SciTech Connect

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

    2009-10-09

    Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26{sup tm1Sor}, revealed that treatment of 1-cell or 2-cell embryos with 3 {mu}M of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

  6. Impairment of preimplantation porcine embryo development by histone demethylase KDM5B knockdown through disturbance of bivalent H3K4me3-H3K27me3 modifications.

    PubMed

    Huang, Jiaojiao; Zhang, Hongyong; Wang, Xianlong; Dobbs, Kyle B; Yao, Jing; Qin, Guosong; Whitworth, Kristin; Walters, Eric M; Prather, Randall S; Zhao, Jianguo

    2015-03-01

    KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for embryonic stem cell self-renewal, cell fate, and murine embryonic development. However, little is known about the role of KDM5B during preimplantation embryo development. Here we show that KDM5B is critical to porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired porcine embryo development to the blastocyst stage. The impairment of embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for porcine embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications.

  7. Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos.

    PubMed

    Filliers, Muriel; Goossens, Karen; Van Soom, Ann; Merlo, Barbara; Pope, Charles Earle; de Rooster, Hilde; Smits, Katrien; Vandaele, Leen; Peelman, Luc J

    2012-01-01

    During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of

  8. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development.

    PubMed

    Yagi, Rieko; Kohn, Matthew J; Karavanova, Irina; Kaneko, Kotaro J; Vullhorst, Detlef; DePamphilis, Melvin L; Buonanno, Andres

    2007-11-01

    Specification of cell lineages in mammals begins shortly after fertilization with formation of a blastocyst consisting of trophectoderm, which contributes exclusively to the placenta, and inner cell mass (ICM), from which the embryo develops. Here we report that ablation of the mouse Tead4 gene results in a preimplantation lethal phenotype, and TEAD4 is one of two highly homologous TEAD transcription factors that are expressed during zygotic gene activation in mouse 2-cell embryos. Tead4(-/-) embryos do not express trophectoderm-specific genes, such as Cdx2, but do express ICM-specific genes, such as Oct4 (also known as Pou5f1). Consequently, Tead4(-/-) morulae do not produce trophoblast stem cells, trophectoderm or blastocoel cavities, and therefore do not implant into the uterine endometrium. However, Tead4(-/-) embryos can produce embryonic stem cells, a derivative of ICM, and if the Tead4 allele is not disrupted until after implantation, then Tead4(-/-) embryos complete development. Thus, Tead4 is the earliest gene shown to be uniquely required for specification of the trophectoderm lineage.

  9. Some process control/design considerations in the development of a microgravity mammalian cell bioreactor

    NASA Technical Reports Server (NTRS)

    Goochee, Charles F.

    1987-01-01

    The purpose is to review some of the physical/metabolic factors which must be considered in the development of an operating strategy for a mammalian cell bioreactor. Emphasis is placed on the dissolved oxygen and carbon dioxide requirements of growing mammalian epithelial cells. Literature reviews concerning oxygen and carbon dioxide requirements are discussed. A preliminary, dynamic model which encompasses the current features of the NASA bioreactor is presented. The implications of the literature survey and modeling effort on the design and operation of the NASA bioreactor are discussed.

  10. Predicting evolutionary patterns of mammalian teeth from development.

    PubMed

    Kavanagh, Kathryn D; Evans, Alistair R; Jernvall, Jukka

    2007-09-27

    One motivation in the study of development is the discovery of mechanisms that may guide evolutionary change. Here we report how development governs relative size and number of cheek teeth, or molars, in the mouse. We constructed an inhibitory cascade model by experimentally uncovering the activator-inhibitor logic of sequential tooth development. The inhibitory cascade acts as a ratchet that determines molar size differences along the jaw, one effect being that the second molar always makes up one-third of total molar area. By using a macroevolutionary test, we demonstrate the success of the model in predicting dentition patterns found among murine rodent species with various diets, thereby providing an example of ecologically driven evolution along a developmentally favoured trajectory. In general, our work demonstrates how to construct and test developmental rules with evolutionary predictability in natural systems.

  11. Epigenetic asymmetry in the zygote and mammalian development.

    PubMed

    Feil, Robert

    2009-01-01

    In mammals, the maternal and the paternal genome are not functionally equivalent and are both required for embryonic and postnatal development. The genome is organised differently in the oocyte as compared to sperm, in which the DNA is tightly packaged with protamines rather than with histones. The requirement of both the parental genomes for normal development is a consequence of differential epigenetic marking in oogenesis and spermatogenesis, at the regulatory elements that control genomic imprinting. These germ line-derived marks of DNA methylation are resistant to the global waves of demethylation that occur following fertilisation, and bring about the parental allele-specific expression of imprinted genes during development and after birth. Perturbation of the differential organisation of the maternally and paternally derived genomes, before fertilisation, or in the early embryo, can give rise to aberrant growth and developmental disorders in humans.

  12. Whole genome amplification in preimplantation genetic diagnosis*

    PubMed Central

    Zheng, Ying-ming; Wang, Ning; Li, Lei; Jin, Fan

    2011-01-01

    Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation, improving the chance of conception for patients at high risk of transmitting specific inherited disorders. This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s. Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD, but there are some inevitable shortcomings limiting the scope of genetic diagnosis. Fortunately, different whole genome amplification (WGA) techniques have been developed to overcome these problems. Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed. Moreover, WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis. In this review, we will focus on the currently available WGA techniques and their applications, as well as the new technical trends from WGA products. PMID:21194180

  13. [Preimplantation genetic diagnosis: technical and ethical considerations].

    PubMed

    Oliva Teles, Natália

    2011-01-01

    Following the advances in the techniques of medically assisted reproduction (ART), 1990 has seen the first born child after the development of pre-implantation genetic diagnosis. In this analysis embryos are tested for the presence of genetic anomalies at three to five days after fertilization and only unaffected embryos are transferred to the maternal uterus. The technique offers good prospects to couples at risk for conventional prenatal diagnosis. It is particularly useful where ART techniques are necessary, in which early embryo selection avoids later termination of pregnancy. In Portugal the application of laws concerning regulation of medically assisted reproduction in 2008 has clarified and formalised the medical and laboratory procedures and in some cases fundamentally changed them, particularly in the requirement to cryopreserve all high quality non-transferred embryos and in specifying conditions in which embryo experimentation may be permitted.

  14. Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos.

    PubMed

    Li, Wenzhi; Wang, Peizhe; Zhang, Bingjie; Zhang, Jing; Ming, Jia; Xie, Wei; Na, Jie

    2017-04-22

    Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B (AurkB) is ubiquitously expressed while Aurora kinase C (AurkC) is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 (H3S10P) and regulate metaphase timing. Using an Oct4-photoactivatable GFP fusion protein (Oct4-paGFP) and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.

  15. Embryonic development of circadian clocks in the mammalian suprachiasmatic nuclei.

    PubMed

    Landgraf, Dominic; Koch, Christiane E; Oster, Henrik

    2014-01-01

    In most species, self-sustained molecular clocks regulate 24-h rhythms of behavior and physiology. In mammals, a circadian pacemaker residing in the hypothalamic suprachiasmatic nucleus (SCN) receives photic signals from the retina and synchronizes subordinate clocks in non-SCN tissues. The emergence of circadian rhythmicity during development has been extensively studied for many years. In mice, neuronal development in the presumptive SCN region of the embryonic hypothalamus occurs on days 12-15 of gestation. Intra-SCN circuits differentiate during the following days and retinal projections reach the SCN, and thus mediate photic entrainment, only after birth. In contrast the genetic components of the clock gene machinery are expressed much earlier and during midgestation SCN explants and isolated neurons are capable of generating molecular oscillations in culture. In vivo metabolic rhythms in the SCN, however, are observed not earlier than the 19th day of rat gestation, and rhythmic expression of clock genes is hardly detectable until after birth. Together these data indicate that cellular coupling and, thus, tissue-wide synchronization of single-cell rhythms, may only develop very late during embryogenesis. In this mini-review we describe the developmental origin of the SCN structure and summarize our current knowledge about the functional initiation and entrainment of the circadian pacemaker during embryonic development.

  16. Lysyl oxidases in mammalian development and certain pathological conditions.

    PubMed

    Mäki, Joni M

    2009-05-01

    Lysyl oxidase (LOX) catalyzes the oxidation of the side chain of a peptidyl lysine converting specific lysine and hydroxylysine residues of alpha-aminoadipic-delta-semialdehydes, which form covalent crosslinks in collagens and elastin. Five different but closely related lysyl oxidase isoenzymes have been identified to date, and they seem to have overlapping functions in many tissues. Modification of the extracellular matrix by lysyl oxidases has been shown to be a critical contributor to the development of various organs and certain pathological conditions.

  17. Vitronectin is not essential for normal mammalian development and fertility.

    PubMed Central

    Zheng, X; Saunders, T L; Camper, S A; Samuelson, L C; Ginsburg, D

    1995-01-01

    Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618914

  18. Chromatin modification and epigenetic reprogramming in mammalian development.

    PubMed

    Li, En

    2002-09-01

    The developmental programme of embryogenesis is controlled by both genetic and epigenetic mechanisms. An emerging theme from recent studies is that the regulation of higher-order chromatin structures by DNA methylation and histone modification is crucial for genome reprogramming during early embryogenesis and gametogenesis, and for tissue-specific gene expression and global gene silencing. Disruptions to chromatin modification can lead to the dysregulation of developmental processes, such as X-chromosome inactivation and genomic imprinting, and to various diseases. Understanding the process of epigenetic reprogramming in development is important for studies of cloning and the clinical application of stem-cell therapy.

  19. Signaling and Gene Regulatory Networks in Mammalian Lens Development.

    PubMed

    Cvekl, Ales; Zhang, Xin

    2017-10-01

    Ocular lens development represents an advantageous system in which to study regulatory mechanisms governing cell fate decisions, extracellular signaling, cell and tissue organization, and the underlying gene regulatory networks. Spatiotemporally regulated domains of BMP, FGF, and other signaling molecules in late gastrula-early neurula stage embryos generate the border region between the neural plate and non-neural ectoderm from which multiple cell types, including lens progenitor cells, emerge and undergo initial tissue formation. Extracellular signaling and DNA-binding transcription factors govern lens and optic cup morphogenesis. Pax6, c-Maf, Hsf4, Prox1, Sox1, and a few additional factors regulate the expression of the lens structural proteins, the crystallins. Extensive crosstalk between a diverse array of signaling pathways controls the complexity and order of lens morphogenetic processes and lens transparency. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Development-Inspired Reprogramming of the Mammalian Central Nervous System

    PubMed Central

    Amamoto, Ryoji; Arlotta, Paola

    2014-01-01

    In 2012, John Gurdon and Shinya Yamanaka shared the Nobel Prize for the exciting demonstration that the identity of differentiated cells is not irreversibly determined but can be changed back to a pluripotent state under appropriate instructive signals. The principle that differentiated cells can revert to an embryonic state and even be converted directly from one cell-type into another not only turns fundamental principles of development on their head but also has profound implications for regenerative medicine. Replacement of diseased tissue with newly reprogrammed cells and modeling of human disease are concrete opportunities. Here, we focus on the central nervous system to consider whether and how reprogramming of cell identity may impact regeneration and modeling of a system historically considered immutable and hardwired. PMID:24482482

  1. Development-inspired reprogramming of the mammalian central nervous system.

    PubMed

    Amamoto, Ryoji; Arlotta, Paola

    2014-01-31

    In 2012, John Gurdon and Shinya Yamanaka shared the Nobel Prize for the demonstration that the identity of differentiated cells is not irreversibly determined but can be changed back to a pluripotent state under appropriate instructive signals. The principle that differentiated cells can revert to an embryonic state and even be converted directly from one cell type into another not only turns fundamental principles of development on their heads but also has profound implications for regenerative medicine. Replacement of diseased tissue with newly reprogrammed cells and modeling of human disease are concrete opportunities. Here, we focus on the central nervous system to consider whether and how reprogramming of cell identity may affect regeneration and modeling of a system historically considered immutable and hardwired.

  2. A methodological overview on molecular preimplantation genetic diagnosis and screening: a genomic future?

    PubMed

    Vendrell, Xavier; Bautista-Llácer, Rosa

    2012-12-01

    The genetic diagnosis and screening of preimplantation embryos generated by assisted reproduction technology has been consolidated in the prenatal care framework. The rapid evolution of DNA technologies is tending to molecular approaches. Our intention is to present a detailed methodological view, showing different diagnostic strategies based on molecular techniques that are currently applied in preimplantation genetic diagnosis. The amount of DNA from one single, or a few cells, obtained by embryo biopsy is a limiting factor for the molecular analysis. In this sense, genetic laboratories have developed molecular protocols considering this restrictive condition. Nevertheless, the development of whole-genome amplification methods has allowed preimplantation genetic diagnosis for two or more indications simultaneously, like the selection of histocompatible embryos plus detection of monogenic diseases or aneuploidies. Moreover, molecular techniques have permitted preimplantation genetic screening to progress, by implementing microarray-based comparative genome hybridization. Finally, a future view of the embryo-genetics field based on molecular advances is proposed. The normalization, cost-effectiveness analysis, and new technological tools are the next topics for preimplantation genetic diagnosis and screening. Concomitantly, these additions to assisted reproduction technologies could have a positive effect on the schedules of preimplantation studies.

  3. Novel technologies emerging for preimplantation genetic diagnosis and preimplantation genetic testing for aneuploidy.

    PubMed

    Sermon, Karen

    2017-01-01

    Preimplantation genetic diagnosis (PGD) was introduced as an alternative to prenatal diagnosis: embryos cultured in vitro were analysed for a monogenic disease and only disease-free embryos were transferred to the mother, to avoid the termination of pregnancy with an affected foetus. It soon transpired that human embryos show a great deal of acquired chromosomal abnormalities, thought to explain the low success rate of IVF - hence preimplantation genetic testing for aneuploidy (PGT-A) was developed to select euploid embryos for transfer. Areas covered: PGD has followed the tremendous evolution in genetic analysis, with only a slight delay due to adaptations for diagnosis on small samples. Currently, next generation sequencing combining chromosome with single-base pair analysis is on the verge of becoming the golden standard in PGD and PGT-A. Papers highlighting the different steps in the evolution of PGD/PGT-A were selected. Expert commentary: Different methodologies used in PGD/PGT-A with their pros and cons are discussed.

  4. Variable imprinting of the MEST gene in human preimplantation embryos

    PubMed Central

    Huntriss, John D; Hemmings, Karen E; Hinkins, Matthew; Rutherford, Anthony J; Sturmey, Roger G; Elder, Kay; Picton, Helen M

    2013-01-01

    There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos. PMID:22763377

  5. Behavioral biology of mammalian reproduction and development for a space station

    NASA Technical Reports Server (NTRS)

    Alberts, J. R.

    1983-01-01

    Space Station research includes two kinds of adaption to space: somatic (the adjustments made by an organism, within its lifetime, in response to local conditions), and transgenerational adaption (continuous exposure across sequential life cycles of genetic descendents). Transgenerational effects are akin to evolutionary process. Areas of a life Sciences Program in a space station address the questions of the behavioral biology of mammalian reproduction and development, using the Norway rat as the focus of experimentation.

  6. Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

    PubMed Central

    Barnea, Eytan R.; Lubman, David M.; Liu, Yan-Hui; Absalon-Medina, Victor; Hayrabedyan, Soren; Todorova, Krassimira; Gilbert, Robert O.; Guingab, Joy; Barder, Timothy J.

    2014-01-01

    Background Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. Methods FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. Results PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Conclusion Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF

  7. Insight into PreImplantation Factor (PIF*) mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP) through essential RIKP [corrected] binding site.

    PubMed

    Barnea, Eytan R; Lubman, David M; Liu, Yan-Hui; Absalon-Medina, Victor; Hayrabedyan, Soren; Todorova, Krassimira; Gilbert, Robert O; Guingab, Joy; Barder, Timothy J

    2014-01-01

    Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel

  8. Preimplantation genetic diagnosis for monogenic diseases: overview and emerging issues.

    PubMed

    Renwick, Pamela; Ogilvie, Caroline Mackie

    2007-01-01

    Preimplantation genetic diagnosis (PGD) is an established reproductive option for couples at risk of conceiving a pregnancy affected with a known genetic disease, who wish to avoid an (additional) affected child, termination of pregnancy or recurrent miscarriages. Early technologies concentrated on different approaches to direct mutation testing for monogenic diseases using single cell PCR protocols, or sex selection by fluorescent in situ hybridization for X-linked monogenic disease. Development of multiplex fluorescent PCR allowed simultaneously testing of linked markers alongside the mutation test, increasing the accuracy by controlling for contamination and identifying allele drop-out. The advent of highly effective whole genome amplification methods has opened the way for new technologies such as preimplantation genetic haplotyping and microarrays, thus increasing the number of genetic defects that can be detected in preimplantation embryos; the number of cases carried out and the new indications tested increases each year. Different countries have taken very different approaches to legislating and regulating PGD, giving rise to the phenomenon of reproductive tourism. PGD is now being performed for scenarios previously not undertaken using prenatal diagnosis, some of which raise significant ethical concerns. While PGD has benefited many couples aiming to have healthy children, ethical concerns remain over inappropriate use of this technology.

  9. Extracellular modulation of Fibroblast Growth Factor signaling through heparan sulfate proteoglycans in mammalian development.

    PubMed

    Matsuo, Isao; Kimura-Yoshida, Chiharu

    2013-08-01

    Fibroblast Growth Factor (FGF) signaling plays crucial roles in multiple cellular processes including cell proliferation, differentiation, survival, and migration during mammalian embryogenesis. In the extracellular matrix, as well as at the cell surface, the movement of FGF ligands to target cells and the subsequent complex formations with their receptors are positively and negatively controlled extracellularly by heparan sulfate proteoglycans (HSPGs) such as syndecans, glypicans, and perlecan. Additionally, spreading of HSPGs by cleavage with sheddases such as proteinases and heparanases, and the overall length and sulfation level of specific heparan sulfate structures further generate a great diversity of FGF signaling outcomes. This review presents our current understanding of the regulatory mechanisms of FGF signaling in extracellular spaces through HSPGs in mammalian development. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Metabolic plasticity during mammalian development is directionally dependent on early nutritional status.

    PubMed

    Gluckman, Peter D; Lillycrop, Karen A; Vickers, Mark H; Pleasants, Anthony B; Phillips, Emma S; Beedle, Alan S; Burdge, Graham C; Hanson, Mark A

    2007-07-31

    Developmental plasticity in response to environmental cues can take the form of polyphenism, as for the discrete morphs of some insects, or of an apparently continuous spectrum of phenotype, as for most mammalian traits. The metabolic phenotype of adult rats, including the propensity to obesity, hyperinsulinemia, and hyperphagia, shows plasticity in response to prenatal nutrition and to neonatal administration of the adipokine leptin. Here, we report that the effects of neonatal leptin on hepatic gene expression and epigenetic status in adulthood are directionally dependent on the animal's nutritional status in utero. These results demonstrate that, during mammalian development, the direction of the response to one cue can be determined by previous exposure to another, suggesting the potential for a discontinuous distribution of environmentally induced phenotypes, analogous to the phenomenon of polyphenism.

  11. Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.

    PubMed

    Mao, Junhao; Kim, Byeong-Moo; Rajurkar, Mihir; Shivdasani, Ramesh A; McMahon, Andrew P

    2010-05-01

    Homeostasis of the vertebrate digestive tract requires interactions between an endodermal epithelium and mesenchymal cells derived from the splanchnic mesoderm. Signaling between these two tissue layers is also crucial for patterning and growth of the developing gut. From early developmental stages, sonic hedgehog (Shh) and indian hedgehog (Ihh) are secreted by the endoderm of the mammalian gut, indicative of a developmental role. Further, misregulated hedgehog (Hh) signaling is implicated in both congenital defects and cancers arising from the gastrointestinal tract. In the mouse, only limited gastrointestinal anomalies arise following removal of either Shh or Ihh. However, given the considerable overlap in their endodermal expression domains, a functional redundancy between these signals might mask a more extensive role for Hh signaling in development of the mammalian gut. To address this possibility, we adopted a conditional approach to remove both Shh and Ihh functions from early mouse gut endoderm. Analysis of compound mutants indicates that continuous Hh signaling is dispensable for regional patterning of the gut tube, but is essential for growth of the underlying mesenchyme. Additional in vitro analysis, together with genetic gain-of-function studies, further demonstrate that Hh proteins act as paracrine mitogens to promote the expansion of adjacent mesenchymal progenitors, including those of the smooth muscle compartment. Together, these studies provide new insights into tissue interactions underlying mammalian gastrointestinal organogenesis and disease.

  12. Development of the diaphragm, a skeletal muscle essential for mammalian respiration

    PubMed Central

    Merrell, Allyson J.; Kardon, Gabrielle

    2013-01-01

    The mammalian diaphragm muscle is essential for respiration, and thus it is among the most critical of the skeletal muscles in the human body. Defects in diaphragm development, leading to congenital diaphragmatic hernias (CDH), are common birth defects and result in severe morbidity or mortality. Given its functional importance and the frequency of congenital defects, an understanding of diaphragm development normally and during herniation is important. We review the current knowledge of the embryological origins of the diaphragm, diaphragm development and morphogenesis, and the genetic and developmental etiology of diaphragm birth defects. PMID:23586979

  13. Effect of zidovudine on preimplantation murine embryos.

    PubMed Central

    Toltzis, P; Mourton, T; Magnuson, T

    1993-01-01

    It previously has been demonstrated that zidovudine (AZT) is lethal to early murine embryos. The effect of the drug on pre- and postimplantation embryos was examined to delineate the timing of this toxicity and to investigate its possible mechanisms. Embryos exposed in the whole mouse during preblastocyst development were unable to proceed beyond the blastocyst stage. Similarly, when two-cell embryos harvested from unexposed females were exposed to low-concentration (1 microM) AZT in vitro over 24 h, development beyond the blastocyst stage was inhibited. In contrast, drug exposure during in vitro blastocyst and postblastocyst development resulted in little or no morphologic toxicity. Further investigation revealed that preblastocyst AZT exposure resulted in the development of blastocysts with significantly lower cell numbers than control embryos. While embryonic exposure to AZT at the blastocyst and postblastocyst stages also resulted in retarded cell division, the effects were milder than those recorded after preblastocyst exposure. These data demonstrate that the critical period of AZT toxicity toward murine embryos is between ovulation and implantation and indicate that AZT directly suppresses cell division in the preimplantation embryo. PMID:8215271

  14. Analysis of chromatin structure in mouse preimplantation embryos by fluorescent recovery after photobleaching

    PubMed Central

    Ooga, Masatoshi; Fulka, Helena; Hashimoto, Satoshi; Suzuki, Masataka G.; Aoki, Fugaku

    2016-01-01

    Abstract Zygotes are totipotent cells that have the ability to differentiate into all cell types. It is believed that this ability is lost gradually and differentiation occurs along with the progression of preimplantation development. Here, we hypothesized that the loose chromatin structure is involved in the totipotency of one-cell stage embryos and that the change from loose to tight chromatin structure is associated with the loss of totipotency. To address this hypothesis, we investigated the mobility of eGFP-tagged histone H2B (eGFP-H2B), which is an index for the looseness of chromatin, during preimplantation development based on fluorescent recovery after photobleaching (FRAP) analysis. The highest mobility of eGFP-H2B was observed in pronuclei in 1-cell stage embryos and mobility gradually decreased during preimplantation development. The decrease in mobility between the 1- and 2-cell stages depended on DNA synthesis in 2-cell stage embryos. In nuclear transferred embryos, chromatin in the pseudopronuclei loosened to a level comparable to the pronuclei in 1-cell stage embryos. These results indicated that the mobility of eGFP-H2B is negatively correlated with the degree of differentiation of preimplantation embryos. Therefore, we suggest that highly loosened chromatin is involved in totipotency of 1-cell embryos and the loss of looseness is associated with differentiation during preimplantation development. PMID:26901819

  15. Sry and SoxE genes: How they participate in mammalian sex determination and gonadal development?

    PubMed

    She, Zhen-Yu; Yang, Wan-Xi

    2017-03-01

    In mammals, sex determination defines the differentiation of the bipotential genital ridge into either testes or ovaries. Sry, the mammalian Y-chromosomal testis-determining gene, is a master regulator of male sex determination. It acts to switch the undifferentiated genital ridge towards testis development, triggering the adoption of a male fate. Sry initiates a cascade of gene networks through the direct regulation of Sox9 expression and promotes supporting cell differentiation, Leydig cell specification, vasculature formation and testis cord development. In the absence of Sry, alternative genetic cascades, including female sex-determining genes RSPO1, Wnt4/β-catenin and Foxl2, are involved in the formation of female genitalia and the maintenance of female ovarian development. The mutual antagonisms between male and female sex-determining pathways are crucial in not just the initiation but also the maintenance of the somatic sex of the gonad throughout the organism's lifetime. Any imbalances in above sex-determining genes can cause disorders of sex development in humans and mice. In this review, we provide a detailed summary of the expression profiles, biochemical properties and developmental functions of Sry and SoxE genes in embryonic testis development and adult gonadal development. We also briefly summarize the dedicate balances between male and female sex-determining genes in mammalian sex development, with particular highlights on the molecular actions of Sry and Sox9 transcription factors.

  16. Antagonist Xist and Tsix co-transcription during mouse oogenesis and maternal Xist expression during pre-implantation development calls into question the nature of the maternal imprint on the X chromosome.

    PubMed

    Deuve, Jane Lynda; Bonnet-Garnier, Amélie; Beaujean, Nathalie; Avner, Philip; Morey, Céline

    2015-01-01

    During the first divisions of the female mouse embryo, the paternal X-chromosome is coated by Xist non-coding RNA and gradually silenced. This imprinted X-inactivation principally results from the apposition, during oocyte growth, of an imprint on the X-inactivation master control region: the X-inactivation center (Xic). This maternal imprint of yet unknown nature is thought to prevent Xist upregulation from the maternal X (X(M)) during early female development. In order to provide further insight into the X(M) imprinting mechanism, we applied single-cell approaches to oocytes and pre-implantation embryos at different stages of development to analyze the expression of candidate genes within the Xic. We show that, unlike the situation pertaining in most other cellular contexts, in early-growing oocytes, Xist and Tsix sense and antisense transcription occur simultaneously from the same chromosome. Additionally, during early development, Xist appears to be transiently transcribed from the X(M) in some blastomeres of late 2-cell embryos concomitant with the general activation of the genome indicating that X(M) imprinting does not completely suppress maternal Xist transcription during embryo cleavage stages. These unexpected transcriptional regulations of the Xist locus call for a re-evaluation of the early functioning of the maternal imprint on the X-chromosome and suggest that Xist/Tsix antagonist transcriptional activities may participate in imprinting the maternal locus as described at other loci subject to parental imprinting.

  17. Preimplantation genetic diagnosis: state of the art.

    PubMed

    Basille, Claire; Frydman, René; El Aly, Abdelwahab; Hesters, Laetitia; Fanchin, Renato; Tachdjian, Gérard; Steffann, Julie; LeLorc'h, Marc; Achour-Frydman, Nelly

    2009-07-01

    Preimplantation genetic diagnosis (PGD) is used to analyze embryos genetically before their transfer into the uterus. It was developed first in England in 1990, as part of progress in reproductive medicine, genetic and molecular biology. PGD offers couples at risk the chance to have an unaffected child, without facing termination of pregnancy. Embryos are obtained by in vitro fertilization with intracytoplasmic sperm injection (ICSI), and are biopsied mostly on day 3; blastocyst biopsy is mentioned as a possible alternative. The genetic analysis is performed on one or two blastomeres, by fluorescent in situ hybridization (FISH) for cytogenetic diagnosis, or polymerase chain reaction (PCR) for molecular diagnosis. Genetic analysis of the first or second polar body can be used to study maternal genetic contribution. Only unaffected embryos are transferred into the uterus. To improve the accuracy of the diagnosis, new technologies are emerging, with comparative genomic hybridization (CGH) and microarrays. In Europe, depending on national regulations, PGD is either prohibited, or allowed, or practiced in the absence of recommendations. The indications are chromosomal abnormalities, X-linked diseases or single gene disorders. The number of disorders being tested increases. In Europe, data collection from the year 2004 reports that globally 69.6% of cycles lead to embryo transfer and implantation rate is 17%. European results from the year 2004 show a clinical pregnancy rate of 18% per oocyte retrieval and 25% per embryo transfer, leading to 528 babies born. The cohort studies concerning the paediatric follow-up of PGD babies show developmental outcomes similar to children conceived after IVF-ICSI. Recent advances include human leucocyte antigen (HLA) typing for PGD embryos, when an elder sibling is affected with a genetic disorder and needs stem cell transplantation. The HLA-matched offspring resulting can give cord blood at birth. Preimplantation genetic screening (PGS

  18. PreImplantation factor promotes neuroprotection by targeting microRNA let-7.

    PubMed

    Mueller, Martin; Zhou, Jichun; Yang, Lihua; Gao, Yuan; Wu, Fuju; Schoeberlein, Andreina; Surbek, Daniel; Barnea, Eytan R; Paidas, Michael; Huang, Yingqun

    2014-09-23

    Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer's disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)-dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development.

  19. Rapamycin ameliorates chitosan nanoparticle-induced developmental defects of preimplantation embryos in mice

    PubMed Central

    Choi, Yun-Jung; Gurunathan, Sangiliyandi; Kim, DaSom; Jang, Hyung Seok; Park, Woo-Jin; Cho, Ssang-Goo; Park, Chankyu; Song, Hyuk; Seo, Han Geuk; Kim, Jin-Hoi

    2016-01-01

    Chitosan nanoparticles (CSNPs) are used as drug or gene delivery vehicles. However, a detailed understanding of the effects of CSNPs on embryonic development remains obscure. Here, we show that CSNPs can be internalized into mouse blastocysts, such as the zona pellucida, the perivitelline space, and the cytoplasm. Consequently, CSNPs-induced endoplasmic reticulum (ER) stress increases both of Bip/Grp78, Chop, Atf4, Perk, and Ire1a mRNAs expression levels, and reactive oxygen species. Moreover, CSNPs show double- and multi-membraned autophagic vesicles, and lead to cell death of blastocoels. Conversely, treatment with rapamycin, which plays an important role as a central regulator of cellular proliferation and stress responses, decreased CSNPs-induced mitochondrial Ca+2 overloading, apoptosis, oxidative stress, ER stress, and autophagy. In vivo studies demonstrated that CSNPs injection has significant toxic effect on primordial and developing follicles. Notably, rapamycin rescued oxidative stress-induced embryonic defects via modulating gene expression of sirtuin and mammalian target of rapamycin. Interestingly, CSNPs treatment alters epigenetic reprogramming in mouse embryos. Overall, these observations suggest that rapamycin treatment could ameliorate CSNPs-induced developmental defects in preimplantation embryos. The data from this study would facilitate to understand the toxicity of these CSNPs, and enable the engineering of safer nanomaterials for therapeutic applications. PMID:27463007

  20. Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development.

    PubMed

    Li, Wei; Xiong, Yiqin; Shang, Ching; Twu, Karen Y; Hang, Calvin T; Yang, Jin; Han, Pei; Lin, Chieh-Yu; Lin, Chien-Jung; Tsai, Feng-Chiao; Stankunas, Kryn; Meyer, Tobias; Bernstein, Daniel; Pan, Minggui; Chang, Ching-Pin

    2013-01-29

    Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21(cip1), to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.

  1. Preimplantation genetic diagnosis: current and future perspectives.

    PubMed

    Cram, David; Pope, Adrianne

    2007-08-01

    Over the last 30 years prenatal diagnosis has been available to couples at genetic risk to determine the genetic health of a naturally conceived pregnancy. Prenatal diagnosis involves fetal cell sampling either by chorionic villous sampling at 10-12 weeks or by amniocentesis at 12-15 weeks gestation and testing for genetic disease. If the fetus is affected, termination of pregnancy is a difficult and emotional issue for the couple. The advent of assisted reproductive technologies (ARTs) and more recently preimplantation genetic diagnosis (PGD) now provides an alternative reproductive option that allows couples to commence a pregnancy knowing that their baby will not be affected with the indicated genetic condition. This article explores the option of PGD, its clinical application now and into the future and the current status of regulation and legislation. With recent changes to national legislation, scientists now have the opportunity to access affected PGD embryos donated by patients to establish disease-specific stem cells that may be useful models of human disease and a means to develop more effective therapies for treatment.

  2. Using the amniotic cavity of the developing chick embryo for the in vivo culture of early-stage mammalian embryos.

    PubMed

    Blakewood, E G; Jaynes, J M; Johnson, W A; Godke, R A

    1989-12-01

    The fertile chicken egg may provide an effective, inexpensive method for promoting the development of early-stage embryos from other species. Presently, the loss of viability associated with the in vitro culture of mammalian embryos is hindering the use of in vitro fertilization with farm animals. Consequently, alternative in vitro laboratory methods are needed for the culture of mammalian embryos. A new method has been developed that involves the culture of mammalian embryos in the amniotic cavity of a developing chick embryo. Chick embryos were placed into shell-less incubation (37 C) at the 72-h developmental stage. After 24 h of shell-less incubation, agarose-embedded mammalian embryos were injected into the amniotic cavity of the chick embryo. The mammalian embryos were first placed into a drop of liquid agarose. One to four embryos were then aspirated into a beveled injection pipette and cooled, allowing the agarose to harden. Following penetration of the amnion with the beveled pipette, the agarose cylinder containing the embryos was expelled into the amniotic cavity. The shell-less culture system was then returned to incubation at 37 C for an additional 72 to 96 h. Following incubation, the amniotic cavity containing both chick and mammalian embryos was isolated and the agarose-embedded mammalian embryos were harvested. Significantly more embryos developed in the chick embryo amnion than in the control medium alone. Results obtained using this method on laboratory animals (mice) and on domestic mammals (goats and cattle) indicate that the chick-embryo amnion can support the development of early-stage, mammalian embryos to the blastocyst stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Functional genomics of HMGN3a and SMARCAL1 in early mammalian embryogenesis

    PubMed Central

    Uzun, Alper; Rodriguez-Osorio, Nelida; Kaya, Abdullah; Wang, Hongfeng; Parrish, John J; Ilyin, Valentin A; Memili, Erdogan

    2009-01-01

    Background Embryonic genome activation (EGA) is a critical event for the preimplantation embryo, which is manifested by changes in chromatin structure, transcriptional machinery, expression of embryonic genes, and degradation of maternal transcripts. The objectives of this study were to determine transcript abundance of HMGN3a and SMARCAL1 in mature bovine oocytes and early bovine embryos, to perform comparative functional genomics analysis of these genes across mammals. Results New annotations of both HMGN3a and SMARCAL1 were submitted to the Bovine Genome Annotation Submission Database at BovineGenome.org. Careful analysis of the bovine SMARCAL1 consensus gene set for this protein (GLEAN_20241) showed that the NCBI protein contains sequencing errors, and that the actual bovine protein has a high degree of homology to the human protein. Our results showed that there was a high degree of structural conservation of HMGN3a and SMARCAL1 in the mammalian species studied. HMGN3a transcripts were present at similar levels in bovine matured oocytes and 2–4-cell embryos but at higher levels in 8–16-cell embryos, morulae and blastocysts. On the other hand, transcript levels of SMARCAL1 decreased throughout preimplantation development. Conclusion The high levels of structural conservation of these proteins highlight the importance of chromatin remodeling in the regulation of gene expression, particularly during early mammalian embryonic development. The greater similarities of human and bovine HMGN3a and SMARCAL1 proteins may suggest the cow as a valuable model to study chromatin remodeling at the onset of mammalian development. Understanding the roles of chromatin remodeling proteins during embryonic development emphasizes the importance of epigenetics and could shed light on the underlying mechanisms of early mammalian development. PMID:19393058

  4. Alterations in sarcomere function modify the hyperplastic to hypertrophic transition phase of mammalian cardiomyocyte development

    PubMed Central

    Nixon, Benjamin R.; Williams, Alexandra F.; Glennon, Michael S.; de Feria, Alejandro E.; Sebag, Sara C.; Baldwin, H. Scott; Becker, Jason R.

    2017-01-01

    It remains unclear how perturbations in cardiomyocyte sarcomere function alter postnatal heart development. We utilized murine models that allowed manipulation of cardiac myosin-binding protein C (MYBPC3) expression at critical stages of cardiac ontogeny to study the response of the postnatal heart to disrupted sarcomere function. We discovered that the hyperplastic to hypertrophic transition phase of mammalian heart development was altered in mice lacking MYBPC3 and this was the critical period for subsequent development of cardiomyopathy. Specifically, MYBPC3-null hearts developed evidence of increased cardiomyocyte endoreplication, which was accompanied by enhanced expression of cell cycle stimulatory cyclins and increased phosphorylation of retinoblastoma protein. Interestingly, this response was self-limited at later developmental time points by an upregulation of the cyclin-dependent kinase inhibitor p21. These results provide valuable insights into how alterations in sarcomere protein function modify postnatal heart development and highlight the potential for targeting cell cycle regulatory pathways to counteract cardiomyopathic stimuli. PMID:28239655

  5. Pre-implantation genetic diagnosis.

    PubMed

    Traeger-Synodinos, Joanne

    2017-02-01

    The aim of pre-implantation genetic diagnosis (PGD) is to characterize the genetic status of the cells (usually single cells) that have been biopsied from oocytes/zygotes or embryos created in vitro during assisted reproductive treatment. PGD is a multi-step procedure that requires close collaboration between gynaecologists who are experts in assisted reproduction, embryologists who are experts in micromanipulation of germ cells and in embryo biopsy and geneticists who are experts in genetic analysis at the single-cell level. PGD can be applied as a form of early pre-natal diagnosis with the aim to establish a pregnancy unaffected by a haemoglobinopathy. In addition, PGD can identify embryos that are human leukocyte antigen compatible with an existing sibling affected by a haemoglobinopathy to support a haematopoietic stem cell transplantation. PGD has an advantage over conventional pre-natal diagnosis as it precludes the need to consider terminating an affected ongoing pregnancy. However, PGD is a multi-step, complex and costly procedure with an unpredictable outcome and thus is most suited for couples with an unsuccessful reproductive history or challenging reproductive status. In addition, PGD supports the cure of an affected child. Couples who decide to undergo a PGD cycle should be fully aware of the advantages and limitations. The three teams of health practitioners involved (gynaecologists, embryologists and geneticists) should thoroughly counsel the couples and provide support at all the stages: the initial evaluation of their genetic and reproductive status, all steps of assisted reproduction, embryo biopsy, genetic analysis and, when relevant, follow-up of pregnancy and baby(ies) delivered. Copyright © 2016. Published by Elsevier Ltd.

  6. Preimplantation Genetic Testing: Indications and Controversies

    PubMed Central

    Cooper, Amber R.; Jungheim, Emily S.

    2014-01-01

    Synopsis In the last two decades, the use of preimplantation genetic testing has increased dramatically. It is used for single gene disorders, chromosomal abnormalities, mitochondrial disorders, gender selection in non-Mendelian disorders with unequal gender distribution, aneuploidy screening, and other preconceptually identified genetic abnormalities in prospective parents. Genetic testing strategies and diagnostic accuracy continues to improve. Yet, it does not come without risks or controversies. In this review we discuss the techniques and clinical application of preimplantation genetic diagnosis and the debate surrounding its associated uncertainty and expanded use. PMID:20638568

  7. Advances in preimplantation genetic diagnosis/screening.

    PubMed

    Yan, LiYing; Wei, Yuan; Huang, Jin; Zhu, XiaoHui; Shi, XiaoDan; Xia, Xi; Yan, Jie; Lu, CuiLing; Lian, Ying; Li, Rong; Liu, Ping; Qiao, Jie

    2014-07-01

    Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS.

  8. Role of H1 linker histones in mammalian development and stem cell differentiation.

    PubMed

    Pan, Chenyi; Fan, Yuhong

    2016-03-01

    H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome.

  9. The evolution of basal progenitors in the developing non-mammalian brain

    PubMed Central

    Nomura, Tadashi; Ohtaka-Maruyama, Chiaki; Yamashita, Wataru; Wakamatsu, Yoshio; Murakami, Yasunori; Calegari, Federico; Suzuki, Kunihiro; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2+ intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2+ cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution. PMID:26732839

  10. A gene network model accounting for development and evolution of mammalian teeth.

    PubMed

    Salazar-Ciudad, Isaac; Jernvall, Jukka

    2002-06-11

    Generation of morphological diversity remains a challenge for evolutionary biologists because it is unclear how an ultimately finite number of genes involved in initial pattern formation integrates with morphogenesis. Ideally, models used to search for the simplest developmental principles on how genes produce form should account for both developmental process and evolutionary change. Here we present a model reproducing the morphology of mammalian teeth by integrating experimental data on gene interactions and growth into a morphodynamic mechanism in which developing morphology has a causal role in patterning. The model predicts the course of tooth-shape development in different mammalian species and also reproduces key transitions in evolution. Furthermore, we reproduce the known expression patterns of several genes involved in tooth development and their dynamics over developmental time. Large morphological effects frequently can be achieved by small changes, according to this model, and similar morphologies can be produced by different changes. This finding may be consistent with why predicting the morphological outcomes of molecular experiments is challenging. Nevertheless, models incorporating morphology and gene activity show promise for linking genotypes to phenotypes.

  11. MicroRNA expression and its association with DNA repair in preimplantation embryos

    PubMed Central

    TULAY, Pinar; SENGUPTA, Sioban B.

    2016-01-01

    Active DNA repair pathways are crucial for preserving genomic integrity and are likely among the complex mechanisms involved in the normal development of preimplantation embryos. MicroRNAs (miRNA), short non-coding RNAs, are key regulators of gene expression through the post-transcriptional and post-translational modification of mRNA. The association of miRNA expression with infertility or polycystic ovarian syndrome has been widely investigated; however, there are limited data regarding the importance of miRNA regulation in DNA repair during preimplantation embryo development. In this article, we review normal miRNA biogenesis and consequences of aberrant miRNA expression in the regulation of DNA repair in gametes and preimplantation embryos. PMID:26853522

  12. The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis

    PubMed Central

    Twohig, Jason P.; Cuff, Simone M.; Yong, Audrey A.; Wang, Eddie C.Y.

    2012-01-01

    Tumor necrosis factor receptor superfamily (TNFRSF) members were initially identified as immunological mediators, and are still commonly perceived as immunological molecules. However, our understanding of the diversity of TNFRSF members’ roles in mammalian physiology has grown significantly since the first discovery of TNFRp55 (TNFRSF1) in 1975. In particular, the last decade has provided evidence for important roles in brain development, function and the emergent field of neuronal homeostasis. Recent evidence suggests that TNFRSF members are expressed in an overlapping regulated pattern during neuronal development, participating in the regulation of neuronal expansion, growth, differentiation and regional pattern development. This review examines evidence for non-immunological roles of TNFRSF members in brain development, function and maintenance under normal physiological conditions. In addition, several aspects of brain function during inflammation will also be described, when illuminating and relevant to the non-immunological role of TNFRSF members. Finally, key questions in the field will be outlined. PMID:21861782

  13. The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis.

    PubMed

    Twohig, Jason P; Cuff, Simone M; Yong, Audrey A; Wang, Eddie C Y

    2011-01-01

    Tumor necrosis factor receptor superfamily (TNFRSF) members were initially identified as immunological mediators, and are still commonly perceived as immunological molecules. However, our understanding of the diversity of TNFRSF members' roles in mammalian physiology has grown significantly since the first discovery of TNFRp55 (TNFRSF1) in 1975. In particular, the last decade has provided evidence for important roles in brain development, function and the emergent field of neuronal homeostasis. Recent evidence suggests that TNFRSF members are expressed in an overlapping regulated pattern during neuronal development, participating in the regulation of neuronal expansion, growth, differentiation and regional pattern development. This review examines evidence for non-immunological roles of TNFRSF members in brain development, function and maintenance under normal physiological conditions. In addition, several aspects of brain function during inflammation will also be described, when illuminating and relevant to the non-immunological role of TNFRSF members. Finally, key questions in the field will be outlined.

  14. The value of mammalian models for duchenne muscular dystrophy in developing therapeutic strategies.

    PubMed

    Banks, Glen B; Chamberlain, Jeffrey S

    2008-01-01

    Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. There is no effective treatment and patients typically die in approximately the third decade. DMD is an X-linked recessive disease caused by mutations in the dystrophin gene. There are three mammalian models of DMD that have been used to understand better the pathogenesis of disease and develop therapeutic strategies. The mdx mouse is the most widely used model of DMD that displays some features of muscle degeneration, but the pathogenesis of disease is comparatively mild. The severity of disease in mice lacking both dystrophin and utrophin is similar to DMD, but one has to account for the discrete functions of utrophin. Canine X-linked muscular dystrophy (cxmd) is the best representation of DMD, but the phenotype of the most widely used golden retriever (GRMD) model is variable, making functional endpoints difficult to ascertain. Although each mammalian model has its limitations, together they have been essential for the development of several treatment strategies for DMD that target dystrophin replacement, disease progression, and muscle regeneration.

  15. Evolutionary modification of development in mammalian teeth: Quantifying gene expression patterns and topography

    PubMed Central

    Jernvall, Jukka; Keränen, Soile V. E.; Thesleff, Irma

    2000-01-01

    The study of mammalian evolution often relies on detailed analysis of dental morphology. For molecular patterning to play a role in dental evolution, gene expression differences should be linkable to corresponding morphological differences. Because teeth, like many other structures, are complex and evolution of new shapes usually involves subtle changes, we have developed topographic methods by using Geographic Information Systems. We investigated how genetic markers for epithelial signaling centers known as enamel knots are associated with evolutionary divergence of molar teeth in two rodent species, mouse and vole. Our analysis of expression patterns of Fgf4, Lef1, p21, and Shh genes in relation to digital elevation models of developing tooth shapes shows that molecular prepatterns predict the lateral cusp topography more than a day in advance. A heterotopic shift in the molecular prepatterns can be implicated in the evolution of mouse molar, changing locations from which historically homologous cusps form. The subtle but measurable heterotopic shifts may play a large role in the evolution of tooth cusp topographies. However, evolutionary increase in the number of longitudinal cusps in vole molar has involved accelerated longitudinal growth and iterative addition of new cusps without changes in lateral cusp topography. The iterative addition of cusps after the establishment of lateral cusp topography may limit the independence of individual morphological features used in evolutionary studies. The diversity of mammalian molar patterns may largely result from the heterotopic and iterative processes. PMID:11121045

  16. Pervasive translational regulation of the cell signalling circuitry underlies mammalian development

    PubMed Central

    Fujii, Kotaro; Shi, Zhen; Zhulyn, Olena; Denans, Nicolas; Barna, Maria

    2017-01-01

    The degree and dynamics of translational control during mammalian development remain poorly understood. Here we monitored translation of the mammalian genome as cells become specified and organize into tissues in vivo. This identified unexpected and pervasive translational regulation of most of the core signalling circuitry including Shh, Wnt, Hippo, PI3K and MAPK pathways. We further identify and functionally characterize a complex landscape of upstream open reading frames (uORFs) across 5′-untranslated regions (UTRs) of key signalling components. Focusing on the Shh pathway, we demonstrate the importance of uORFs within the major SHH receptor, Ptch1, in control of cell signalling and neuronal differentiation. Finally, we show that the expression of hundreds of mRNAs underlying critical tissue-specific developmental processes is largely regulated at the translation but not transcript levels. Altogether, this work reveals a new layer of translational control to major signalling components and gene regulatory networks that diversifies gene expression spatially across developing tissues. PMID:28195124

  17. The role of BAF (mSWI/SNF) complexes in mammalian neural development.

    PubMed

    Son, Esther Y; Crabtree, Gerald R

    2014-09-01

    The BAF (mammalian SWI/SNF) complexes are a family of multi-subunit ATP-dependent chromatin remodelers that use ATP hydrolysis to alter chromatin structure. Distinct BAF complex compositions are possible through combinatorial assembly of homologous subunit families and can serve non-redundant functions. In mammalian neural development, developmental stage-specific BAF assemblies are found in embryonic stem cells, neural progenitors and postmitotic neurons. In particular, the neural progenitor-specific BAF complexes are essential for controlling the kinetics and mode of neural progenitor cell division, while neuronal BAF function is necessary for the maturation of postmitotic neuronal phenotypes as well as long-term memory formation. The microRNA-mediated mechanism for transitioning from npBAF to nBAF complexes is instructive for the neuronal fate and can even convert fibroblasts into neurons. The high frequency of BAF subunit mutations in neurological disorders underscores the rate-determining role of BAF complexes in neural development, homeostasis, and plasticity. © 2014 Wiley Periodicals, Inc.

  18. Functional synergy between cholecystokinin receptors CCKAR and CCKBR in mammalian brain development.

    PubMed

    Nishimura, Sayoko; Bilgüvar, Kaya; Ishigame, Keiko; Sestan, Nenad; Günel, Murat; Louvi, Angeliki

    2015-01-01

    Cholecystokinin (CCK), a peptide hormone and one of the most abundant neuropeptides in vertebrate brain, mediates its actions via two G-protein coupled receptors, CCKAR and CCKBR, respectively active in peripheral organs and the central nervous system. Here, we demonstrate that the CCK receptors have a dynamic and largely reciprocal expression in embryonic and postnatal brain. Using compound homozygous mutant mice lacking the activity of both CCK receptors, we uncover their additive, functionally synergistic effects in brain development and demonstrate that CCK receptor loss leads to abnormalities of cortical development, including defects in the formation of the midline and corpus callosum, and cortical interneuron migration. Using comparative transcriptome analysis of embryonic neocortex, we define the molecular mechanisms underlying these defects. Thus we demonstrate a developmental, hitherto unappreciated, role of the two CCK receptors in mammalian neocortical development.

  19. Functional Synergy between Cholecystokinin Receptors CCKAR and CCKBR in Mammalian Brain Development

    PubMed Central

    Nishimura, Sayoko; Bilgüvar, Kaya; Ishigame, Keiko; Sestan, Nenad; Günel, Murat; Louvi, Angeliki

    2015-01-01

    Cholecystokinin (CCK), a peptide hormone and one of the most abundant neuropeptides in vertebrate brain, mediates its actions via two G-protein coupled receptors, CCKAR and CCKBR, respectively active in peripheral organs and the central nervous system. Here, we demonstrate that the CCK receptors have a dynamic and largely reciprocal expression in embryonic and postnatal brain. Using compound homozygous mutant mice lacking the activity of both CCK receptors, we uncover their additive, functionally synergistic effects in brain development and demonstrate that CCK receptor loss leads to abnormalities of cortical development, including defects in the formation of the midline and corpus callosum, and cortical interneuron migration. Using comparative transcriptome analysis of embryonic neocortex, we define the molecular mechanisms underlying these defects. Thus we demonstrate a developmental, hitherto unappreciated, role of the two CCK receptors in mammalian neocortical development. PMID:25875176

  20. Intracytoplasmic sperm injection combined with preimplantation genetic diagnosis for the prevention of recurrent gestational trophoblastic disease.

    PubMed

    Reubinoff, B E; Lewin, A; Verner, M; Safran, A; Schenker, J G; Abeliovich, D

    1997-04-01

    A strategy for the prevention of repeated molar pregnancies by using intracytoplasmic sperm injection (ICSI) coupled with preimplantation genetic diagnosis (PGD) with fluorescence in-situ hybridization (FISH) was developed. In this approach, complete moles which arise from dispermic fertilization are avoided by the use of ICSI. ICSI is followed by preimplantation selection against the transfer of 46,XX embryos, thus preventing complete moles resulting from a fertilization of an inactive oocyte, by a haploid X-bearing spermatozoon which subsequently duplicates. Triploid partial moles which arise mainly from dispermic fertilization may also be prevented by ICSI. The preimplantation confirmation of diploidy by FISH guards against triploid partial moles which may result from mechanisms other than dispermic fertilization. The employment of this strategy in an attempt to prevent a repeated event of molar pregnancy in a patient with a history of two previous episodes of gestational trophoblastic disease is reported.

  1. Preimplantation genetic screening: who benefits?

    PubMed

    Kang, Hey-Joo; Melnick, Alexis P; Stewart, Joshua D; Xu, Kangpu; Rosenwaks, Zev

    2016-09-01

    To compare IVF outcomes between women undergoing frozen transfers of blastocysts verified as euploid by preimplantation genetic screening (PGS) with patients undergoing fresh nonbiopsied blastocyst transfers. Retrospective cohort study. Academic medical center. All patients undergoing IVF-PGS cycles between January 2010 and November 2014 were included (n = 274). Patients were compared with a control group consisting of all fresh blastocyst transfers that occurred during the same period (n = 863). Patients underwent IVF-PGS with 24-chromosome screening. Patients with euploid embryos had transfer of one to two embryos in a subsequent frozen ET cycle. Implantation, clinical intrauterine gestation (CIG), miscarriage, biochemical pregnancy (BC), and live birth (LB) rates were compared. Odds ratios (ORs) were estimated for outcomes in women undergoing PGS versus controls. Among patients ≤37 years old, there were no differences in CIG and LB rates for single (adjusted ORs [aORs], 1.20 [95 %confidence interval {CI}, 0.66-2.21]; 1.21 [95% CI, 0.66-2.2]) and double ETs (aORs, 1.09 [95% CI, 0.54-2.18]; 0.87 [95% CI, 0.44-1.7]). BC and miscarriage rates were also similar. For patients >37 years old, CIG and LB rates were increased for single (aORs, 3.86 [95% CI, 1.25-11.9]; 8.2 [95% CI, 2.28-29.5]) and double ETs (aORs, 9.91 [95% CI, 2.0-49.6]; 8.67 [95% CI, 2.08-36.2]) with no difference in BC and miscarriage rates. A per-retrieval analysis of the >37 group failed to demonstrate any difference in CIG or LB rates. Among patients ≤37, IVF-PGS does not improve CIG, LB, and miscarriage rates. IVF-PGS in women >37 improved CIG and LB rates. However, per cycle, the PGS advantage in this age group does not persist. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  2. Methionine requirements for the preimplantation bovine embryo.

    PubMed

    BONILLA, Luciano; LUCHINI, Daniel; DEVILLARD, Estelle; HANSEN, Peter J

    2010-10-01

    The early embryo's nutritional environment plays an important role in establishing its developmental potential. However, little is known about the specific nutrient requirements of the embryo. The objective of the present study was to determine requirements of the in vitro produced bovine embryo for the essential amino acid methionine. In addition to serving as a precursor for polypeptides, methionine plays roles in regulation of translation, DNA methylation, and antioxidant balance. In the first experiment, embryos were cultured in potassium simplex optimized medium - bovine embryo modification 2 containing 0, 35, 50, 100, 200 or 400 µmol/l L-methionine for 8 days. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 than other groups but was similar for embryos cultured with 35-400 µmol/l. Neither total cell number, allocation of cells to trophectoderm or inner cell mass, or frequency of apoptosis was affected by methionine concentration. In the second experiment, embryos were cultured with 0, 7, 14, 21, 28 or 35 µmol/l methionine. There was no effect of methionine concentration on cleavage rate. The percent of oocytes that developed to blastocyst was lower for embryos without methionine at Day 7 and 8 but was not different between embryos cultured with 7-35 µmol/l methionine. However, the proportion of blastocysts that were expanded, hatching or hatched on Day 7 was reduced at lower concentrations of methionine (7 and 14). DNA methylation of blastocyst nuclei was unaffected by methionine concentration but intracellular glutathione content was higher for embryos cultured without methionine. In conclusion, the methionine requirement for preimplantation development is between 14 and 21 µmol/l. These concentrations are lower or similar to those found in the reproductive tract and suggest that methionine deficiency is not a common cause of

  3. Friend or Foe: Epigenetic Regulation of Retrotransposons in Mammalian Oogenesis and Early Development

    PubMed Central

    Evsikov, Alexei V.; Marín de Evsikova, Caralina

    2016-01-01

    Epigenetics is the study of phenotypic variation arising from developmental and environmental factors regulating gene transcription at molecular, cellular, and physiological levels. A naturally occurring biological process driven by epigenetics is the egg-to-embryo developmental transition when two fully differentiated adult cells – egg and sperm – revert to an early stem cell type with totipotency but subsequently differentiates into pluripotent embryonic stem cells that give rise to any cell type. Transposable elements (TEs) are active in mammalian oocytes and early embryos, and this activity, albeit counterintuitive because TEs can lead to genomic instability in somatic cells, correlates to successful development. TEs bridge genetic and epigenetic landscapes because TEs are genetic elements whose silencing and de-repression are regulated by epigenetic mechanisms that are sensitive to environmental factors. Ultimately, transposition events can change size, content, and function of mammalian genomes. Thus, TEs act beyond mutagenic agents reshuffling the genomes, and epigenetic regulation of TEs may act as a proximate mechanism by which evolutionary forces increase a species’ hidden reserve of epigenetic and phenotypic variability facilitating the adaptation of genomes to their environment. PMID:28018140

  4. Microarray analysis of microRNA expression in the developing mammalian brain

    PubMed Central

    Miska, Eric A; Alvarez-Saavedra, Ezequiel; Townsend, Matthew; Yoshii, Akira; Šestan, Nenad; Rakic, Pasko; Constantine-Paton, Martha; Horvitz, H Robert

    2004-01-01

    Background MicroRNAs are a large new class of tiny regulatory RNAs found in nematodes, plants, insects and mammals. MicroRNAs are thought to act as post-transcriptional modulators of gene expression. In invertebrates microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death and fat metabolism. Little is known about the roles of microRNAs in mammals. Results We isolated 18-26 nucleotide RNAs from developing rat and monkey brains. From the sequences of these RNAs and the sequences of the rat and human genomes we determined which of these small RNAs are likely to have derived from stem-loop precursors typical of microRNAs. Next, we developed a microarray technology suitable for detecting microRNAs and printed a microRNA microarray representing 138 mammalian microRNAs corresponding to the sequences of the microRNAs we cloned as well as to other known microRNAs. We used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain. Conclusion We describe a microarray technology that can be used to analyze the expression of microRNAs and of other small RNAs. MicroRNA microarrays offer a new tool that should facilitate studies of the biological roles of microRNAs. We used this method to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs. PMID:15345052

  5. Exposure of preimplantation embryos to low-dose bisphenol A impairs testes development and suppresses histone acetylation of StAR promoter to reduce production of testosterone in mice.

    PubMed

    Hong, Juan; Chen, Fang; Wang, Xiaoli; Bai, Yinyang; Zhou, Rong; Li, Yingchun; Chen, Ling

    2016-05-15

    Previous studies have shown that bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. The present study focused on exploring the impact of exposure to low dose of BPA on male reproductive development during the early embryo stage and the underlying mechanisms. BPA (20 μg/kg/day) was orally administered to female mice on days 1-5 of gestation. The male offspring were euthanized at PND10, 20, 24, 35 or PND50. We found that the mice exposed to BPA before implantation (BPA-mice) displayed retardation of testicular development with reduction of testosterone level. The diameter and epithelium height of seminiferous tubules were reduced in BPA-mice at PND35. The numbers of spermatogenic cells at different stages were significantly reduced in BPA-mice at PND50. BPA-mice showed a persistent reduction in serum and testicular testosterone levels starting from PND24, whereas GnRH mRNA was significantly increased at PND35 and PND50. The expressions of testicular StAR and P450scc in BPA-mice also decreased relative to those of the controls at PND35 and PND50. Further analysis found that the levels of histone H3 and H3K14 acetylation (Ac-H3 and H3K14ac) in the promoter of StAR were decreased relative to those of control mice, whereas the level of Ac-H3 in the promoter of P450scc was not significantly different between the groups. These results provide evidence that exposure to BPA in preimplantation embryo retards the development of testes by reducing histone acetylation of the StAR promoter to disrupt the testicular testosterone synthesis.

  6. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development

    PubMed Central

    Guan, Yingjie; Yang, Xu; Yang, Wentian; Charbonneau, Cherie; Chen, Qian

    2014-01-01

    Mechanical stress regulates development by modulating cell signaling and gene expression. However, the cytoplasmic components mediating mechanotransduction remain unclear. In this study, elimination of muscle contraction during chicken embryonic development resulted in a reduction in the activity of mammalian target of rapamycin (mTOR) in the cartilaginous growth plate. Inhibition of mTOR activity led to significant inhibition of chondrocyte proliferation, cartilage tissue growth, and expression of chondrogenic genes, including Indian hedgehog (Ihh), a critical mediator of mechanotransduction. Conversely, cyclic loading (1 Hz, 5% matrix deformation) of embryonic chicken growth plate chondrocytes in 3-dimensional (3D) collagen scaffolding induced sustained activation of mTOR. Mechanical activation of mTOR occurred in serum-free medium, indicating that it is independent of growth factor or nutrients. Treatment of chondrocytes with Rapa abolished mechanical activation of cell proliferation and Ihh gene expression. Cyclic loading of chondroprogenitor cells deficient in SH2-containing protein tyrosine phosphatase 2 (Shp2) further enhanced mechanical activation of mTOR, cell proliferation, and chondrogenic gene expression. This result suggests that Shp2 is an antagonist of mechanotransduction through inhibition of mTOR activity. Our data demonstrate that mechanical activation of mTOR is necessary for cell proliferation, chondrogenesis, and cartilage growth during bone development, and that mTOR is an essential mechanotransduction component modulated by Shp2 in the cytoplasm.—Guan, Y., Yang, X., Yang, W., Charbonneau, C., Chen, Q. Mechanical activation of mammalian target of rapamycin pathway is required for cartilage development. PMID:25002119

  7. Placental, Matrilineal, and Epigenetic Mechanisms Promoting Environmentally Adaptive Development of the Mammalian Brain

    PubMed Central

    Broad, Kevin D.; Rocha-Ferreira, Eridan; Hristova, Mariya

    2016-01-01

    The evolution of intrauterine development, vivipary, and placentation in eutherian mammals has introduced new possibilities and constraints in the regulation of neural plasticity and development which promote neural function that is adaptive to the environment that a developing brain is likely to encounter in the future. A range of evolutionary adaptations associated with placentation transfers disproportionate control of this process to the matriline, a period unique in mammalian development in that there are three matrilineal genomes interacting in the same organism at the same time (maternal, foetal, and postmeiotic oocytes). The interactions between the maternal and developing foetal hypothalamus and placenta can provide a template by which a mother can transmit potentially adaptive information concerning potential future environmental conditions to the developing brain. In conjunction with genomic imprinting, it also provides a template to integrate epigenetic information from both maternal and paternal lineages. Placentation also hands ultimate control of genomic imprinting and intergenerational epigenetic information transfer to the matriline as epigenetic markers undergo erasure and reprogramming in the developing oocyte. These developments, in conjunction with an expanded neocortex, provide a unique evolutionary template by which matrilineal transfer of maternal care, resources, and culture can be used to promote brain development and infant survival. PMID:27069693

  8. Analysis of the sex ratio in preimplantation embryos from B6.K1 and B6.K2 Ped gene congenic mice

    PubMed Central

    Byrne, Michael J.; Newmark, Judith A.

    2008-01-01

    Purpose The mouse preimplantation embryo development (Ped) gene product, Qa-2, which is the homolog of human HLA-G, influences the rate of preimplantation embryonic development and overall reproductive success. The sex ratio in preimplantation embryos from Ped gene congenic mice was examined in order to determine whether embryo sex is a confounding factor in the control of the rate of preimplantation development. Methods B6.K1 (Ped slow) and B6.K2 (Ped fast) congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the sex of B6.K1 (n=221) and B6.K2 (n=260) preimplantation embryos by using Real-Time PCR with primers specific for the X and Y chromosomes. Results We found that there was no statistically significant difference in the ratio of male to female preimplantation embryos in either strain. Conclusions We conclude that the sex of the embryos is not a confounding factor that affects the Ped gene control of the rate of preimplantation development. Therefore, the Ped gene is entirely responsible for mediating the faster development of B6.K2 embryos compared to B6.K1 embryos. PMID:16902830

  9. Every amino acid matters: essential contributions of histone variants to mammalian development and disease

    PubMed Central

    Maze, Ian; Noh, Kyung-Min; Soshnev, Alexey A.; Allis, C. David

    2014-01-01

    Despite a conserved role for histones as general DNA packaging agents, it is now clear that another key function of these proteins is to confer variations in chromatin structure to ensure dynamic patterns of transcriptional regulation in eukaryotes. The incorporation of histone variants is particularly important to this process. Recent knockdown and knockout studies in various cellular systems, as well as direct mutational evidence from human cancers, now suggest a crucial role for histone variant regulation in processes as diverse as differentiation and proliferation, meiosis and nuclear reprogramming. In this Review, we provide an overview of histone variants in the context of their unique functions during mammalian germ cell and embryonic development, and examine the consequences of aberrant histone variant regulation in human disease. PMID:24614311

  10. Evolution and development of the mammalian dentition: insights from the marsupial Monodelphis domestica.

    PubMed

    Moustakas, Jacqueline E; Smith, Kathleen K; Hlusko, Leslea J

    2011-01-01

    To understand developmental mechanisms of evolutionary change, we must first know how different morphologies form. The vast majority of our knowledge on the developmental genetics of tooth formation derives from studies in mice, which have relatively derived mammalian dentitions. The marsupial Monodelphis domestica has a more plesiomorphic heterodont dentition with incisors, canines, premolars, and molars on both the upper and the lower jaws, and a deciduous premolar. The complexity of the M. domestica dentition ranges from simple, unicusped incisors to conical, sharp canines to multicusped molars. We examine the development of the teeth in M. domestica, with a specific focus on the enamel knot, a signaling center in the embryonic tooth that controls shape. We show that the tooth germs of M. domestica express fibroblast growth factor (FGF) genes and Sprouty genes in a manner similar to wild-type mouse molar germs, but with a few key differences.

  11. Primary cilia and signaling pathways in mammalian development, health and disease

    PubMed Central

    VELAND, IBEN R.; AWAN, AASHIR; PEDERSEN, LOTTE B.; YODER, BRADLEY K.; CHRISTENSEN, SØREN T.

    2010-01-01

    SUMMARY Although first described 1898 and long considered a vestigial organelle of little functional importance, the primary cilium has become one of the hottest research topics in modern cell biology and physiology. Primary cilia are non-motile sensory organelles present in a single copy on the surface of most growth-arrested or differentiated mammalian cells, and defects in their assembly or function are tightly coupled to many developmental defects, diseases and disorders. In normal tissues the primary cilium coordinates a series of signal transduction pathways, including Hedgehog, Wnt, PDGFRα and integrin signaling. In the kidney the primary cilium may function as a mechano-, chemo- and osmosensing unit that probes the extracellular environment and transmits signals to the cell via e.g. polycystins, which depend on ciliary localization for appropriate function. Indeed, hypomorphic mutations in the mouse ift88 (previously called Tg737) gene, which encodes a ciliogenic intraflagellar transport (IFT) protein, result in malformation of primary cilia, and in the collecting ducts of kidney tubules this is accompanied by development of autosomal recessive polycystic kidney disease (PKD; (1)). While PKD was one of the first diseases to be linked to dysfunctional primary cilia, defects in this organelle have subsequently been associated with many other phenotypes, including cancer, obesity, diabetes as well as a number of developmental defects. Collectively, these disorders of the cilium are now referred to as the ciliopathies. In this review we provide a brief overview of the structure and function of primary cilia and some of their roles in coordinating signal transduction pathways in mammalian development, health and disease. This review was written in conjunction with the Takis Anagnostopoulos Symposium on Renal and Epithelial Physiology and Physiopathology at Faculté de Médecine Necker in Paris, June 26-27, 2008. PMID:19276629

  12. Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria.

    PubMed

    Kawahara, Manabu; Koyama, Shiori; Iimura, Satomi; Yamazaki, Wataru; Tanaka, Aiko; Kohri, Nanami; Sasaki, Keisuke; Takahashi, Masashi

    2015-09-29

    Mitochondria, cellular organelles playing essential roles in eukaryotic cell metabolism, are thought to have evolved from bacteria. The organization of mtDNA is remarkably uniform across species, reflecting its vital and conserved role in oxidative phosphorylation (OXPHOS). Our objectives were to evaluate the compatibility of xenogeneic mitochondria in the development of preimplantation embryos in mammals. Mouse embryos harbouring bovine mitochondria (mtB-M embryos) were prepared by the cell-fusion technique employing the haemagglutinating virus of Japan (HVJ). The mtB-M embryos showed developmental delay at embryonic days (E) 3.5 after insemination. Furthermore, none of the mtB-M embryos could implant into the maternal uterus after embryo transfer, whereas control mouse embryos into which mitochondria from another mouse had been transferred developed as well as did non-manipulated embryos. When we performed quantitative PCR (qPCR) of mouse and bovine ND5, we found that the mtB-M embryos contained 8.3% of bovine mitochondria at the blastocyst stage. Thus, contamination with mitochondria from another species induces embryonic lethality prior to implantation into the maternal uterus. The heteroplasmic state of these xenogeneic mitochondria could have detrimental effects on preimplantation development, leading to preservation of species-specific mitochondrial integrity in mammals.

  13. Neurotrophins and their receptors in early development of the mammalian nervous system.

    PubMed

    Bartkowska, Katarzyna; Turlejski, Kris; Djavadian, Rouzanna L

    2010-01-01

    Neurotrophins belonging to the class of growth factors and including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are widely recognized as essential factors in the developing central nervous system (CNS). Neurotrophins are synthesized as precursor forms (proneurotrophins). Mature forms of neurotrophins exert their effect by binding to specific tyrosine kinases receptors (TrkA, TrkB and TrkC) as well as via the p75 receptor, a member of the tumor necrosis factor receptor superfamily while proneurotrophins interact with the receptor p75 or co-receptor complex of p75 and sortilin, that is a Vps10p domain-containing transmembrane protein. Expression of neurotrophins corresponds with the onset of neurogenesis in developing mammalian species. BDNF is low in early embryonic stages of development, while NT-3 highly expresses in the developing CNS. Expression of neurotrophins receptors mainly overlaps at early development. Data concerning early distribution of neurotrophins and their receptors in the nervous system and results in mice with targeted disruptions of neurotrophin or receptor genes show that neurotrophins and their receptors play distinct roles in control and regulation of the most crucial developmental processes such as proliferation, migration, differentiation, survival, apoptosis and synaptic plasticity.

  14. Practices and ethical concerns regarding preimplantation diagnosis. Who regulates preimplantation genetic diagnosis in Brazil?

    PubMed Central

    Damian, B.B.; Bonetti, T.C.S.; Horovitz, D.D.G.

    2014-01-01

    Preimplantation genetic diagnosis (PGD) was originally developed to diagnose embryo-related genetic abnormalities for couples who present a high risk of a specific inherited disorder. Because this technology involves embryo selection, the medical, bioethical, and legal implications of the technique have been debated, particularly when it is used to select features that are not related to serious diseases. Although several initiatives have attempted to achieve regulatory harmonization, the diversity of healthcare services available and the presence of cultural differences have hampered attempts to achieve this goal. Thus, in different countries, the provision of PGD and regulatory frameworks reflect the perceptions of scientific groups, legislators, and society regarding this technology. In Brazil, several texts have been analyzed by the National Congress to regulate the use of assisted reproduction technologies. Legislative debates, however, are not conclusive, and limited information has been published on how PGD is specifically regulated. The country requires the development of new regulatory standards to ensure adequate access to this technology and to guarantee its safe practice. This study examined official documents published on PGD regulation in Brazil and demonstrated how little direct oversight of PGD currently exists. It provides relevant information to encourage reflection on a particular regulation model in a Brazilian context, and should serve as part of the basis to enable further reform of the clinical practice of PGD in the country. PMID:25493379

  15. Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney

    PubMed Central

    Nagalakshmi, Vidya K.; Ren, Qun; Pugh, Margaret M.; Valerius, M. Todd; McMahon, Andrew P.; Yu, Jing

    2011-01-01

    MicroRNAs (miRNAs) are a large and growing class of small, non-coding, regulatory RNAs that control gene expression predominantly at the post-transcriptional level. The production of most functional miRNAs depends on the enzymatic activity of Dicer, an RNase III class enzyme. To address the potential action of Dicer-dependent miRNAs in mammalian kidney development, we conditionally ablated Dicer function within cells of nephron lineage and the ureteric bud-derived collecting duct system. Six2Cre-mediated removal of Dicer activity from the progenitors of the nephron epithelium led to elevated apoptosis and premature termination of nephrogenesis. Thus, Dicer action is important for maintaining the viability of this critical self-renewing progenitor pool and, consequently, development of a normal nephron complement. HoxB7Cre-mediated removal of Dicer function from the ureteric bud epithelium led to the development of renal cysts. This was preceded by excessive cell proliferation and apoptosis, and accompanied by disrupted ciliogenesis within the ureteric bud epithelium. Dicer removal also disrupted branching morphogenesis with the phenotype correlating with downregulation of Wnt11 and c-Ret expression at ureteric tips. Thus Dicer, and by inference Dicer-dependent miRNA activity, have distinct regulatory roles within different components of the developing mouse kidney. Furthermore, an understanding of miRNA action may provide new insights into the etiology and pathogenesis of renal cyst-based kidney disease. PMID:20944551

  16. Preimplantation Genetic Diagnosis for Stargardt Disease

    PubMed Central

    Sohrab, Mahsa A.; Allikmets, Rando; Guarnaccia, Michael M.; Smith, R. Theodore

    2010-01-01

    Purpose To report the first use of in vitro fertilization (IVF) and preimplantation genetic diagnosis to achieve an unaffected pregnancy in an autosomal-recessive retinal dystrophy. Design Case report. Methods An affected male with Stargardt disease and his carrier wife underwent IVF. Embryos obtained by intracytoplasmic sperm injection underwent single-cell DNA testing via polymerase chain reaction and restriction enzyme analysis to detect the presence of ABCA4 mutant alleles. Embryos were diagnosed as being either affected by or carriers for Stargardt disease. A single carrier embryo was implanted. Results Chorionic villus sampling performed during the first trimester verified that the fetus possessed only one mutant paternal allele and one normal maternal allele, thus making her an unaffected carrier of the disease. A healthy, live-born female was delivered. Conclusion IVF and preimplantation genetic diagnosis can assist couples with an affected spouse and a carrier spouse with recessive retinal dystrophies to have an unaffected child. PMID:20149343

  17. [Custom-made medicine: preimplantation genetic diagnosis].

    PubMed

    Sperling, Karl

    2006-01-01

    Who wants Preimplantation Genetic Diagnosis (PGD), who rejects it? The implications resulting from biological facts and legal regulations are addressed here. It is discussed under which conditions the restrictions of the Embryo Protection Act (Embryonenschutzgesetz, EschG) should be relaxed in order to be able to perform assisted reproduction technologies in Germany under international quality standards and also PGD in cases of stringent medical-genetic conditions.

  18. [Progress in proteomics of mammalian oocyte and early embryo].

    PubMed

    Chen, Lingsheng; Xu, Ping; Shi, Deshun; Li, Xiangping

    2014-07-01

    The development of female germ cell is the cornerstone for animal reproduction. Mammalian oocyte and early embryo have many distinct phenomena and mechanisms during their growth and development, involving series dynamic changes of protein synthesis/degradation and phosphorylation. Research on the regulatory mechanism of oocyte division, maturation, and developmental principle of pre-implantation embryo is an important topic in the field of animal developmental biology. Proteomics using all of proteins expressed by a cell or tissue as research object, systematically identify, quantify and study the function of all these proteins. With the rapid development of protein separation and identification technology, proteomics provide some new methods and the research contents on fields of oogenesis, differentiation, maturation and quality control, such as protein quantification, modification, location and interaction important information which other omics technology can not provide. These information will contribute to uncover the molecular mechanisms of mammalian oocyte maturation and embryonic development. And it is great significant for improving the culture system of oocyte in vitro maturation, the efficiency of embryo production in vitro, somatic cell clone and transgenic animal production.

  19. Preimplantation genetic diagnosis guided by single-cell genomics

    PubMed Central

    2013-01-01

    Preimplantation genetic diagnosis (PGD) aims to help couples with heritable genetic disorders to avoid the birth of diseased offspring or the recurrence of loss of conception. Following in vitro fertilization, one or a few cells are biopsied from each human preimplantation embryo for genetic testing, allowing diagnosis and selection of healthy embryos for uterine transfer. Although classical methods, including single-cell PCR and fluorescent in situ hybridization, enable PGD for many genetic disorders, they have limitations. They often require family-specific designs and can be labor intensive, resulting in long waiting lists. Furthermore, certain types of genetic anomalies are not easy to diagnose using these classical approaches, and healthy offspring carrying the parental mutant allele(s) can result. Recently, state-of-the-art methods for single-cell genomics have flourished, which may overcome the limitations associated with classical PGD, and these underpin the development of generic assays for PGD that enable selection of embryos not only for the familial genetic disorder in question, but also for various other genetic aberrations and traits at once. Here, we discuss the latest single-cell genomics methodologies based on DNA microarrays, single-nucleotide polymorphism arrays or next-generation sequence analysis. We focus on their strengths, their validation status, their weaknesses and the challenges for implementing them in PGD. PMID:23998893

  20. De novo DNA methylation during monkey pre-implantation embryogenesis

    PubMed Central

    Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

    2017-01-01

    Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis. PMID:28233770

  1. Kremen1 regulates mechanosensory hair cell development in the mammalian cochlea and the zebrafish lateral line

    PubMed Central

    Mulvaney, Joanna F.; Thompkins, Cathrine; Noda, Teppei; Nishimura, Koji; Sun, Willy W.; Lin, Shuh-Yow; Coffin, Allison; Dabdoub, Alain

    2016-01-01

    Here we present spatio-temporal localization of Kremen1, a transmembrane receptor, in the mammalian cochlea, and investigate its role in the formation of sensory organs in mammal and fish model organisms. We show that Kremen1 is expressed in prosensory cells during cochlear development and in supporting cells of the adult mouse cochlea. Based on this expression pattern, we investigated whether Kremen1 functions to modulate cell fate decisions in the prosensory domain of the developing cochlea. We used gain and loss-of-function experiments to show that Kremen1 is sufficient to bias cells towards supporting cell fate, and is implicated in suppression of hair cell formation. In addition to our findings in the mouse cochlea, we examined the effects of over expression and loss of Kremen1 in the zebrafish lateral line. In agreement with our mouse data, we show that over expression of Kremen1 has a negative effect on the number of mechanosensory cells that form in the zebrafish neuromasts, and that fish lacking Kremen1 protein develop more hair cells per neuromast compared to wild type fish. Collectively, these data support an inhibitory role for Kremen1 in hair cell fate specification. PMID:27550540

  2. Transitory cystic cavities in the developing mammalian brain - normal or anomalous?

    PubMed

    Kaur, Charanjit; Ling, Eng-Ang

    2017-02-01

    Transitory cavities associated with the ventricular system represent probably one of the most unique features in the developing mammalian brain. In rodents, the cavities exist transiently in the developing brain and do not appear to be associated with any pathological events. Among the various cavities, the pyramidal-shaped cavum septum pellucidum (CSP) located beneath the corpus callosum and between the lateral ventricles is most well defined. In addition to the CSP are the bilateral subependymal cysts that are consistently associated with the third and fourth ventricles as well as the aqueduct. The cavities/cysts contain a large number of amoeboid microglia expressing surface receptors and hydrolytic enzymes common to tissue macrophages. The significance of these cavities in the developing brain remains a conjecture. Firstly, the cavity walls are free of an apparent epithelial lining; hence, it is speculated that the cavities that appear to communicate with the widened neighboring interstitial tissue spaces may have resulted from physical traction due to the rapid growth of the perinatal brain. Secondly, the cavities contain prominent clusters of amoeboid microglia that may be involved in clearing the debris of degenerating axons and cells resulting from the early brain tissue remodeling. With the increase in brain tissue compactness following the beginning of myelination in the second postnatal week, all cavities are obliterated; concomitantly, the number of amoeboid microglia in them diminishes and all this might signal further maturation of the brain.

  3. Master regulators in development: Views from the Drosophila retinal determination and mammalian pluripotency gene networks.

    PubMed

    Davis, Trevor L; Rebay, Ilaria

    2017-01-15

    Among the mechanisms that steer cells to their correct fate during development, master regulatory networks are unique in their sufficiency to trigger a developmental program outside of its normal context. In this review we discuss the key features that underlie master regulatory potency during normal and ectopic development, focusing on two examples, the retinal determination gene network (RDGN) that directs eye development in the fruit fly and the pluripotency gene network (PGN) that maintains cell fate competency in the early mammalian embryo. In addition to the hierarchical transcriptional activation, extensive positive transcriptional feedback, and cooperative protein-protein interactions that enable master regulators to override competing cellular programs, recent evidence suggests that network topology must also be dynamic, with extensive rewiring of the interactions and feedback loops required to navigate the correct sequence of developmental transitions to reach a final fate. By synthesizing the in vivo evidence provided by the RDGN with the extensive mechanistic insight gleaned from the PGN, we highlight the unique regulatory capabilities that continual reorganization into new hierarchies confers on master control networks. We suggest that deeper understanding of such dynamics should be a priority, as accurate spatiotemporal remodeling of network topology will undoubtedly be essential for successful stem cell based therapeutic efforts.

  4. Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy

    PubMed Central

    Uno, Narumi; Uno, Katsuhiro; Komoto, Shinya; Suzuki, Teruhiko; Hiratsuka, Masaharu; Osaki, Mitsuhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo

    2015-01-01

    The development of a safeguard system to remove tumorigenic cells would allow safer clinical applications of stem cells for the treatment of patients with an intractable disease including genetic disorders. Such safeguard systems should not disrupt the host genome and should have long-term stability. Here, we attempted to develop a tumor-suppressing mammalian artificial chromosome containing a safeguard system that uses the immune rejection system against allogeneic tissue from the host. For proof-of-concept of the safeguard system, B16F10 mouse melanoma cells expressing the introduced H2-K(d) major histocompatibility complex (MHC class I)-allogenic haplotype were transplanted into recipient C57BL/6J mice expressing MHC H2-K(b). Subcutaneous implantation of B16F10 cells into C57BL/6J mice resulted in high tumorigenicity. The volume of tumors derived from B16F10 cells expressing allogenic MHC H2-K(d) was decreased significantly (P < 0.01). Suppression of MHC H2-K(d)-expressing tumors in C57BL/6J mice was enhanced by immunization with MHC H2-K(d)-expressing splenocytes (P < 0.01). These results suggest that the safeguard system is capable of suppressing tumor formation by the transplanted cells. PMID:26670279

  5. Involvement of miRNAs and Cell-Secreted Vesicles in Mammalian Ovarian Antral Follicle Development.

    PubMed

    da Silveira, Juliano C; de Andrade, Gabriella M; Nogueira, Marcelo F G; Meirelles, Flávio V; Perecin, Felipe

    2015-12-01

    Ovarian follicular development is a controlled series of events culminating with an ovulatory or atretic follicle. MicroRNAs (miRNAs) are small noncoding RNAs involved in translational regulation of genes in different developmental processes. Deletion of Dicer in mice ovaries demonstrated the importance of miRNAs in reproduction, which led to infertility. The miRNAs were thought to act only within host cells; however, these molecules are also present in cell-secreted vesicles. These vesicles are present in body fluids such as milk, serum, and ovarian follicular fluid. Vesicles are secreted in extracellular fluids and travel from donor to target cells, mediating transfer of bioactive material. Herein we discuss the role of hormonal-regulated miRNAs within different ovarian follicular cells as well as cell-secreted vesicles participation in mammalian ovarian follicular fluid. Furthermore, we discuss the possibility of miRNAs transference mediated by cell-secreted vesicles present in ovarian follicular fluid, increasing the versatility of miRNA functions during antral follicle development. © The Author(s) 2015.

  6. Mammalian Brain Development is Accompanied by a Dramatic Increase in Bipolar DNA Methylation

    PubMed Central

    Sun, Ming-an; Sun, Zhixiong; Wu, Xiaowei; Rajaram, Veena; Keimig, David; Lim, Jessica; Zhu, Hongxiao; Xie, Hehuang

    2016-01-01

    DNA methylation is an epigenetic mechanism critical for tissue development and cell specification. Mammalian brains consist of many different types of cells with assumedly distinct DNA methylation profiles, and thus some genomic loci may demonstrate bipolar DNA methylation pattern, i.e. hypermethylated in one cell subset but hypomethylated in others. Currently, how extensive methylation patterns vary among brain cells is unknown and bipolar methylated genomic loci remain largely unexplored. In this study, we implemented a procedure to infer cell-subset specific methylated (CSM) loci from the methylomes of human and mouse frontal cortices at different developmental stages. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Correlated with enhancer-associated histone modifications, putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. Altogether, this study provides a procedure to identify genomic regions showing methylation differences in a mixed cell population and our results suggest that a set of cis-regulatory elements are primed in early postnatal life whose functions may be compromised in human neurological disorders. PMID:27585862

  7. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo.

    PubMed

    Watson, A J; Hogan, A; Hahnel, A; Wiemer, K E; Schultz, G A

    1992-02-01

    The sensitive technique of mRNA phenotyping with the reverse transcription-polymerase chain reaction was employed to determine the patterns of gene expression for several growth factor ligand and receptor genes during bovine preimplantation development. Several thousand bovine embryos encompassing a developmental series from one-cell zygotes to hatched blastocysts were produced by the application of in vitro maturation, fertilization, and oviductal epithelial cell embryo coculture methods. Transcripts for transforming growth factor (TGF-alpha) and platelet-derived growth factor (PDGF-A) are detectable in all preimplantation bovine stages as observed in the mouse. Transcripts for TGF-beta 2 and insulin-like growth factor (IGF-II) and the receptors for PDGF-alpha, insulin, IGF-I, and IGF-II are also detectable throughout bovine preimplantation development, suggesting that these mRNAs are products of both the maternal and the embryonic genomes in the cow, whereas in the mouse they are present only following the activation of the embryonic genome at the two-cell stage. In contrast to the mouse embryo, IGF-I mRNA was detected within preimplantation bovine embryos. Basic fibroblast growth factor (bFGF) is a maternal message in the bovine embryo, since it is only detectable up until the eight-cell embryo stage. Bovine trophoblast protein (bTP) mRNA was detectable within day 8 bovine blastocysts. As was observed in the mouse, the transcripts for insulin, epidermal growth factor (EGF), or nerve growth factor (NGF) were not detectable in any bovine embryo stage. Analyses of this type should aid the development of a completely defined culture medium for the more efficient production of preimplantation bovine embryos.

  8. Expanding the test set: Chemicals with potential to disrupt mammalian brain development.

    PubMed

    Mundy, William R; Padilla, Stephanie; Breier, Joseph M; Crofton, Kevin M; Gilbert, Mary E; Herr, David W; Jensen, Karl F; Radio, Nicholas M; Raffaele, Kathleen C; Schumacher, Kelly; Shafer, Timothy J; Cowden, John

    2015-01-01

    High-throughput test methods including molecular, cellular, and alternative species-based assays that examine critical events of normal brain development are being developed for detection of developmental neurotoxicants. As new assays are developed, a "training set" of chemicals is used to evaluate the relevance of individual assays for specific endpoints. Different training sets are necessary for each assay that would comprise a developmental neurotoxicity test battery. In contrast, evaluation of the predictive ability of a comprehensive test battery requires a set of chemicals that have been shown to alter brain development after in vivo exposure ("test set"). Because only a small number of substances have been well documented to alter human neurodevelopment, we have proposed an expanded test set that includes chemicals demonstrated to adversely affect neurodevelopment in animals. To compile a list of potential developmental neurotoxicants, a literature review of compounds that have been examined for effects on the developing nervous system was conducted. The search was limited to mammalian studies published in the peer-reviewed literature and regulatory studies submitted to the U.S. EPA. The definition of developmental neurotoxicity encompassed changes in behavior, brain morphology, and neurochemistry after gestational or lactational exposure. Reports that indicated developmental neurotoxicity was observed only at doses that resulted in significant maternal toxicity or were lethal to the fetus or offspring were not considered. As a basic indication of reproducibility, we only included a chemical if data on its developmental neurotoxicity were available from more than one laboratory (defined as studies originating from laboratories with a different senior investigator). Evidence from human studies was included when available. Approximately 100 developmental neurotoxicity test set chemicals were identified, with 22% having evidence in humans. Published by Elsevier

  9. Evidence Supporting a Functional Requirement of SMAD4 for Bovine Preimplantation Embryonic Development: A Potential Link to Embryotrophic Actions of Follistatin1

    PubMed Central

    Lee, Kyung-Bon; Zhang, Kun; Folger, Joseph K.; Knott, Jason G.; Smith, George W.

    2014-01-01

    ABSTRACT Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent. PMID:25031360

  10. Evidence supporting a functional requirement of SMAD4 for bovine preimplantation embryonic development: a potential link to embryotrophic actions of follistatin.

    PubMed

    Lee, Kyung-Bon; Zhang, Kun; Folger, Joseph K; Knott, Jason G; Smith, George W

    2014-09-01

    Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.

  11. Addition of erythrocytes to in vitro culture medium attenuates the detrimental effects of reactive oxygen species on bovine preimplantation embryo development.

    PubMed

    Ideta, Atsushi; Tsuchiya, Kanami; Aoyagi, Yoshito

    2012-01-01

    Erythrocytes were recently found to improve the early development of mice embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine in vitro fertilized (IVF) embryos in medium supplemented with reactive oxygen species (ROS). IVF embryos were cultured in CR1aa medium supplemented with oxidizing agents, 0.5mmol/L hypoxanthine and 0.01U/mL xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5×10(4) , 5×10(5) , 5×10(6) and 5×10(7) erythrocytes/mL). After 8 days, blastocysts were examined with a stereomicroscope. HX/XOD blocked development to the blastocyst stage (HX/XOD: 0%, control: 33%), but in the presence of both erythrocytes and HX/XOD, blastocyst development was restored to about one-third to two-thirds the normal rate (5×10(5) to 5×10(7) erythrocytes/mL: 12 to 23%). Furthermore, adding erythrocytes or erythrocyte hemolysate to medium without HX/XOD increased the blastocyst rate. These results suggest that the addition of erythrocytes can attenuate the detrimental effects of ROS on embryo development in bovine species as well as in mice.

  12. Actions of activin A, connective tissue growth factor, hepatocyte growth factor and teratocarcinoma-derived growth factor 1 on the development of the bovine preimplantation embryo.

    PubMed

    Kannampuzha-Francis, Jasmine; Tribulo, Paula; Hansen, Peter J

    2016-05-17

    The reproductive tract secretes bioactive molecules collectively known as embryokines that can regulate embryonic growth and development. In the present study we tested four growth factors expressed in the endometrium for their ability to modify the development of the bovine embryo to the blastocyst stage and alter the expression of genes found to be upregulated (bone morphogenetic protein 15 (BMP15) and keratin 8, type II (KRT8)) or downregulated (NADH dehydrogenase 1 (ND1) and S100 calcium binding protein A10 (S100A10)) in embryos competent to develop to term. Zygotes were treated at Day 5 with 0.01, 0.1 or 1.0 nM growth factor. The highest concentration of activin A increased the percentage of putative zygotes that developed to the blastocyst stage. Connective tissue growth factor (CTGF) increased the number of cells in the inner cell mass (ICM), decreased the trophectoderm : ICM ratio and increased blastocyst expression of KRT8 and ND1. The lowest concentration of hepatocyte growth factor (HGF) reduced the percentage of putative zygotes becoming blastocysts. Teratocarcinoma-derived growth factor 1 increased total cell number at 0.01 nM and expression of S100A10 at 1.0 nM, but otherwise had no effects. Results confirm the prodevelopmental actions of activin A and indicate that CTGF may also function as an embryokine by regulating the number of ICM cells in the blastocyst and altering gene expression. Low concentrations of HGF were inhibitory to development.

  13. Analysis of gene–environment interactions in postnatal development of the mammalian intestine

    PubMed Central

    Rakoff-Nahoum, Seth; Kong, Yong; Kleinstein, Steven H.; Subramanian, Sathish; Ahern, Philip P.; Gordon, Jeffrey I.; Medzhitov, Ruslan

    2015-01-01

    Unlike mammalian embryogenesis, which takes place in the relatively predictable and stable environment of the uterus, postnatal development can be affected by a multitude of highly variable environmental factors, including diet, exposure to noxious substances, and microorganisms. Microbial colonization of the intestine is thought to play a particularly important role in postnatal development of the gastrointestinal, metabolic, and immune systems. Major changes in environmental exposure occur right after birth, upon weaning, and during pubertal maturation into adulthood. These transitions include dramatic changes in intestinal contents and require appropriate adaptations to meet changes in functional demands. Here, we attempt to both characterize and provide mechanistic insights into postnatal intestinal ontogeny. We investigated changes in global intestinal gene expression through postnatal developmental transitions. We report profound alterations in small and large intestinal transcriptional programs that accompany both weaning and puberty in WT mice. Using myeloid differentiation factor 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) double knockout littermates, we define the role of toll-like receptors (TLRs) and interleukin (IL)-1 receptor family member signaling in postnatal gene expression programs and select ontogeny-specific phenotypes, such as vascular and smooth muscle development and neonatal epithelial and mast cell homeostasis. Metaanalysis of the effect of the microbiota on intestinal gene expression allowed for mechanistic classification of developmentally regulated genes by TLR/IL-1R (TIR) signaling and/or indigenous microbes. We find that practically every aspect of intestinal physiology is affected by postnatal transitions. Developmental timing, microbial colonization, and TIR signaling seem to play distinct and specific roles in regulation of gene-expression programs throughout postnatal development. PMID:25691701

  14. Epigenetic regulation of Atoh1 guides hair cell development in the mammalian cochlea

    PubMed Central

    Stojanova, Zlatka P.; Kwan, Tao; Segil, Neil

    2015-01-01

    In the developing cochlea, sensory hair cell differentiation depends on the regulated expression of the bHLH transcription factor Atoh1. In mammals, if hair cells die they do not regenerate, leading to permanent deafness. By contrast, in non-mammalian vertebrates robust regeneration occurs through upregulation of Atoh1 in the surviving supporting cells that surround hair cells, leading to functional recovery. Investigation of crucial transcriptional events in the developing organ of Corti, including those involving Atoh1, has been hampered by limited accessibility to purified populations of the small number of cells present in the inner ear. We used µChIP and qPCR assays of FACS-purified cells to track changes in the epigenetic status of the Atoh1 locus during sensory epithelia development in the mouse. Dynamic changes in the histone modifications H3K4me3/H3K27me3, H3K9ac and H3K9me3 reveal a progression from poised, to active, to repressive marks, correlating with the onset of Atoh1 expression and its subsequent silencing during the perinatal (P1 to P6) period. Inhibition of acetylation blocked the increase in Atoh1 mRNA in nascent hair cells, as well as ongoing hair cell differentiation during embryonic organ of Corti development ex vivo. These results reveal an epigenetic mechanism of Atoh1 regulation underlying hair cell differentiation and subsequent maturation. Interestingly, the H3K4me3/H3K27me3 bivalent chromatin structure observed in progenitors persists at the Atoh1 locus in perinatal supporting cells, suggesting an explanation for the latent capacity of these cells to transdifferentiate into hair cells, and highlighting their potential as therapeutic targets in hair cell regeneration. PMID:26487780

  15. Analysis of gene-environment interactions in postnatal development of the mammalian intestine.

    PubMed

    Rakoff-Nahoum, Seth; Kong, Yong; Kleinstein, Steven H; Subramanian, Sathish; Ahern, Philip P; Gordon, Jeffrey I; Medzhitov, Ruslan

    2015-02-17

    Unlike mammalian embryogenesis, which takes place in the relatively predictable and stable environment of the uterus, postnatal development can be affected by a multitude of highly variable environmental factors, including diet, exposure to noxious substances, and microorganisms. Microbial colonization of the intestine is thought to play a particularly important role in postnatal development of the gastrointestinal, metabolic, and immune systems. Major changes in environmental exposure occur right after birth, upon weaning, and during pubertal maturation into adulthood. These transitions include dramatic changes in intestinal contents and require appropriate adaptations to meet changes in functional demands. Here, we attempt to both characterize and provide mechanistic insights into postnatal intestinal ontogeny. We investigated changes in global intestinal gene expression through postnatal developmental transitions. We report profound alterations in small and large intestinal transcriptional programs that accompany both weaning and puberty in WT mice. Using myeloid differentiation factor 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) double knockout littermates, we define the role of toll-like receptors (TLRs) and interleukin (IL)-1 receptor family member signaling in postnatal gene expression programs and select ontogeny-specific phenotypes, such as vascular and smooth muscle development and neonatal epithelial and mast cell homeostasis. Metaanalysis of the effect of the microbiota on intestinal gene expression allowed for mechanistic classification of developmentally regulated genes by TLR/IL-1R (TIR) signaling and/or indigenous microbes. We find that practically every aspect of intestinal physiology is affected by postnatal transitions. Developmental timing, microbial colonization, and TIR signaling seem to play distinct and specific roles in regulation of gene-expression programs throughout postnatal development.

  16. Essential role of retinoblastoma protein in mammalian hair cell development and hearing.

    PubMed

    Sage, Cyrille; Huang, Mingqian; Vollrath, Melissa A; Brown, M Christian; Hinds, Philip W; Corey, David P; Vetter, Douglas E; Chen, Zheng-Yi

    2006-05-09

    The retinoblastoma protein pRb is required for cell-cycle exit of embryonic mammalian hair cells but not for their early differentiation. However, its role in postnatal hair cells is unknown. To study the function of pRb in mature animals, we created a new conditional mouse model, with the Rb gene deleted primarily in the inner ear. Progeny survive up to 6 months. During early postnatal development, pRb(-/-) hair cells continue to divide and can transduce mechanical stimuli. However, adult pRb(-/-) mice exhibit profound hearing loss due to progressive degeneration of the organ of Corti. We show that pRb is required for the full maturation of cochlear outer hair cells, likely in a gene-specific manner, and is also essential for their survival. In addition, lack of pRb results in cell division in postnatal auditory supporting cells. In contrast, many pRb(-/-) vestibular hair cells survive and continue to divide in adult mice. Significantly, adult pRb(-/-) vestibular hair cells are functional, and pRb(-/-) mice maintain partial vestibular function. Therefore, the functional adult vestibular pRb(-/-) hair cells, derived from proliferation of postnatal hair cells, are largely integrated into vestibular pathways. This study reveals essential yet distinct roles of pRb in cochlear and vestibular hair cell maturation, function, and survival and suggests that transient block of pRb function in mature hair cells may lead to propagation of functional hair cells.

  17. Axotomy of single fluorescent nerve fibers in developing mammalian spinal cord by photoconversion of diaminobenzidine.

    PubMed

    De-Miguel, Francisco F; Muller, Kenneth J; Adams, William B; Nicholls, John G

    2002-05-30

    A technique has been developed for cutting single nerve fibers in mammalian spinal cord. In the presence of diaminobenzidine (DAB), a laser microbeam was applied to carbocyanine (Dil) stained sensory fibers in cultured spinal cords of the newly born opossum Monodelphis domestica. Digital images of fluorescent fibers were acquired with an intensified video CCD-camera coupled to an image processor. Laser illumination of two spots on a fiber in the presence of 3 mg/ml DAB cut it, so that following DAB wash out, Dil fluorescence did not return after the intermediate segment was bleached. In contrast, when a similar procedure was carried out without DAB, fluorescence of the bleached segment was recovered within minutes in darkness, by dye diffusion from adjacent regions of the uncut fiber. After exposure to DAB, through-conduction of compound action potentials continued in undamaged fibers. The DAB reaction product remained as a dark precipitate, helping to localize the lesion sites. By illuminating a continuous series of spots it was possible to cut whole nerve roots. Fluorescent fibers extended across the cut segment 24 h later. With minor modifications, the procedure described here allows a precise lesioning of single fibers within an intact nervous system.

  18. Product quality considerations for mammalian cell culture process development and manufacturing.

    PubMed

    Gramer, Michael J

    2014-01-01

    The manufacturing of a biologic drug from mammalian cells results in not a single substance, but an array of product isoforms, also known as variants. These isoforms arise due to intracellular or extracellular events as a result of biological or chemical modification. The most common examples related to biomanufacturing include amino acid modifications (glycosylation, isomerization, oxidation, adduct formation, pyroglutamate formation, phosphorylation, sulfation, amidation), amino acid sequence variants (genetic mutations, amino acid misincorporation, N- and C-terminal heterogeneity, clipping), and higher-order structure modifications (misfolding, aggregation, disulfide pairing). Process-related impurities (HCP, DNA, media components, viral particles) are also important quality attributes related to product safety. The observed ranges associated with each quality attribute define the product quality profile. A biologic drug must have a correct and consistent quality profile throughout clinical development and scale-up to commercial production to ensure product safety and efficacy. In general, the upstream process (cell culture) defines the quality of product-related substances, whereas the downstream process (purification) defines the residual level of process- and product-related impurities. The purpose of this chapter is to review the impact of the cell culture process on product quality. Emphasis is placed on studies with industrial significance and where the direct mechanism of product quality impact was determined. Where possible, recommendations for maintaining consistent or improved quality are provided.

  19. Mechanisms of Long Non-coding RNAs in Mammalian Nervous System Development, Plasticity, Disease, and Evolution.

    PubMed

    Briggs, James A; Wolvetang, Ernst J; Mattick, John S; Rinn, John L; Barry, Guy

    2015-12-02

    Only relatively recently has it become clear that mammalian genomes encode tens of thousands of long non-coding RNAs (lncRNAs). A striking 40% of these are expressed specifically in the brain, where they show precisely regulated temporal and spatial expression patterns. This begs the question, what is the functional role of these many lncRNA transcripts in the brain? Here we canvass a growing number of mechanistic studies that have elucidated central roles for lncRNAs in the regulation of nervous system development and function. We also survey studies indicating that neurological and psychiatric disorders may ensue when these mechanisms break down. Finally, we synthesize these insights with evidence from comparative genomics to argue that lncRNAs may have played important roles in brain evolution, by virtue of their abundant sequence innovation in mammals and plausible mechanistic connections to the adaptive processes that occurred recently in the primate and human lineages. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Birth of a healthy infant following preimplantation PKHD1 haplotyping for autosomal recessive polycystic kidney disease using multiple displacement amplification

    PubMed Central

    Janson, Marleen M.; Roesler, Mark R.; Avner, Ellis D.; Strawn, Estil Y.; Bick, David P.

    2010-01-01

    Purpose To develop a reliable preimplantation genetic diagnosis protocol for couples who both carry a mutant PKHD1 gene wishing to conceive children unaffected with autosomal recessive polycystic kidney disease (ARPKD). Methods Development of a unique protocol for preimplantation genetic testing using whole genome amplification of single blastomeres by multiple displacement amplification (MDA), and haplotype analysis with novel short tandem repeat (STR) markers from the PKHD1 gene and flanking sequences, and a case report of successful utilization of the protocol followed by successful IVF resulting in the birth of an infant unaffected with ARPKD. Results We have developed 20 polymorphic STR markers suitable for linkage analysis of ARPKD. These linked STR markers have enabled unambiguous identification of the PKHD1 haplotypes of embryos produced by at-risk couples. Conclusions We have developed a reliable protocol for preimplantation genetic diagnosis of ARPKD using single-cell MDA products for PKHD1 haplotyping. PMID:20490649

  1. Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

    PubMed

    Vireque, A A; Camargo, L S A; Serapião, R V; Rosa E Silva, A A M; Watanabe, Y F; Ferreira, E M; Navarro, P A A S; Martins, W P; Ferriani, R A

    2009-03-01

    In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

  2. Survival implications of the development of behavioural responsiveness and awareness in different groups of mammalian young.

    PubMed

    Mellor, D J; Lentle, R G

    2015-05-01

    This paper focuses on the development of behaviours that are critical for the survival of newborn and juvenile mammals of veterinary and wider biological interest. It provides an updated, integrated and comparative analysis of how postnatal maturation of sensory, motor and perceptual capacities support and constrain behavioural interactions between mammalian young and the mother, any littermates and the environment. Young that are neurologically exceptionally immature, moderately immature and mature at birth are compared, and include, for example, marsupial joeys, rodent pups and ruminant offspring. Mothers in these three groups exhibit distinctive patterns of birthing and postnatal care behaviours. To secure survival of the young, maternal care must compensate for behavioural inadequacies imposed by the limited sensory capacities the young possess at each stage. These sensory capacities develop in a predictable sequence in most mammals such that before birth the sequence progresses to an extent that parallels the degree of neurological maturity reached at birth. The extent of neurological maturity is likewise reflected in how long it takes after birth for the necessary brain circuit connectivity to develop sufficiently to support cortically based cognitive modulation of behaviour. This takes several months, days-to-weeks or minutes-to-hours in young that are, respectively, neurologically exceptionally immature, moderately immature, or mature at birth. Once achieved, cognitive awareness confers a high degree of behavioural flexibility that allows the young to respond more effectively to the unpredictability of their postnatal environments. It is shown that the onset of this cognitively based flexibility in the young of each group coincides with their first exposure to a variable environment that requires such behavioural flexibility.

  3. Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

    PubMed

    Schroeder, Diane I; Jayashankar, Kartika; Douglas, Kory C; Thirkill, Twanda L; York, Daniel; Dickinson, Pete J; Williams, Lawrence E; Samollow, Paul B; Ross, Pablo J; Bannasch, Danika L; Douglas, Gordon C; LaSalle, Janine M

    2015-08-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo.

  4. The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development.

    PubMed

    Kim, Kyeoung-Hwa; Seo, You-Mi; Kim, Eun-Young; Lee, Su-Yeon; Kwon, Jini; Ko, Jung-Jae; Lee, Kyung-Ah

    2016-11-01

    Previously, we reported that Sebox is a new maternal effect gene (MEG) that is required for early embryo development beyond the two-cell (2C) stage because this gene orchestrates the expression of important genes for zygotic genome activation (ZGA). However, regulators of Sebox expression remain unknown. Therefore, the objectives of the present study were to use bioinformatics tools to identify such regulatory microRNAs (miRNAs) and to determine the effects of the identified miRNAs on Sebox expression. Using computational algorithms, we identified a motif within the 3'UTR of Sebox mRNA that is specific to the seed region of the miR-125 family, which includes miR-125a-5p, miR-125b-5p and miR-351-5p. During our search for miRNAs, we found that the Lin28a 3'UTR also contains the same binding motif for the seed region of the miR-125 family. In addition, we confirmed that Lin28a also plays a role as a MEG and affects ZGA at the 2C stage, without affecting oocyte maturation or fertilization. Thus, we provide the first report indicating that the miR-125 family plays a crucial role in regulating MEGs related to the 2C block and in regulating ZGA through methods such as affecting Sebox and Lin28a in oocytes and embryos. © 2016 The Authors.

  5. The miR-125 family is an important regulator of the expression and maintenance of maternal effect genes during preimplantational embryo development

    PubMed Central

    Kim, Kyeoung-Hwa; Seo, You-Mi; Kim, Eun-Young; Lee, Su-Yeon; Kwon, Jini; Ko, Jung-Jae

    2016-01-01

    Previously, we reported that Sebox is a new maternal effect gene (MEG) that is required for early embryo development beyond the two-cell (2C) stage because this gene orchestrates the expression of important genes for zygotic genome activation (ZGA). However, regulators of Sebox expression remain unknown. Therefore, the objectives of the present study were to use bioinformatics tools to identify such regulatory microRNAs (miRNAs) and to determine the effects of the identified miRNAs on Sebox expression. Using computational algorithms, we identified a motif within the 3′UTR of Sebox mRNA that is specific to the seed region of the miR-125 family, which includes miR-125a-5p, miR-125b-5p and miR-351-5p. During our search for miRNAs, we found that the Lin28a 3′UTR also contains the same binding motif for the seed region of the miR-125 family. In addition, we confirmed that Lin28a also plays a role as a MEG and affects ZGA at the 2C stage, without affecting oocyte maturation or fertilization. Thus, we provide the first report indicating that the miR-125 family plays a crucial role in regulating MEGs related to the 2C block and in regulating ZGA through methods such as affecting Sebox and Lin28a in oocytes and embryos. PMID:27906131

  6. Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus Scrofa).

    PubMed

    Zhang, Ting-Yu; Dai, Jian-Jun; Wu, Cai-Feng; Gu, Xiao-Long; Liu, Liang; Wu, Zhi-Qiang; Xie, Yi-Ni; Wu, Bin; Chen, Hui-Lan; Li, Yao; Chen, Xue-Jin; Zhang, De-Fu

    2012-02-01

    To optimize somatic cell nuclear transfer (SCNT) procedures in mini-pigs, the present study was designed to examine the effects of donor cell types and aphidicolin (APC) treatment on in vitro development of reconstructed embryos. Oviduct epithelial cells (OEC), ear fibroblast cells (EFC) and cumulus cells (CC) derived from mini-pigs were treated with serum starvation only or serum starvation followed by treatment of 0.1 µg/mL APC. The reconstructed embryos were cultured for 7 days to evaluate their developmental competency. Cleavage and blastocyst formation rates of reconstructed embryos derived from the OEC by APC treatment were significantly higher than the serum starvation (61.82% vs. 56.25%, 24.55% vs. 17.86%; P < 0.05). The cleavage rate from the EFC was significantly increased by APC treatment compared to serum starvation only (63.36% vs. 57.01%; P < 0.05). In the ooctyes with the CC, the reconstructed embryos could yield high blastocyst formation rate by APC treatment (29.63%; P < 0.05). In the presence of APC, CC gave rise to the highest cleavage and blastocyst formation rates among the three cell types. Therefore, our results suggest that treatment of CC with serum starvation plus APC prior to nuclear transfer is more suitable in SCNT of mini-pigs.

  7. The Turing-Child energy field as a driver of early mammalian development.

    PubMed

    Schiffmann, Yoram

    2008-09-01

    The equivalence of the early mammalian cells, of importance in assisted reproductive technologies (ART), is considered. It is suggested that this controversial topic can be settled by finding whether the cells are distinguished by the Turing-Child (TC) field, as expressed for example by patterns of mitochondrial activity. The division of the pronuclear embryo is driven by a symmetrical bipolar TC pattern whose experimental shape and chemical nature is predicted by TC theory. This bipolar pattern drives the subsequent cell divisions too, and according to present experimental results all cells are equivalent until compaction since they are not distinguished by the TC field in normal development. Interphase cells exhibit homogeneous mitochondrial activity, or perinuclear, or perinuclear and cortical activity, and these patterns too and the rotational symmetry observed are predicted by TC theory. The first differentiation, into an inner mass cell and the trophectoderm, as well as the formation of cell polarity in the trophectoderm are considered. It is suggested that these two events are driven by a peripheral spherical shell of high energy metabolism in the morula; such a shell is predicted by TC theory in a compacted multicellular sphere whose cells are connected by gap junctions. The experimental patterns of mitochondrial activity in unfertilized oocytes exhibit rotational symmetry or polarity. The shape and the chemical nature of these patterns also are predicted and explained by TC theory in a sphere. The change in the spatial pattern of mitochondrial activity with development is attributed to a change in the spatial pattern of mitochondrial activity and not to physical translocation of mitochondria. The experimental finding that these spatial patterns of mitochondrial activity are observed only in live and not in dead biological material is explained by the TC pattern being biology's unique and universal dissipative structure that requires ongoing specific

  8. FISH for pre-implantation genetic diagnosis.

    PubMed

    Scriven, Paul N; Kirby, Toby L; Ogilvie, Caroline Mackie

    2011-02-23

    Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

  9. Psychosocial development of full term singletons, born after preimplantation genetic diagnosis (PGD) at preschool age and family functioning: a prospective case-controlled study and multi-informant approach.

    PubMed

    Winter, C; Van Acker, F; Bonduelle, M; Desmyttere, S; Nekkebroeck, J

    2015-05-01

    Do full term singletons born after preimplantation genetic diagnosis (PGD) differ in their psychosocial functioning from children born after intracytoplasmic sperm injection (ICSI) and spontaneous conceived controls (SC)? The psychosocial maturation process of 5-6-year-old PGD children is comparable between the three conception groups (PGD, ICSI and SC). In general, a lot of research has been published regarding follow-up of children born after artificial reproductive technologies (ART), which mainly is reassuring. But the ART population itself is marked by broad diversity [IVF, ICSI, gamete donation, preimplantation genetic screening (PGS) or PGD] which complicates comparisons. Some literature concerning the socio-emotional development of PGD/PGS children is available and it suggests a normal maturation process. However, the complex reality of PGD families (e.g. safety of the technique and psychological burden of genetic histories) asks for an exclusive PGD sample with matched control groups and a multi-informant approach. Between April 2011 and May 2013, the psychosocial wellbeing of preschoolers and their families born after PGD was assessed in a prospective case-controlled, matched follow-up study, with a multi-informant approach. A group of 47 PGD, 50 ICSI and 55 SC 5-6-year-old children participated in a follow-up study performed at the Centre for Medical Genetics of the Universitair Ziekenhuis Brussel (UZ Brussel). Assessments took place in the hospital and in kindergartens. Children performed the Bene-Anthony family relations test (FRT), yielding their perceptions upon family relationships. Parents and teachers completed the child behaviour checklist (CBCL) and Caregiver Teacher Report Form (C-/TRF), respectively. Parental and family functioning were measured by the NEO-FFi, the parenting stress index (PSI), the Greenberger Work-Parenting Investment Questionnaire and the Marlowe-Crowne Social Desirability Scale (MCSDS). Statistical analysis was performed by

  10. New Advances of Preimplantation and Prenatal Genetic Screening and Noninvasive Testing as a Potential Predictor of Health Status of Babies

    PubMed Central

    2014-01-01

    The current morphologically based selection of human embryos for transfer cannot detect chromosome aneuploidies. So far, only biopsy techniques have been able to screen for chromosomal aneuploidies in the in vitro fertilization (IVF) embryos. Preimplantation genetic diagnosis (PGD) or screening (PGS) involves the biopsy of oocyte polar bodies or embryonic cells and has become a routine clinical procedure in many IVF clinics worldwide, including recent development of comprehensive chromosome screening of all 23 pairs of chromosomes by microarrays for aneuploidy screening. The routine preimplantation and prenatal genetic diagnosis (PND) require testing in an aggressive manner. These procedures may be invasive to the growing embryo and fetus and potentially could compromise the clinical outcome. Therefore the aim of this review is to summarize not only the new knowledge on preimplantation and prenatal genetic diagnosis in humans, but also on the development of potential noninvasive embryo and fetal testing that might play an important role in the future. PMID:24783200

  11. New advances of preimplantation and prenatal genetic screening and noninvasive testing as a potential predictor of health status of babies.

    PubMed

    Milachich, Tanya

    2014-01-01

    The current morphologically based selection of human embryos for transfer cannot detect chromosome aneuploidies. So far, only biopsy techniques have been able to screen for chromosomal aneuploidies in the in vitro fertilization (IVF) embryos. Preimplantation genetic diagnosis (PGD) or screening (PGS) involves the biopsy of oocyte polar bodies or embryonic cells and has become a routine clinical procedure in many IVF clinics worldwide, including recent development of comprehensive chromosome screening of all 23 pairs of chromosomes by microarrays for aneuploidy screening. The routine preimplantation and prenatal genetic diagnosis (PND) require testing in an aggressive manner. These procedures may be invasive to the growing embryo and fetus and potentially could compromise the clinical outcome. Therefore the aim of this review is to summarize not only the new knowledge on preimplantation and prenatal genetic diagnosis in humans, but also on the development of potential noninvasive embryo and fetal testing that might play an important role in the future.

  12. A Rosetta stone of mammalian genetics.

    PubMed

    Nadeau, J H; Grant, P L; Mankala, S; Reiner, A H; Richardson, J E; Eppig, J T

    1995-01-26

    The Mammalian Comparative Database provides genetic maps of mammalian species. Comparative maps are valuable aids for predicting linkages, developing animal models and studying genome organization and evolution.

  13. Inhibiting the Mammalian Target of Rapamycin Blocks the Development of Experimental Cerebral Malaria

    PubMed Central

    Gordon, Emile B.; Hart, Geoffrey T.; Tran, Tuan M.; Waisberg, Michael; Akkaya, Munir; Skinner, Jeff; Zinöcker, Severin; Pena, Mirna; Yazew, Takele; Qi, Chen-Feng; Miller, Louis H.

    2015-01-01

    ABSTRACT Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host’s immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. PMID:26037126

  14. Recent advances in preimplantation genetic diagnosis and screening.

    PubMed

    Lu, Lina; Lv, Bo; Huang, Kevin; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-09-01

    Preimplantation genetic diagnosis/screening (PGD/PGS) aims to help couples lower the risks of transmitting genetic defects to their offspring, implantation failure, and/or miscarriage during in vitro fertilization (IVF) cycles. However, it is still being debated with regard to the practicality and diagnostic accuracy of PGD/PGS due to the concern of invasive biopsy and the potential mosaicism of embryos. Recently, several non-invasive and high-throughput assays have been developed to help overcome the challenges encountered in the conventional invasive biopsy and low-throughput analysis in PGD/PGS. In this mini-review, we will summarize the recent progresses of these new methods for PGD/PGS and discuss their potential applications in IVF clinics.

  15. Preimplantation Genetic Testing in the 21st Century: Uncharted Territory

    PubMed Central

    Brezina, Paul R.

    2013-01-01

    The past hundred years have given birth to arguably the most profound changes in society, medicine, and technology the world has ever witnessed. Genetics is one such field that has enjoyed a meteoric rise during this time. Progressing from Mendelian genetics to the discovery of DNA to the ability to sequence the human genome, perhaps no other discipline holds more promise to affect future change than genetics. Technology currently exists to evaluate some of the genetic information held by developing embryos in the context of an in vitro fertilization (IVF) cycle. This information is then used to determine which embryos are selected for uterine transfer. Many societies have enacted legislation to protect against possible abuses utilizing this technology. However, it is incumbent upon society to continue ensuring that preimplantation genetic diagnosis (PGD)—and genetic testing in general—is applied in a way that utilizes its potential in a responsible manner to improve health care. PMID:24453515

  16. Preimplantation genetic testing in the 21st century: uncharted territory.

    PubMed

    Brezina, Paul R

    2013-02-10

    The past hundred years have given birth to arguably the most profound changes in society, medicine, and technology the world has ever witnessed. Genetics is one such field that has enjoyed a meteoric rise during this time. Progressing from Mendelian genetics to the discovery of DNA to the ability to sequence the human genome, perhaps no other discipline holds more promise to affect future change than genetics. Technology currently exists to evaluate some of the genetic information held by developing embryos in the context of an in vitro fertilization (IVF) cycle. This information is then used to determine which embryos are selected for uterine transfer. Many societies have enacted legislation to protect against possible abuses utilizing this technology. However, it is incumbent upon society to continue ensuring that preimplantation genetic diagnosis (PGD)-and genetic testing in general-is applied in a way that utilizes its potential in a responsible manner to improve health care.

  17. The roles of endoplasmic reticulum stress response in female mammalian reproduction.

    PubMed

    Yang, Yanzhou; Pei, Xiuying; Jin, Yaping; Wang, Yanrong; Zhang, Cheng

    2016-03-01

    Endoplasmic reticulum stress (ERS) activates a protective pathway, called the unfold protein response, for maintaining cellular homeostasis, but cellular apoptosis is triggered by excessive or persistent ERS. Several recent studies imply that the ERS response might have broader physiological roles in the various reproductive processes of female mammals, including embryo implantation, decidualization, preimplantation embryonic development, follicle atresia, and the development of the placenta. This review summarizes the existing data concerning the molecular and biological roles of the ERS response. The study of the functions of the ERS response in mammalian reproduction might provide novel insights into and an understanding of reproductive cell survival and apoptosis under physiological and pathological conditions. The ERS response is a novel signaling pathway for reproductive cell survival and apoptosis. Infertility might be a result of disturbing the ERS response during the process of female reproduction.

  18. Inhibiting the Mammalian target of rapamycin blocks the development of experimental cerebral malaria.

    PubMed

    Gordon, Emile B; Hart, Geoffrey T; Tran, Tuan M; Waisberg, Michael; Akkaya, Munir; Skinner, Jeff; Zinöcker, Severin; Pena, Mirna; Yazew, Takele; Qi, Chen-Feng; Miller, Louis H; Pierce, Susan K

    2015-06-02

    Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host's immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4(+) and CD8(+) T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection

  19. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    SciTech Connect

    Zhang, Pengpeng; Shan, Tizhong; Liang, Xinrong; Deng, Changyan; Kuang, Shihuan

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  20. Preimplantation genetic testing for Marfan syndrome.

    PubMed

    Harton, G L; Tsipouras, P; Sisson, M E; Starr, K M; Mahoney, B S; Fugger, E F; Schulman, J D; Kilpatrick, M W; Levinson, G; Black, S H

    1996-09-01

    Marfan syndrome (MFS) is an autosomal dominant disease that affects the skeletal, ocular and cardiovascular systems. Defects in the gene that codes for fibrillin (FBN-1) are responsible for MFS. Here we report the world's first use of preimplantation genetic testing (PGT) to achieve a clinical pregnancy and live birth of a baby free of a Marfan mutation. One or two blastomeres from each embryo were tested for a CA repeat within the FBN-1 gene. The prospective mother is homozygous for the CA repeat (2/2) and has two normal copies of the FBN-1 gene, while the prospective father is heterozygous for the CA repeat (1/2), and is affected with the Marfan syndrome. In the father's family, allele 2 segregates with the mutated FBN-1 gene. For PGT, any embryo diagnosed as heterozygous for the CA repeat (1/2) would be presumed to have inherited normal FBN-1 genes from the father and the mother and be unaffected. One in-vitro fertilization (IVF) cycle yielded 12 embryos for preimplantation testing; six of the embryos were heterozygous for the CA repeat (1/2) and presumed to be free of the Marfan mutation. Five of the six embryos were subsequently transferred into the uterus. The fetus was tested by chorionic villus sampling and found to be free of the Marfan mutation by the same linkage analysis, had a normal fetal echocardiogram, and was normal at birth.

  1. Update in preimplantation genetic diagnosis: successes, advances, and problems.

    PubMed

    Grifo, J A; Tang, Y X; Munne, S; Krey, L

    1996-04-01

    The field of preimplantation genetic diagnosis has undergone significant advances since the report of the first birth from this method in 1990. The first birth in the USA was reported in 1992, as was the first successful diagnosis and delivery of a baby free of a single gene defect disorder (cystic fibrosis and then Tay Sachs). Investigators have now reported approximately 40 births worldwide from preimplantation genetic diagnosis using the polymerase chain reaction and fluorescent in-situ hybridization methods to analyze single cells removed from early cleavage stage preimplantation embryos. The International Working Group on Preimplantation Genetics meets annually to discuss progress and pitfalls in this field. Although preimplantation genetic diagnosis offers hope to patients at risk of transmitting disease, there are many technical hazards of this experimental procedure. Technical difficulties must be overcome in order for preimplantation genetic diagnosis to become a standard clinical tool. This review will highlight some of the recent advances and problems in the field of preimplantation genetic diagnosis.

  2. The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution

    PubMed Central

    Bestor, Timothy H.; Woodfine, Kathryn; Lin, Chyuan-Sheng; Lin, Shau-Ping; Prissette, Marine; Oakey, Rebecca J.; Bourc'his, Déborah

    2010-01-01

    In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two

  3. Reactive oxygen species-mediated unfolded protein response pathways in preimplantation embryos

    PubMed Central

    Ali, Ihsan; Shah, Syed Zahid Ali; Jin, Yi; Li, Zhong-Shu; Ullah, Obaid

    2017-01-01

    Excessive production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress-mediated responses are critical to embryonic development in the challenging in vitro environment. ROS production increases during early embryonic development with the increase in protein requirements for cell survival and growth. The ER is a multifunctional cellular organelle responsible for protein folding, modification, and cellular homeostasis. ER stress is activated by a variety of factors including ROS. Such stress leads to activation of the adaptive unfolded protein response (UPR), which restores homeostasis. However, chronic stress can exceed the toleration level of the ER, resulting in cellular apoptosis. In this review, we briefly describe the generation and impact of ROS in preimplantation embryo development, the ROS-mediated activation mechanism of the UPR via the ER, and the subsequent activation of signaling pathways following ER stress in preimplantation embryos. PMID:28057903

  4. Effects of Simulated Weightlessness on Mammalian Development. Part 2: Meiotic Maturation of Mouse Oocytes During Clinostat Rotation

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1985-01-01

    In order to understand the role of gravity in basic cellular processes that are important during development, the effects of a simulated microgravity environment on mammalian gametes and early embryos cultured in vitro are examined. A microgravity environment is simulated by use of a clinostat, which essentially reorients cells relative to the gravity vector. Initial studies have focused on assessing the effects of clinostat rotation on the meiotic progression of mouse oocytes. Modifications centered on providing the unique in vitro culture of the clinostat requirements of mammalian oocytes and embryos: 37 C temperature, constant humidity, and a 5% CO2 in air environment. The oocytes are observed under the dissecting microscope for polar body formation and gross morphological appearance. They are then processed for cytogenetic analysis.

  5. Vitrified/warmed single blastocyst transfer in preimplantation genetic diagnosis/preimplantation genetic screening cycles.

    PubMed

    Huang, Jin; Li, Rong; Lian, Ying; Chen, Lixue; Shi, Xiaodan; Qiao, Jie; Liu, Ping

    2015-01-01

    To investigate the single blastocyst transfer in preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles. 80 PGD/PGS cycles undergoing blastocyst biopsy were studied. There were 88 warming cycles during the study period. Only one warmed blastocyst was transferred per cycle. The outcomes were followed up to the infants were born. The embryo implantation rate was 54.55% (48/88). The clinical pregnancy rate was 54.55% (48/88) per transfer cycle and 60% (48/80) per initial PGD/PGS cycle. There was no multi-pregnant in this study. The live birth rate was 42.05% (37/88) per transfer cycle and 46.25% (37/80) per initial PGD/PGS cycle. In PGD/PGS cycles, single blastocyst transfer reduces the multiple pregnancy rate without affecting the clinical outcomes.

  6. The interferon α-responsive gene, Ifrg15, plays vital roles during mouse early embryonic development.

    PubMed

    Yang, Ye; Wang, Jiayi; Zhao, Chun; Chen, Xiaojiao; Chen, Li; Zhang, Junqiang; Huo, Ran; Liu, Chang; Tong, Hua; Ling, Xiufeng

    2016-08-01

    The interferon alpha-responsive gene (Ifrg15) mRNA is highly expressed in various stages during preimplantation mammalian embryo development. Unfortunately, few studies have investigated the effect of Ifrg15 in this process. In mammals, the fusion of male and female pronuclei generates a diploid zygote, and is an important step for subsequent cleavage and blastocyst formation. Here, by using RNA interference, rescue experiments, immunofluorescence staining and live cell observations, we found that preimplantation embryo development was arrested at the 1-cell stage after knocking down Ifrg15 expression. This induced DNA damage and prevented the cleavage of embryos. Furthermore, the effect of Ifrg15 deficiency in arresting preimplantation embryo development produced by specific short interfering RNA microinjection was concentration-dependent. Using transcriptome expression profiles, gene ontogeny functional annotation and enrichment analysis, we gained 197 enriched pathways based on 1445 differentially expressed genes (DEGs). Of these, 12 pathways and about one third of the DEGs were involved in DNA damage, DNA repair, cell cycle, and developmental processes. Thus, the IFRG15 protein might be an important molecule for maintaining genomic integrity and stability through upregulating or downregulating a cascade of genes to permit normal preimplantation embryo development.

  7. Mammalian sleep

    NASA Astrophysics Data System (ADS)

    Staunton, Hugh

    2005-05-01

    This review examines the biological background to the development of ideas on rapid eye movement sleep (REM sleep), so-called paradoxical sleep (PS), and its relation to dreaming. Aspects of the phenomenon which are discussed include physiological changes and their anatomical location, the effects of total and selective sleep deprivation in the human and animal, and REM sleep behavior disorder, the latter with its clinical manifestations in the human. Although dreaming also occurs in other sleep phases (non-REM or NREM sleep), in the human, there is a contingent relation between REM sleep and dreaming. Thus, REM is taken as a marker for dreaming and as REM is distributed ubiquitously throughout the mammalian class, it is suggested that other mammals also dream. It is suggested that the overall function of REM sleep/dreaming is more important than the content of the individual dream; its function is to place the dreamer protagonist/observer on the topographical world. This has importance for the developing infant who needs to develop a sense of self and separateness from the world which it requires to navigate and from which it is separated for long periods in sleep. Dreaming may also serve to maintain a sense of ‘I’ness or “self” in the adult, in whom a fragility of this faculty is revealed in neurological disorders.

  8. [Advance in the methods of preimplantation genetic diagnosis for single gene diseases].

    PubMed

    Ren, Yixin; Qiao, Jie; Yan, Liying

    2017-06-10

    More than 7000 single gene diseases have been identified and most of them lack effective treatment. As an early form of prenatal diagnosis, preimplantation genetic diagnosis (PGD) is a combination of in vitro fertilization and genetic diagnosis. PGD has been applied in clinics for more than 20 years to avoid the transmission of genetic defects through analysis of embryos at early stages of development. In this paper, a review for the recent advances in PGD for single gene diseases is provided.

  9. [Update on preimplantation genetic diagnosis and screening].

    PubMed

    Kőrösi, Tamás; Török, Olga; Vajta, Gábor

    2014-08-31

    Recent advancement in both human embryology and genomics has created a completely new situation for practical and widespread application of preimplantation genetic diagnosis and screening with a dramatic effect on assisted reproduction. The mapping of the first human genome and the advancement in sequencing technology and bioinformatics has led to the discovery of the exact genetic background of exponentially increasing number of diseases. In parallel, methods for culturing human embryos have also radically improved, enabling the late transfer, and the procedure of vitrification the safe cryopreservation. In consequence, refined genetic analyses have become available from blastocyst biopsy followed by the application of novel genomic methods. Furthermore, some studies suggest that by the selection of aneuploid embryos the pregnancy- and birth-rates can be increased. The amount and the depth of information obtainable from the embryos raise several technical and ethical questions that can be answered by further prospective randomized trials.

  10. Preimplantation genetic diagnosis and the 'new' eugenics.

    PubMed Central

    King, D S

    1999-01-01

    Preimplantation genetic diagnosis (PID) is often seen as an improvement upon prenatal testing. I argue that PID may exacerbate the eugenic features of prenatal testing and make possible an expanded form of free-market eugenics. The current practice of prenatal testing is eugenic in that its aim is to reduce the numbers of people with genetic disorders. Due to social pressures and eugenic attitudes held by clinical geneticists in most countries, it results in eugenic outcomes even though no state coercion is involved. I argue that technological advances may soon make PID widely accessible. Because abortion is not involved, and multiple embryos are available, PID is radically more effective as a tool of genetic selection. It will also make possible selection on the basis of non-pathological characteristics, leading, potentially, to a full-blown free-market eugenics. For these reasons, I argue that PID should be strictly regulated. PMID:10226925

  11. Preimplantation genetic diagnosis to improve pregnancy outcomes in subfertility.

    PubMed

    Simpson, Joe Leigh

    2012-12-01

    Pre-implantation genetic diagnosis provides prenatal genetic diagnosis before implantation, thus allowing detection of chromosomal abnormalities and their exclusion from embryo transfer in assisted reproductive technologies. Polar body, blastomere or trophectoderm can each be used to obtain requisite genetic or embryonic DNA. Pre-implantation genetic diagnosis for excluding unbalanced translocations is well accepted, and pre-implantation genetic diagnosis aneuploidy testing to avoid repeated pregnancy losses in couples having recurrent aneuploidy is efficacious in reducing miscarriages. Controversy remains about whether pre-implantation genetic diagnosis aneuploidy testing improves take home pregnancy rates, for which reason adherence to specific indications is recommended while the issue is being adjudicated. Current recommendations are for obligatory 24 chromosome testing, most readily using array comparative genome hybridisation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos.

    PubMed

    Nishioka, Noriyuki; Yamamoto, Shinji; Kiyonari, Hiroshi; Sato, Hiroko; Sawada, Atsushi; Ota, Mitsunori; Nakao, Kazuki; Sasaki, Hiroshi

    2008-01-01

    During pre-implantation mouse development, embryos form blastocysts with establishment of the first two cell lineages: the trophectoderm (TE) which gives rise to the placenta, and the inner cell mass (ICM) which will form the embryo proper. Differentiation of TE is regulated by the transcription factor Caudal-related homeobox 2 (Cdx2), but the mechanisms which act upstream of Cdx2 expression remain unknown. Here we show that the TEA domain family transcription factor, Tead4, is required for TE development. Tead1, Tead2 and Tead4 were expressed in pre-implantation embryos, and at least Tead1 and Tead4 were expressed widely in both TE and ICM lineages. Tead4-/- embryos died at pre-implantation stages without forming the blastocoel. The mutant embryos continued cell proliferation, and adherens junction and cell polarity were not significantly affected. In Tead4-/- embryos, Cdx2 was weakly expressed at the morula stage but was not expressed in later stages. None of the TE specific genes, including Eomes and a Cdx2 independent gene, Fgfr2, was detected in Tead4-/- embryos. Instead, the ICM specific transcription factors, Oct3/4 and Nanog, were expressed in all the blastomeres. Tead4-/- embryos also failed to differentiate trophoblast giant cells when they were cultured in vitro. ES cells with normal in vitro differentiation abilities were established from Tead4-/- embryos. These results suggest that Tead4 has a distinct role from Tead1 and Tead2 in trophectoderm specification of pre-implantation embryos, and that Tead4 is an early transcription factor required for specification and development of the trophectoderm lineage, which includes expression of Cdx2.

  13. Comparison of gene expression in fresh and frozen-thawed human preimplantation embryos.

    PubMed

    Shaw, Lisa; Sneddon, Sharon F; Brison, Daniel R; Kimber, Susan J

    2012-11-01

    Identification and characterisation of differentially regulated genes in preimplantation human embryonic development are required to improve embryo quality and pregnancy rates in IVF. In this study, we examined expression of a number of genes known to be critical for early development and compared expression profiles in individual preimplantation human embryos to establish any differences in gene expression in fresh compared to frozen-thawed embryos used routinely in IVF. We analysed expression of 19 genes by cDNA amplification followed by quantitative real-time PCR in a panel of 44 fresh and frozen-thawed human preimplantation embryos. Fresh embryos were obtained from surplus early cleavage stage embryos and frozen-thawed embryos from cryopreserved 2PN embryos. Our aim was to determine differences in gene expression between fresh and frozen-thawed human embryos, but we also identified differences in developmental expression patterns for particular genes. We show that overall gene expression among embryos of the same stage is highly variable and our results indicate that expression levels between groups did differ and differences in expression of individual genes was detected. Our results show that gene expression from frozen-thawed embryos is more consistent when compared with fresh, suggesting that cryopreserved embryos may represent a reliable source for studying the molecular events underpinning early human embryo development.

  14. Following the course of pre-implantation embryo patterning by non-linear microscopy.

    PubMed

    Kyvelidou, Christiana; Tserevelakis, George J; Filippidis, George; Ranella, Anthi; Kleovoulou, Anastasia; Fotakis, Costas; Athanassakis, Irene

    2011-12-01

    Embryo patterning is subject to intense investigation. So far only large, microscopically obvious structures like polar body, cleavage furrow, pro-nucleus shape can be evaluated in the intact embryo. Using non-linear microscopic techniques, the present work describes new methodologies to evaluate pre-implantation mouse embryo patterning. Third Harmonic Generation (THG) imaging, by detecting mitochondrial/lipid body structures, could provide valuable and complementary information as to the energetic status of pre-implantation embryos, time evolution of different developmental stages, embryo polarization prior to mitotic division and blastomere equivalence. Quantification of THG imaging detected highest signalling in the 2-cell stage embryos, while evaluating a 12-18% difference between blastomeres at the 8-cell stage embryos. Such a methodology provides novel, non-intrusive imaging assays to follow up intracellular structural patterning associated with the energetic status of a developing embryo, which could be successfully used for embryo selection during the in vitro fertilization process.

  15. In vitro maturation and in vitro fertilization of mouse oocytes and preimplantation embryo culture.

    PubMed

    Kidder, Benjamin L

    2014-01-01

    Epigenetic regulation of gene expression in the germline is important for reproductive success of mammals. Misregulation of genes whose expression is correlated with reproductive success may result in subfertility or infertility. To study epigenetic events that occur during oocyte maturation and preimplantation embryo development, it is important to generate large numbers of ovarian follicles and embryos. Oocyte maturation can be modeled using in vitro maturation (IVM), which is a system of maturing ovarian follicles in a culture dish. In addition, fertilization and early embryogenesis can be modeled using in vitro fertilization (IVF), which involves the fertilization of mature oocytes with capacitated sperm in a culture dish. Here, we describe protocols for in vitro maturation (IVM) and in vitro fertilization (IVF) of mouse oocytes and preimplantation embryo culture. These protocols are suitable for the study of oocyte and embryo biology and the production of embryonic mice.

  16. Expression of Variant Ribosomal RNA Genes in Mouse Oocytes and Preimplantation Embryos1

    PubMed Central

    Ihara, Motomasa; Tseng, Hung; Schultz, Richard M.

    2011-01-01

    Ribosomal DNA (rDNA) is not composed of multiple copies of identical transcription units, as commonly believed, but rather of at least seven rDNA variant subtypes that are expressed in somatic cells. This finding raises the possibility that ribosome function may be modulated as proposed by the ribosome filter hypothesis. We report here that mouse oocytes and preimplantation embryos express all the rDNA variants except variant V and that there is no marked developmental change in the qualitative pattern of variant expression. The maternal and embryonic ribosome pools are therefore quite similar, minimizing the likelihood that developmental changes in composition of the ribosome population are critical for preimplantation development. PMID:21209414

  17. Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

    SciTech Connect

    Yukawa, Masashi; Oda, Shoji; Mitani, Hiroshi; Nagata, Masao; Aoki, Fugaku . E-mail: aokif@k.u-tokyo.ac.jp

    2007-06-29

    DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by {gamma}-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX ({gamma}-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to {gamma}-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of {gamma}-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.

  18. Application of single-needle blastomere biopsy in human preimplantation genetic diagnosis.

    PubMed

    Inzunza, J; Iwarsson, E; Fridström, M; Rosenlund, B; Sjöblom, P; Hillensjö, T; Blennow, E; Jones, B; Nordenskjöld, M; Ahrlund-Richter, L

    1998-12-01

    We have tested and subsequently successfully applied a single-needle approach to obtain blastomere biopsies from human preimplantation embryos for preimplantation genetic diagnosis (PGD). The method was first evaluated in a mouse system and shown to be compatible with a high degree of in vitro and in vivo development of biopsied mouse embryos. Furthermore, we showed that biopsied mouse embryos after transfer to recipient mice underwent implantation, normal development and delivery. Litters were followed through puberty and adulthood and shown to be normal with regard to sexual function and also a panel of biochemical and morphological parameters including organ histology. Successful human preimplantation diagnosis, followed by pregnancies and birth of healthy babies, was established with two out of three couples carrying a risk to transmit chromosomal abnormalities leading to severe disease. This is the first report of the successful use of a single-needle approach in human PGD. Considering its simplicity, we conclude that the single-needle approach is an attractive alternative for biopsies in PGD.

  19. Recent advances in developing molecular tools for targeted genome engineering of mammalian cells.

    PubMed

    Lim, Kwang-il

    2015-01-01

    Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future.

  20. Evolution, development, and initial function of the mammalian neocortex: response of the germinal zones to endothermy.

    PubMed

    Smart, I H M

    2008-01-01

    In the mouse the release of neocortical neurons from the periventricular germinal layers of the forebrain commences towards the ventral margin of the lateral pallium at the level of the interventricular foramen and is propagated from there across the lateral wall of the hemisphere. In the adult cortex the origin of the gradient corresponded to the ventral portion of the somatotopic map of the body, that is, to the area representating structures derived from the embryonic branchial arches, namely, the peri-oral region and laryngo-pharyngeal masticatory apparatus. Branchial arch nerves also innervate the fore- and mid-gut and all the related exocrine and endocrine glands. This suggests that the mammalian neocortex evolved from a visceral integration area in a positionally equivalent area in the pallium of a reptilian ancestor which expanded in relation to extensive changes taking place in the visceral and branchial systems of the body during the transition from reptilian ectothermy to mammalian endothermy. The practical problem facing early mammals was to acquire and process the extra energy required to sustain a continuously high metabolic rate. Improvements to the food processing capabilities of the visceral and branchial systems and the expansion of their neural control were important components in the conglomerate of changes required to sustain the increased energy demands of endothermic tissues. Endothermy also bestowed the ability to sustain greater numbers of metabolically expensive neurons and this, in turn, required an appropriate response from the cell production mechanisms in the periventricular germinal layers.

  1. Carbon dioxide starvation, the development of C4 ecosystems, and mammalian evolution.

    PubMed Central

    Cerling, T E; Ehleringer, J R; Harris, J M

    1998-01-01

    The decline of atmospheric CO2 over the last 65 million years (Ma) resulted in the 'CO2-starvation' of terrestrial ecosystems and led to the widespread distribution of C4 plants, which are less sensitive to CO2 levels than are C3 plants. Global expansion of C4 biomass is recorded in the diets of mammals from Asia, Africa, North America, and South America during the interval from about 8 to 5 Ma. This was accompanied by the most significant Cenozoic faunal turnover on each of these continents, indicating that ecological changes at this time were an important factor in mammalian extinction. Further expansion of tropical C4 biomass in Africa also occurred during the last glacial interval confirming the link between atmospheric CO2 levels and C4 biomass response. Changes in fauna and flora at the end of the Miocene, and between the last glacial and interglacial, have previously been attributed to changes in aridity; however, an alternative explanation for a global expansion of C4 biomass is CO2 starvation of C3 plants when atmospheric CO2 levels dropped below a threshold significant to C3 plants. Aridity may also have been a factor in the expansion of C4 ecosystems but one that was secondary to, and perhaps because of, gradually decreasing CO2 concentrations in the atmosphere. Mammalian evolution in the late Neogene, then, may be related to the CO2 starvation of C3 ecosystems. PMID:9507562

  2. Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development

    PubMed Central

    2011-01-01

    Background We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. Results The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. Conclusions Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution. PMID:21854559

  3. Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

    PubMed

    Renfree, Marilyn B; Papenfuss, Anthony T; Deakin, Janine E; Lindsay, James; Heider, Thomas; Belov, Katherine; Rens, Willem; Waters, Paul D; Pharo, Elizabeth A; Shaw, Geoff; Wong, Emily S W; Lefèvre, Christophe M; Nicholas, Kevin R; Kuroki, Yoko; Wakefield, Matthew J; Zenger, Kyall R; Wang, Chenwei; Ferguson-Smith, Malcolm; Nicholas, Frank W; Hickford, Danielle; Yu, Hongshi; Short, Kirsty R; Siddle, Hannah V; Frankenberg, Stephen R; Chew, Keng Yih; Menzies, Brandon R; Stringer, Jessica M; Suzuki, Shunsuke; Hore, Timothy A; Delbridge, Margaret L; Patel, Hardip R; Mohammadi, Amir; Schneider, Nanette Y; Hu, Yanqiu; O'Hara, William; Al Nadaf, Shafagh; Wu, Chen; Feng, Zhi-Ping; Cocks, Benjamin G; Wang, Jianghui; Flicek, Paul; Searle, Stephen M J; Fairley, Susan; Beal, Kathryn; Herrero, Javier; Carone, Dawn M; Suzuki, Yutaka; Sugano, Sumio; Toyoda, Atsushi; Sakaki, Yoshiyuki; Kondo, Shinji; Nishida, Yuichiro; Tatsumoto, Shoji; Mandiou, Ion; Hsu, Arthur; McColl, Kaighin A; Lansdell, Benjamin; Weinstock, George; Kuczek, Elizabeth; McGrath, Annette; Wilson, Peter; Men, Artem; Hazar-Rethinam, Mehlika; Hall, Allison; Davis, John; Wood, David; Williams, Sarah; Sundaravadanam, Yogi; Muzny, Donna M; Jhangiani, Shalini N; Lewis, Lora R; Morgan, Margaret B; Okwuonu, Geoffrey O; Ruiz, San Juana; Santibanez, Jireh; Nazareth, Lynne; Cree, Andrew; Fowler, Gerald; Kovar, Christie L; Dinh, Huyen H; Joshi, Vandita; Jing, Chyn; Lara, Fremiet; Thornton, Rebecca; Chen, Lei; Deng, Jixin; Liu, Yue; Shen, Joshua Y; Song, Xing-Zhi; Edson, Janette; Troon, Carmen; Thomas, Daniel; Stephens, Amber; Yapa, Lankesha; Levchenko, Tanya; Gibbs, Richard A; Cooper, Desmond W; Speed, Terence P; Fujiyama, Asao; Graves, Jennifer A M; O'Neill, Rachel J; Pask, Andrew J; Forrest, Susan M; Worley, Kim C

    2011-08-29

    We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

  4. Transcript Expression Analysis of Putative Trypanosoma brucei GPI-Anchored Surface Proteins during Development in the Tsetse and Mammalian Hosts

    PubMed Central

    Savage, Amy F.; Cerqueira, Gustavo C.; Regmi, Sandesh; Wu, Yineng; El Sayed, Najib M.; Aksoy, Serap

    2012-01-01

    Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of

  5. [BETWEEN USAGE AND POLEMIC, AN ARGUMENT IN FAVOUR OF CLARIFYING THE TERMINOLOGY FOR PREIMPLANTATION GENETIC DIAGNOSIS].

    PubMed

    Côté, Stéphanie; Ravitsky, Vardit; Hamet, Pavel; Bouffard, Chantal

    2015-12-01

    Over 30 years ago, preimplantation genetic diagnosis (PGD) was developed to help couples at risk of transmitting a serious genetic disease to their offspring. Today, the range of medical and non-medical uses of PGD has expanded considerably and some raise much controversy. This is the case, for example, with In-Vitro Fertilization to select embryos as 'saviour siblings' or to screen for susceptibility and predisposition to late onset diseases or conditions of variable penetrance. The situation is even more problematic in the case of sex selection or selection of traits that are culturally valued or discredited (such as deafness, behavioral traits, or height). The debate surrounding PGD has been employing terms to describe these particular uses that have contributed to a focus on the negative effects, thus preventing a distinction between the abuses and the benefits of this reproductive technology. In this context, this paper proposes a terminological clarification that would allow distinguishing medical and non-medical use and, therefore, the issues relevant to each. A more accurate and less generic nomenclature could prevent a conflation of different levels of ethical, clinical and social issues under the single term 'PGD'. For the vast majority of medical uses, we propose to keep: 'preimplantation genetic diagnosis (PGD)', which emphasizes that it is a genetic diagnosis. For non-medical uses, we suggest: 'preimplantation genetic trait selection (PGTS)'.

  6. Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

    PubMed Central

    Saiz, Nestor; Kang, Minjung; Schrode, Nadine; Lou, Xinghua; Hadjantonakis, Anna-Katerina

    2016-01-01

    This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.). PMID:26967230

  7. Maternal restraint stress negatively influences growth capacity of preimplantation mouse embryos.

    PubMed

    Burkuš, Ján; Cikoš, Stefan; Fabian, Dušan; Kubandová, Janka; Czikková, Soňa; Koppel, Juraj

    2013-03-01

    In our study we investigated the effect of maternal restraint stress on preimplantation embryo development using a mouse model. We exposed hormonally stimulated (superovulated) and unstimulated (i.e. spontaneously ovulating) mouse females to restraint stress for 30 min three times a day during the preimplantation period. The stress exposure caused significant increase in blood plasma corticosterone concentration. Microscopical evaluation of embryos isolated from spontaneously ovulating females showed that maternal stress significantly increased the proportion of embryos with lower cell numbers (≤32 cells) and decreased the proportion of embryos with higher cell numbers (65-96 cells and 97-128 cells). Moreover maternal restraint stress decreased the cell counts per embryo and per blastocyst. After an additional 24 h in vitro culture we did not find any difference in the embryo distribution or in the cell counts per embryo/blastocyst between embryos isolated from stressed and control mothers. The exposure to restraint stress did not affect the incidence of apoptosis in blastocysts isolated from spontaneously ovulated dams. In gonadotropin stimulated dams, the hormonal treatment itself notably changed embryo distribution (increasing the proportion of degenerated embryos) and increased the occurrence of apoptotic cells. Our results indicate that psychical stress exposure in very early pregnancy can significantly influence the developmental capacity of preimplantation embryos.

  8. Self-correction of chromosomal abnormalities in human preimplantation embryos and embryonic stem cells.

    PubMed

    Bazrgar, Masood; Gourabi, Hamid; Valojerdi, Mojtaba Rezazadeh; Yazdi, Poopak Eftekhari; Baharvand, Hossein

    2013-09-01

    Aneuploidy is commonly seen in human preimplantation embryos, most particularly at the cleavage stage because of genome activation by third cell division. Aneuploid embryos have been used for the derivation of normal embryonic stem cell (ESC) lines and developmental modeling. This review addresses aneuploidies in human preimplantation embryos and human ESCs and the potential of self-correction of these aberrations. Diploid-aneuploid mosaicism is the most frequent abnormality observed; hence, embryos selected by preimplantation genetic diagnosis at the cleavage or blastocyst stage could be partly abnormal. Differentiation is known as the barrier for eliminating mosaic embryos by death and/or decreased division of abnormal cells. However, some mosaicisms, such as copy number variations could be compatible with live birth. Several reasons have been proposed for self-correction of aneuploidies during later stages of development, including primary misdiagnosis, allocation of the aneuploidy in the trophectoderm, cell growth advantage of diploid cells in mosaic embryos, lagging of aneuploid cell division, extrusion or duplication of an aneuploid chromosome, and the abundance of DNA repair gene products. Although more studies are needed to understand the mechanisms of self-correction as a rare phenomenon, most likely, it is related to overcoming mosaicism.

  9. Transcriptional program of Kpna2/Importin-α2 regulates cellular differentiation-coupled circadian clock development in mammalian cells

    PubMed Central

    Umemura, Yasuhiro; Koike, Nobuya; Matsumoto, Tsuguhiro; Yoo, Seung-Hee; Chen, Zheng; Yasuhara, Noriko; Takahashi, Joseph S.; Yagita, Kazuhiro

    2014-01-01

    The circadian clock in mammalian cells is cell-autonomously generated during the cellular differentiation process, but the underlying mechanisms are not understood. Here we show that perturbation of the transcriptional program by constitutive expression of transcription factor c-Myc and DNA methyltransferase 1 (Dnmt1) ablation disrupts the differentiation-coupled emergence of the clock from mouse ESCs. Using these model ESCs, 484 genes are identified by global gene expression analysis as factors correlated with differentiation-coupled circadian clock development. Among them, we find the misregulation of Kpna2 (Importin-α2) during the differentiation of the c-Myc-overexpressed and Dnmt1−/− ESCs, in which sustained cytoplasmic accumulation of PER proteins is observed. Moreover, constitutive expression of Kpna2 during the differentiation culture of ESCs significantly impairs clock development, and KPNA2 facilitates cytoplasmic localization of PER1/2. These results suggest that the programmed gene expression network regulates the differentiation-coupled circadian clock development in mammalian cells, at least in part via posttranscriptional regulation of clock proteins. PMID:25389311

  10. Temporally Distinct Six2-Positive Second Heart Field Progenitors Regulate Mammalian Heart Development and Disease.

    PubMed

    Zhou, Zhengfang; Wang, Jingying; Guo, Chaoshe; Chang, Weiting; Zhuang, Jian; Zhu, Ping; Li, Xue

    2017-01-24

    The embryonic process of forming a complex structure such as the heart remains poorly understood. Here, we show that Six2 marks a dynamic subset of second heart field progenitors. Six2-positive (Six2(+)) progenitors are rapidly recruited and assigned, and their descendants are allocated successively to regions of the heart from the right ventricle (RV) to the pulmonary trunk. Global ablation of Six2(+) progenitors resulted in RV hypoplasia and pulmonary atresia. An early stage-specific ablation of a small subset of Six2(+) progenitors did not cause any apparent structural defect at birth but rather resulted in adult-onset cardiac hypertrophy and dysfunction. Furthermore, Six2 expression depends in part on Shh signaling, and Shh deletion resulted in severe deficiency of Six2(+) progenitors. Collectively, these findings unveil the chronological features of cardiogenesis, in which the mammalian heart is built sequentially by temporally distinct populations of cardiac progenitors, and provide insights into late-onset congenital heart disease.

  11. Early mammalian development under conditions of reorientation relative to the gravity vector

    NASA Technical Reports Server (NTRS)

    Wolgemuth, D. J.; Grills, G. S.

    1985-01-01

    A clinostat was used to assess the effects of reorientation relative to the gravity vector on mammalian germ cells cultured in vitro. Previous studies using this system revealed an inhibition of meiotic maturation of mouse oocytes. In the present study, the effects of clinostat rotation on in vitro fertilization were examined. The frequency of fertilization of experimental cultures did not vary from that of the clinostat vertical control cultures at either of the rotation rates examined. Importantly, no abnormalities of fertilization, such as parthenogenetic activation, fragmentation, or polyspermy were seen. It is concluded that the initial events of fertilization were unaffected by this treatment, although the developmental potential of these embryos remains to be assessed.

  12. Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

    PubMed Central

    Reijns, Martin A.M.; Rabe, Björn; Rigby, Rachel E.; Mill, Pleasantine; Astell, Katy R.; Lettice, Laura A.; Boyle, Shelagh; Leitch, Andrea; Keighren, Margaret; Kilanowski, Fiona; Devenney, Paul S.; Sexton, David; Grimes, Graeme; Holt, Ian J.; Hill, Robert E.; Taylor, Martin S.; Lawson, Kirstie A.; Dorin, Julia R.; Jackson, Andrew P.

    2012-01-01

    Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells. PMID:22579044

  13. BMP-FGF signaling axis mediates Wnt-induced epidermal stratification in developing mammalian skin.

    PubMed

    Zhu, Xiao-Jing; Liu, YuDong; Dai, Zhong-Min; Zhang, Xiaoyun; Yang, XueQin; Li, Yan; Qiu, Mengsheng; Fu, Jiang; Hsu, Wei; Chen, YiPing; Zhang, Zunyi

    2014-10-01

    Epidermal stratification of the mammalian skin requires proliferative basal progenitors to generate intermediate cells that separate from the basal layer and are replaced by post-mitotic cells. Although Wnt signaling has been implicated in this developmental process, the mechanism underlying Wnt-mediated regulation of basal progenitors remains elusive. Here we show that Wnt secreted from proliferative basal cells is not required for their differentiation. However, epidermal production of Wnts is essential for the formation of the spinous layer through modulation of a BMP-FGF signaling cascade in the dermis. The spinous layer defects caused by disruption of Wnt secretion can be restored by transgenically expressed Bmp4. Non-cell autonomous BMP4 promotes activation of FGF7 and FGF10 signaling, leading to an increase in proliferative basal cell population. Our findings identify an essential BMP-FGF signaling axis in the dermis that responds to the epidermal Wnts and feedbacks to regulate basal progenitors during epidermal stratification.

  14. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function

    PubMed Central

    Garza-Lombó, Carla; Gonsebatt, María E.

    2016-01-01

    The kinase mammalian target of rapamycin (mTOR) integrates signals triggered by energy, stress, oxygen levels, and growth factors. It regulates ribosome biogenesis, mRNA translation, nutrient metabolism, and autophagy. mTOR participates in various functions of the brain, such as synaptic plasticity, adult neurogenesis, memory, and learning. mTOR is present during early neural development and participates in axon and dendrite development, neuron differentiation, and gliogenesis, among other processes. Furthermore, mTOR has been shown to modulate lifespan in multiple organisms. This protein is an important energy sensor that is present throughout our lifetime its role must be precisely described in order to develop therapeutic strategies and prevent diseases of the central nervous system. The aim of this review is to present our current understanding of the functions of mTOR in neural development, the adult brain and aging. PMID:27378854

  15. Mammalian PNLDC1 is a novel poly(A) specific exonuclease with discrete expression during early development.

    PubMed

    Anastasakis, Dimitrios; Skeparnias, Ilias; Shaukat, Athanasios-Nasir; Grafanaki, Katerina; Kanellou, Alexandra; Taraviras, Stavros; Papachristou, Dionysios J; Papakyriakou, Athanasios; Stathopoulos, Constantinos

    2016-10-14

    PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported recently to participate in piRNA biogenesis in silkworm and C. elegans, respectively. To get insights on the role of mammalian PNLDC1, we characterized the human and mouse enzymes. PNLDC1 shows limited conservation compared to PARN and represents an evolutionary related but distinct group of enzymes. It is expressed specifically in mouse embryonic stem cells, human and mouse testes and during early mouse embryo development, while it fades during differentiation. Its expression in differentiated cells, is suppressed through methylation of its promoter by the de novo methyltransferase DNMT3B. Both enzymes are localized mainly in the ER and exhibit in vitro specificity restricted solely to 3' RNA or DNA polyadenylates. Knockdown of Pnldc1 in mESCs and subsequent NGS analysis showed that although the expression of the remaining deadenylases remains unaffected, it affects genes involved mainly in reprogramming, cell cycle and translational regulation. Mammalian PNLDC1 is a novel deadenylase expressed specifically in cell types which share regulatory mechanisms required for multipotency maintenance. Moreover, it could be involved both in posttranscriptional regulation through deadenylation and genome surveillance during early development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Mammalian PNLDC1 is a novel poly(A) specific exonuclease with discrete expression during early development

    PubMed Central

    Anastasakis, Dimitrios; Skeparnias, Ilias; Shaukat, Athanasios-Nasir; Grafanaki, Katerina; Kanellou, Alexandra; Taraviras, Stavros; Papachristou, Dionysios J.; Papakyriakou, Athanasios; Stathopoulos, Constantinos

    2016-01-01

    PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported recently to participate in piRNA biogenesis in silkworm and C. elegans, respectively. To get insights on the role of mammalian PNLDC1, we characterized the human and mouse enzymes. PNLDC1 shows limited conservation compared to PARN and represents an evolutionary related but distinct group of enzymes. It is expressed specifically in mouse embryonic stem cells, human and mouse testes and during early mouse embryo development, while it fades during differentiation. Its expression in differentiated cells, is suppressed through methylation of its promoter by the de novo methyltransferase DNMT3B. Both enzymes are localized mainly in the ER and exhibit in vitro specificity restricted solely to 3′ RNA or DNA polyadenylates. Knockdown of Pnldc1 in mESCs and subsequent NGS analysis showed that although the expression of the remaining deadenylases remains unaffected, it affects genes involved mainly in reprogramming, cell cycle and translational regulation. Mammalian PNLDC1 is a novel deadenylase expressed specifically in cell types which share regulatory mechanisms required for multipotency maintenance. Moreover, it could be involved both in posttranscriptional regulation through deadenylation and genome surveillance during early development. PMID:27515512

  17. The biology and dynamics of mammalian cortical granules.

    PubMed

    Liu, Min

    2011-11-17

    Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.

  18. The biology and dynamics of mammalian cortical granules

    PubMed Central

    2011-01-01

    Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals. PMID:22088197

  19. The Cytological Events and Molecular Control of Life Cycle Development of Trypanosoma brucei in the Mammalian Bloodstream.

    PubMed

    Silvester, Eleanor; McWilliam, Kirsty R; Matthews, Keith R

    2017-06-28

    African trypanosomes cause devastating disease in sub-Saharan Africa in humans and livestock. The parasite lives extracellularly within the bloodstream of mammalian hosts and is transmitted by blood-feeding tsetse flies. In the blood, trypanosomes exhibit two developmental forms: the slender form and the stumpy form. The slender form proliferates in the bloodstream, establishes the parasite numbers and avoids host immunity through antigenic variation. The stumpy form, in contrast, is non-proliferative and is adapted for transmission. Here, we overview the features of slender and stumpy form parasites in terms of their cytological and molecular characteristics and discuss how these contribute to their distinct biological functions. Thereafter, we describe the technical developments that have enabled recent discoveries that uncover how the slender to stumpy transition is enacted in molecular terms. Finally, we highlight new understanding of how control of the balance between slender and stumpy form parasites interfaces with other components of the infection dynamic of trypanosomes in their mammalian hosts. This interplay between the host environment and the parasite's developmental biology may expose new vulnerabilities to therapeutic attack or reveal where drug control may be thwarted by the biological complexity of the parasite's lifestyle.

  20. Development of polymer based cryogel matrix for transportation and storage of mammalian cells

    PubMed Central

    Kumari, Jyoti; Kumar, Ashok

    2017-01-01

    We studied the potential of polymeric cryogel matrices such as 2-hydroxyethyl methacrylate (HEMA)-agarose (HA) and gelatin matrix as a transporting and storage material for mammalian cells. Both the HA and gelatin matrices were found to possess a homogenous distribution of pores as shown by scanning electron microscopic (SEM) images and flow rate of 8 and 5 mL/min, respectively. In the case of HA cryogel, after 5 days of simulated transportation, C2C12 cells kept in cryogel matrix showed higher percentage viability (89%) as compared to 64.5% viability of cells kept in suspension culture. The cells recovered from the HA cryogel were able to proliferate as revealed by the microscopic analysis. In the case of gelatin cryogel, it was shown that C2C12 cells seeded on the cryogel under simulated transportation condition were found to proliferate over the period of 5 days. It was also observed that the cells after simulation can be cryopreserved and the duration of cryopreservation does not affect their viability. Furthermore, gelatin cryogel was used for cryopreservation of HepG2 and HUVEC cells to extend the system for other cell types. These results show the potential of cryogels as efficient, low-cost transporting matrix at room temperature and in cryo-conditions. PMID:28139669

  1. GABA transporters in the mammalian cerebral cortex: localization, development and pathological implications.

    PubMed

    Conti, Fiorenzo; Minelli, Andrea; Melone, Marcello

    2004-07-01

    The extracellular levels of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian cerebral cortex, are regulated by specific high-affinity, Na+/Cl- dependent transporters. Four distinct genes encoding GABA transporters (GATs), named GAT-1, GAT-2, GAT-3, and BGT-1 have been identified using molecular cloning. Of these, GAT-1 and -3 are expressed in the cerebral cortex. Studies of the cortical distribution, cellular localization, ontogeny and relationships of GATs with GABA-releasing elements using a variety of light and electron microscopic immunocytochemical techniques have shown that: (i) a fraction of GATs is strategically placed to mediate GABA uptake at fast inhibitory synapses, terminating GABA's action and shaping inhibitory postsynaptic responses; (ii) another fraction may participate in functions such as the regulation of GABA's diffusion to neighboring synapses and of GABA levels in cerebrospinal fluid; (iii) GATs may play a role in the complex processes regulating cortical maturation; and (iv) GATs may contribute to the dysregulation of neuronal excitability that accompanies at least two major human diseases: epilepsy and ischemia.

  2. BMP-FGF Signaling Axis Mediates Wnt-Induced Epidermal Stratification in Developing Mammalian Skin

    PubMed Central

    Zhu, Xiao-Jing; Liu, YuDong; Dai, Zhong-Min; Zhang, Xiaoyun; Yang, XueQin; Li, Yan; Qiu, Mengsheng; Fu, Jiang; Hsu, Wei; Chen, YiPing; Zhang, Zunyi

    2014-01-01

    Epidermal stratification of the mammalian skin requires proliferative basal progenitors to generate intermediate cells that separate from the basal layer and are replaced by post-mitotic cells. Although Wnt signaling has been implicated in this developmental process, the mechanism underlying Wnt-mediated regulation of basal progenitors remains elusive. Here we show that Wnt secreted from proliferative basal cells is not required for their differentiation. However, epidermal production of Wnts is essential for the formation of the spinous layer through modulation of a BMP-FGF signaling cascade in the dermis. The spinous layer defects caused by disruption of Wnt secretion can be restored by transgenically expressed Bmp4. Non-cell autonomous BMP4 promotes activation of FGF7 and FGF10 signaling, leading to an increase in proliferative basal cell population. Our findings identify an essential BMP-FGF signaling axis in the dermis that responds to the epidermal Wnts and feedbacks to regulate basal progenitors during epidermal stratification. PMID:25329657

  3. The mammalian Sin3 proteins are required for muscle development and sarcomere specification.

    PubMed

    van Oevelen, Chris; Bowman, Christopher; Pellegrino, Jessica; Asp, Patrik; Cheng, Jemmie; Parisi, Fabio; Micsinai, Mariann; Kluger, Yuval; Chu, Alphonse; Blais, Alexandre; David, Gregory; Dynlacht, Brian D

    2010-12-01

    The highly related mammalian Sin3A and Sin3B proteins provide a versatile platform for chromatin-modifying activities. Sin3-containing complexes play a role in gene repression through deacetylation of nucleosomes. Here, we explore a role for Sin3 in myogenesis by examining the phenotypes resulting from acute somatic deletion of both isoforms in vivo and from primary myotubes in vitro. Myotubes ablated for Sin3A alone, but not Sin3B, displayed gross defects in sarcomere structure that were considerably enhanced upon simultaneous ablation of both isoforms. Massively parallel sequencing of Sin3A- and Sin3B-bound genomic loci revealed a subset of target genes directly involved in sarcomere function that are positively regulated by Sin3A and Sin3B proteins. Both proteins were coordinately recruited to a substantial number of genes. Interestingly, depletion of Sin3B led to compensatory increases in Sin3A recruitment at certain target loci, but Sin3B was never found to compensate for Sin3A loss. Thus, our analyses describe a novel transcriptional role for Sin3A and Sin3B proteins associated with maintenance of differentiated muscle cells.

  4. Development of Cell-Defined Lentivirus-Based Microarray for Mammalian Cells.

    PubMed

    Kim, Hi Chul; Shum, David; Seol, Hyang Sook; Jang, Se Jin; Cho, Ssang-Goo; Kwon, Yong-Jun

    2017-01-01

    Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing. We selected poly-l-lysine (PLL) with Matrigel as the best combination of biomaterials for cell-defined culture. We used 2 µL PLL to determine by titration the optimum concentration required (0.04% stock, 0.005% final concentration). We also determined the optimum concentration of 10 µL of lentivirus particles for maximum reverse infection efficiency (1 × 10(8) infectious units [IFU]/mL stock, 62.5% final concentration) and established the best combination of components for the lentivirus mixture (10 µL of lentivirus particles and 2 µL each of siGLO Red dye, Matrigel, and 0.04% PLL). Finally, we validated both the effect of reverse infection in various cell lines and lentivirus spot activity in CDLM by storage period. This method provides an effective lentivirus-infected cell microarray for large-scale gene function studies.

  5. Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers.

    PubMed

    Iwarsson, E; Malmgren, H; Inzunza, J; Ahrlund-Richter, L; Sjöblom, P; Rosenlund, B; Fridström, M; Hovatta, O; Nordenskjöld, M; Blennow, E

    2000-12-01

    We have developed preimplantation genetic diagnosis (PGD) for carriers of chromosomal abnormalities using fluorescent in situ hybridisation (FISH). Here we present the detailed analysis of 64 biopsied, normally developing embryos obtained from four Robertsonian and three reciprocal translocation carriers in 11 treatment cycles of which four resulted in normal pregnancies (three simplex, one duplex). In order to investigate the degree of mosaicism and segregation mode in the embryos, the primary analysis of the biopsied cells was extended with the analysis of all cells from the non-transferred embryos. The analysis also included a second hybridisation with two additional probes, not involved in the translocation (chromosomes 1 and 9), in order to investigate the overall degree of mosaicism. Seventeen out of 64 analysed embryos were balanced for the chromosomes involved in the translocation and 14 of these were transferred. Forty-seven out of 64 embryos (73%) were mosaic regarding the chromosomes involved in the translocation and alternate segregation mode was the most common mode of segregation. Moreover, we have found a higher degree of mosaicism for the chromosomes involved in translocations as compared to control chromosomes. This difference was more pronounced for the embryos from reciprocal translocation carriers. The results, mechanisms, significance and implications of our findings are discussed. Copyright 2000 John Wiley & Sons, Ltd.

  6. [Extending preimplantation genetic diagnosis to HLA typing: the Paris experience].

    PubMed

    Steffann, J; Frydman, N; Burlet, P; Gigarel, N; Feyereisen, E; Kerbrat, V; Tachdjian, G; Munnich, A; Frydman, R

    2005-10-01

    Preimplantation genetic diagnosis (PGD) consists in the genetic analysis of one or two cells. These cells (blastomeres) are sampled from embryos, obtained by in vitro fertilization, at the third day of development. Since 1998, the bioethical laws (1994) and their decrees restricted PGD practices in France, strictly to the avoidance of the birth of a child affected with a genetic defect. In parallel, works on blood cord transplantation, taken at the birth of a compatible HLA sibling, showed very encouraging results, particularly for the treatment of Fanconi anemia. In 2001, Verlinsky et al., have reported the first PGD for Fanconi anaemia combined with HLA typing, allowing the birth of a healthy child, HLA-identical with his affected sister. The "designer baby" concept was born. The French law, which allowed PGD under specific conditions, i.e. when the genetic defect has been characterized in one parent at least, recently extended PGD to HLA typing when embryos are at risk of a genetic disorder. Article L.2131-4-1 (August 2004) allows the practice of HLA typing for PGD embryos when an elder sibling is affected with a genetic disorder and need stem cell transplantation. The HLA-matched offspring resulting from PGD can give cord blood at birth to supply the necessary therapy. This double selection give rise to serious ethical problems, but technical difficulties and legal restrictions will probably limit the development of such a procedure.

  7. X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells

    PubMed Central

    Lee, Jeannie T.

    2013-01-01

    X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells. PMID:21667284

  8. X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells.

    PubMed

    Payer, Bernhard; Lee, Jeannie T; Namekawa, Satoshi H

    2011-08-01

    X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.

  9. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors.

  10. Mammalian Pheromones

    PubMed Central

    Liberles, Stephen D.

    2015-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d ) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  11. Successful polar body-based preimplantation genetic diagnosis for achondroplasia.

    PubMed

    Altarescu, G; Renbaum, P; Brooks P, B; Margalioth, E J; Ben Chetrit, A; Munter, G; Levy-Lahad, E; Eldar-Geva, T

    2008-02-01

    Achondroplasia, the most common form of dwarfism, is a candidate for preimplantation genetic diagnosis (PGD) because a single mutation accounts for almost all cases. Multiplex fluorescent assay including the common G380R mutation in the FGFR3 gene and eight close polymorphic markers was developed. First and second polar bodies (PB) were used for PGD analysis. An affected woman was treated with routine long-protocol ovarian stimulation and puncture. In the first PGD cycle, out of four fertilized oocytes, PB analysis revealed two mutant oocytes, one with total amplification failure of the maternal allele and one with inconclusive results. In the second PGD cycle, 14 oocytes were retrieved following a higher FSH dose and by performing oocyte retrieval and by placing the patient in the anti-Trendelenburg position using abdominal pressure to allow all follicles to be drained. Following PB analysis, two embryos containing the wild-type FGFR3 allele were transferred. This led to an uncomplicated pregnancy and delivery by Caesarean section at week 38 of a healthy boy, carrying the FGFR3 wild-type maternal allele. In conclusion, oocyte retrieval, while difficult in patients with achondroplasia, can be successfully performed. PB analysis is a reliable and sensitive method for PGD for maternal achondroplasia.

  12. A simplified technique for embryo biopsy for preimplantation genetic diagnosis.

    PubMed

    Wang, Wei-Hua; Kaskar, Khalied; Gill, Jimmy; DeSplinter, Traci

    2008-08-01

    To report a simplified embryo biopsy method for preimplantation genetic diagnosis (PGD). Technique and method. A regional hospital in vitro fertilization (IVF) laboratory and private reproductive medicine clinic. Women undergoing IVF and PGD. Blastomeres were successfully isolated from day-3 embryos at various stages. Blastomere integrity after biopsy, time of biopsy procedure, and subsequent blastocyst developmental rate. Twenty embryos derived from abnormally fertilized oocytes (one pronucleus or three pronuclei) were used for biopsy at four-cell to 10-cell stages (day 3) by a laser zona drilling and assisted hatching micropipette delivery of culture medium inside the zona to push one blastomere out. Biopsies of all embryos using this method were successful. In two cases for PGD, fourteen 6-9-cell and four 3-4-cell stage embryos were successfully biopsied by this method. Ten out of 14 embryos from the 6-9-cell stage developed to hatching or hatched blastocysts. When two hatched blastocysts were vitrified, warmed, and cultured, both reexpanded, showing normal morphologic features. This technique is easy to learn, less damaging to the embryos, and less time consuming. It can be used for all stages of embryos without damage to either embryos or isolated blastomeres. It is an alternative method for embryo biopsy in PGD.

  13. Is embryo research and preimplantation genetic diagnosis ethical?

    PubMed

    Beyleveld, D

    2000-09-11

    The legal position in the UK on embryo research and preimplantation genetic diagnosis (PGD) is outlined and contrasted with the position in other EU countries. The "gradualist" position of the UK on the moral status of the embryo is defended on the basis of an argument that precaution must be applied in proportion to the degree to which the embryo has developed to display components of agency, on the assumption that mortality is categorically binding and requires agents to be granted rights and that it cannot be known with certainty that the embryo is not an agent. The extent to which this argument, when combined with vicarious protections that the embryo should receive in order to protect the rights of other agents, limits embryo research and PGD, is discussed. It is concluded that the complexities that attend deliberation about the moral problems attending embryo research and PGD are such that the proper response to these problems is via the procedures of political democracy to achieve accountable answers rather than "correct" answers. This allows for a variety of judgements.

  14. Sperm as a noninvasive gene delivery system for preimplantation embryos.

    PubMed

    Chan, P J; Kalugdan, T; Su, B C; Whitney, E A; Perrott, W; Tredway, D R; King, A

    1995-05-01

    To determine if sperm could be manipulated to be a noninvasive transport carrier for the delivery of gene fragments to the blastocyst. Sperm cells carrying foreign DNA fragments from human papillomavirus (HPV) types 16, 18, 31, and 33 were allowed to migrate from one end of an artificial reproductive tube and to come in contact with hatching mouse blastocysts at the other end of the tube. The blastocysts were then washed and analyzed for the presence of the foreign DNA fragments. Clinical and academic research environment. Detection of amplified products from transferred foreign DNA using the polymerase chain reaction and primers targeted at the E6-E7 region for different HPV types. Polymerase chain reaction analyses showed transference of DNA HPV type 18 to the blastocysts. Not all types of DNA fragments were transferred equally. The results suggested the possibility of using sperm as a noninvasive gene delivery system for passing on gene fragments to preimplantation embryos. It was demonstrated that certain DNA fragments were easier to deliver than others, indicating the necessity for exploring all the factors involved in the mechanism of the transference process. The study also serves to highlight the possibility of unintentional transmission of viral or bacterial DNA to the developing embryo via the sperm.

  15. Neonatal outcome after preimplantation genetic diagnosis.

    PubMed

    Eldar-Geva, Talia; Srebnik, Naama; Altarescu, Gheona; Varshaver, Irit; Brooks, Baruch; Levy-Lahad, Ephrat; Bromiker, Ruben; Schimmel, Michael S

    2014-10-01

    To examine whether embryo biopsy for preimplantation genetic diagnosis (PGD) influences neonatal outcomes. Prospective follow-up cohort. Tertiary university-affiliated medical center. 242 children born after PGD, 242 children born after intracytoplasmic sperm injection (ICSI) (158 singletons and 42 twins pairs in each group), and 733 children born after a spontaneous conception (SC) (493 singletons, 120 twins pairs), matched for maternal age, parity, and body mass index. None. Gestational age, birth weight, prematurity (<37 and <34 weeks), low birth weight (<2,500 g, very low birth weight, <1,500 g), and intrauterine growth restriction (<10th percentile for gestational age). For singletons, the mean birth weight was higher after SC compared with ICSI but not compared with PGD. Mean gestational ages were lower after PGD and ICSI compared with SC. The low birth weight and intrauterine growth restriction rates were 4.4%, 12.0%, and 5.7% and 5.1%, 9.5%, and 5.5% for PGD, ICSI, and SC, respectively. Similar results were found when controlled for the number of embryos transferred and cryopreservation. The results for twins exhibited similar but less statistically significant trends. Polar body and blastomere biopsies provided similar outcomes. Embryo biopsy itself did not cause intrauterine growth restriction or low birth weight compared with SC, despite lower gestational ages with PGD. The worsened outcomes in ICSI compared with PGD pregnancies may be due to the infertility itself. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Toward Understanding the Mammalian Zygoma: Insights From Comparative Anatomy, Growth and Development, and Morphometric Analysis.

    PubMed

    Márquez, Samuel; Pagano, Anthony S; Schwartz, Jeffrey H; Curtis, Abigail; Delman, Bradley N; Lawson, William; Laitman, Jeffrey T

    2017-01-01

    The zygoma, or jugum, is a cranial element that was present in Mesozoic tetrapods, well before the appearance of mammals. Although as an entity the zygoma is a primitive retention among mammals, it has assumed myriad configurations as this group diversified. As the zygoma is located at the intersection of the visual, respiratory, and masticatory apparatuses, it is potentially of great importance in systematic, phylogenetic, and functional studies focused on this region. For example, the facial component of the zygoma and its contribution to a postorbital bar (POB) appear to be relevant to the systematics of a number of mammalian subclades, and the formation of a bony postorbital septum (POS) that separates the orbit from the infratemporal fossa is unique to, and thus potentially phylogenetically significant for uniting anthropoid primates, while the zygoma itself appears to serve to resist tension and bending forces during mastication. In order to better understand the zygoma in the context of its contributions to the circumorbital region, we documented its morphological expression in specimens representing 10 orders of mammals. Since the presence of a POB and of a POS has long been used to justify uniting extant primates and anthropoid primates as respective clades, and because postorbital closure (POC) is morphologically more complex than a POB, we provide detail necessary to address these claims. Our taxically broad overview also allowed us to provide for the first time definitions of configurations that can be applied to future studies. Using a different, but also taxically broad sample of mammals, and of primates in particular, we performed two geometric morphometric analyses that were geared toward testing long-held interpretations of the functional role of the zygoma, especially with regard to mastication and in the context of orbital frontation (to which the zygoma contributes). Further, overall, zygomatic morphology tends not to scale with allometry

  17. Discovery of a Novel Prolactin in Non-Mammalian Vertebrates: Evolutionary Perspectives and Its Involvement in Teleost Retina Development

    PubMed Central

    Huang, Xigui; Hui, Michelle N. Y.; Liu, Yun; Yuen, Don S. H.; Zhang, Yong; Chan, Wood Yee; Lin, Hao Ran; Cheng, Shuk Han; Cheng, Christopher H. K.

    2009-01-01

    Background The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence. Principal Findings In this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost. Significance The discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates. PMID:19584915

  18. Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single-tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta-thalassemia.

    PubMed

    Chen, Min; Tan, Arnold S C; Cheah, Felicia S H; Saw, Eugene E L; Chong, Samuel S

    2015-12-01

    Beta (β)-thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β-globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage-based PGD for this disorder. To increase the available markers closely flanking the HBB gene, an in silico search was performed to identify all markers within 1 Mb flanking the HBB gene. Fifteen markers with potentially high polymorphism information content (PIC) and heterozygosity values were selected and optimized into a single-tube pentadecaplex PCR panel. Allele frequencies and polymorphism and heterozygosity indices of each marker were assessed in five populations. A total of 238 alleles were observed from the 15 markers. PIC was >0.7 for all markers, with expected heterozygosity and observed heterozygosity values ranging from 0.74 to 0.90 and 0.72 to 0.88, respectively. Greater than 99% of individuals were heterozygous for at least seven markers, with at least two heterozygous markers on either side of the HBB gene. The pentadecaplex marker assay also performed reliably on single cells either directly or after whole genome amplification, thus validating its use in standalone linkage-based β-thalassemia PGD or in conjunction with HBB mutation detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Four simple rules that are sufficient to generate the mammalian blastocyst.

    PubMed

    Nissen, Silas Boye; Perera, Marta; Gonzalez, Javier Martin; Morgani, Sophie M; Jensen, Mogens H; Sneppen, Kim; Brickman, Joshua M; Trusina, Ala

    2017-07-01

    Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs in the absence of maternal information and in the presence of transcriptional stochasticity. How does the preimplantation embryo ensure robust, reproducible development in this context? It utilizes a versatile toolbox that includes complex intracellular networks coupled to cell-cell communication, segregation by differential adhesion, and apoptosis. Here, we ask whether a minimal set of developmental rules based on this toolbox is sufficient for successful blastocyst development, and to what extent these rules can explain mutant and experimental phenotypes. We implemented experimentally reported mechanisms for polarity, cell-cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells' competence of fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network.

  20. Four simple rules that are sufficient to generate the mammalian blastocyst

    PubMed Central

    Nissen, Silas Boye; Perera, Marta; Gonzalez, Javier Martin; Morgani, Sophie M.; Jensen, Mogens H.; Sneppen, Kim; Brickman, Joshua M.

    2017-01-01

    Early mammalian development is both highly regulative and self-organizing. It involves the interplay of cell position, predetermined gene regulatory networks, and environmental interactions to generate the physical arrangement of the blastocyst with precise timing. However, this process occurs in the absence of maternal information and in the presence of transcriptional stochasticity. How does the preimplantation embryo ensure robust, reproducible development in this context? It utilizes a versatile toolbox that includes complex intracellular networks coupled to cell—cell communication, segregation by differential adhesion, and apoptosis. Here, we ask whether a minimal set of developmental rules based on this toolbox is sufficient for successful blastocyst development, and to what extent these rules can explain mutant and experimental phenotypes. We implemented experimentally reported mechanisms for polarity, cell—cell signaling, adhesion, and apoptosis as a set of developmental rules in an agent-based in silico model of physically interacting cells. We find that this model quantitatively reproduces specific mutant phenotypes and provides an explanation for the emergence of heterogeneity without requiring any initial transcriptional variation. It also suggests that a fixed time point for the cells’ competence of fibroblast growth factor (FGF)/extracellular signal—regulated kinase (ERK) sets an embryonic clock that enables certain scaling phenomena, a concept that we evaluate quantitatively by manipulating embryos in vitro. Based on these observations, we conclude that the minimal set of rules enables the embryo to experiment with stochastic gene expression and could provide the robustness necessary for the evolutionary diversification of the preimplantation gene regulatory network. PMID:28700688

  1. Absence of SOX3 in the developing marsupial gonad is not consistent with a conserved role in mammalian sex determination.

    PubMed

    Pask, A J; Harry, J L; Renfree, M B; Marshall Graves, J A

    2000-08-01

    Expression of Sox3 has been detected in the testes of humans and of developing and adult mice at the same time as Sox9 and Sry. The co-expression of these three related Sox genes in the mouse indifferent gonadal ridge led to the hypothesis that these three genes, encoding transcription factors with similar DNA target binding sites, may interact with each other in initiating testis differentiation. The location of SOX3 on the marsupial Dunnart X chromosome also makes it a candidate for the marsupial X-linked gene responsible for the SRY- and hormone-independent initiation of scrotum or mammary gland development. Here we show that although marsupial SOX3 is highly conserved at the genetic level and appears to have a conserved role in CNS development, its expression during sexual differentiation differs from that of mice and humans. SOX3 expression is absent from the developing marsupial genital ridge and from the scrotal and mammary primordia during the critical time of differentiation and throughout the time that SRY is expressed. The absence of expression in the developing gonad strongly suggests that SOX3 does not have a conserved role in mammalian sexual determination or differentiation.

  2. Essential role for Max in early embryonic growth and development

    PubMed Central

    Shen-Li, Hong; O'Hagan, Rónán C.; Hou, Harry; Horner, James W.; Lee, Han-Woong; DePinho, Ronald A.

    2000-01-01

    Loss of Max function in the mouse resulted in generalized developmental arrest of both embryonic and extraembryonic tissues at early postimplantation (∼E5.5–6.5), coincident with loss or dilution of maternal Max stores in the expanding embryo in vivo and in blastocyst outgrowths in vitro. Developmentally arrested embryos were reduced in size and exhibited widespread cytological degeneration and feeble BrdU incorporation. Max and, by extension, the Myc superfamily, serve essential roles in early mammalian development and a maternal reservoir of Max exists in sufficient amount to sustain Myc superfamily function through preimplantation stages of development. PMID:10640271

  3. Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses

    SciTech Connect

    Van Winkle, L.J.; Campione, A.L. )

    1991-07-01

    The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptuses at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity.

  4. Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

    PubMed

    Liu, Honglin; Kim, Jin-Moon; Aoki, Fugaku

    2004-05-01

    Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic

  5. Cell membrane and cell junctions in differentiation of preimplanted mouse embryos.

    PubMed

    Izquierdo, L; Fernández, S; López, T

    1976-12-01

    Cell membrane and cell junctions in differentiation of preimplanted mouse embryos, (membrana celular y uniones celulares en la diferenciación del embrión de ratón antes de la implantación). Arch. Biol. Med. Exper. 10: 130-134, 1976. The development of cell junctions that seal the peripheral blastomeres could be a decisive step in the differentiation of morulae into blastocysts. The appearance of these junctions is studied by electron microscopy of late morulae and initial blastocysts. Zonulae occludentes as well as impermeability to lanthanum emulsion precedes the appearance of the blastocel and hence might be considered as one of its necessary causes.

  6. From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

    PubMed

    Khodiyar, Varsha K; Howe, Doug; Talmud, Philippa J; Breckenridge, Ross; Lovering, Ruth C

    2013-01-01

    For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer's vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer's vesicle determine asymmetry in the developing heart, the direction of 'heart jogging' and the direction of 'heart looping'.  'Heart jogging' is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward 'jog'. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish 'heart jogging orthologs' are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach.

  7. From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

    PubMed Central

    Lovering, Ruth C

    2014-01-01

    For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, the direction of ‘heart jogging’ and the direction of ‘heart looping’.  ‘Heart jogging’ is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish ‘heart jogging orthologs’ are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach. PMID:24627794

  8. Development of transgenic animals for optogenetic manipulation of mammalian nervous system function: Progress and prospects for behavioral neuroscience

    PubMed Central

    Ting, Jonathan T.; Feng, Guoping

    2014-01-01

    Here we review the rapidly growing toolbox of transgenic mice and rats that exhibit functional expression of engineered opsins for neuronal activation and silencing with light. Collectively, these transgenic animals are enabling neuroscientists to access and manipulate the many diverse cell types in the mammalian nervous system in order to probe synaptic and circuitry connectivity, function, and dysfunction. The availability of transgenic lines affords important advantages such as stable and heritable transgene expression patterns across experimental cohorts. As such, the use of transgenic lines precludes the need for other costly and labor-intensive procedures to achieve functional transgene expression in each individual experimental animal. This represents an important consideration when large cohorts of experimental animals are desirable as in many common behavioral assays. We describe the diverse strategies that have been implemented for developing transgenic mouse and rat lines and highlight recent advances that have led to dramatic improvements in achieving functional transgene expression of engineered opsins. Furthermore, we discuss considerations and caveats associated with implementing recently developed transgenic lines for optogenetics-based experimentation. Lastly, we propose strategies that can be implemented to develop and refine the next generation of genetically modified animals for behaviorally-focused optogenetics-based applications. PMID:23473879

  9. Development of a UHPLC-MS/MS method for the determination of plasma histamine in various mammalian species.

    PubMed

    Liu, Jia; Wang, Lei; Hu, Wenjuan; Chen, Xiaoyan; Zhong, Dafang

    2014-11-15

    Histamine is an important mediator of anaphylactic reactions. Although several methods have been developed to measure histamine levels, each has its limitations. In this study, we developed and validated a convenient bioanalytical method for the qualitative and quantitative determination of histamine in plasma samples from humans, beagle dogs, Sprague-Dawley rats, and imprinting control region mice. A simple plasma protein precipitation method using acetonitrile was selected, and hydrophilic interaction liquid chromatography coupled with mass spectrometry was used for sample separation and detection. Histamine was subjected to gradient elution with acetonitrile, ammonium acetate buffer, and formic acid. A mass spectrometer equipped with an electrospray ionization source was operated in the positive-ion multiple reaction monitoring mode for the detection of histamine and the internal standard. The [M+H](+) transitions were m/z 112→95 for histamine and m/z 116→99 for d4-histamine, which was used as the internal standard. The lower limit of quantification was 0.2μg/L and the calibration range was 0.2-500μg/L. The overall recovery ranged from 93.6% to 102.8%. The intra- and inter-run precision and accuracy were <15% for plasma samples from all four species. The method was validated by measuring the plasma histamine concentrations in five healthy human volunteers. In conclusion, we have developed and validated a novel bioanalytical method for the quantification of histamine levels in plasma samples from various mammalian species.

  10. Development of transgenic animals for optogenetic manipulation of mammalian nervous system function: progress and prospects for behavioral neuroscience.

    PubMed

    Ting, Jonathan T; Feng, Guoping

    2013-10-15

    Here we review the rapidly growing toolbox of transgenic mice and rats that exhibit functional expression of engineered opsins for neuronal activation and silencing with light. Collectively, these transgenic animals are enabling neuroscientists to access and manipulate the many diverse cell types in the mammalian nervous system in order to probe synaptic and circuitry connectivity, function, and dysfunction. The availability of transgenic lines affords important advantages such as stable and heritable transgene expression patterns across experimental cohorts. As such, the use of transgenic lines precludes the need for other costly and labor-intensive procedures to achieve functional transgene expression in each individual experimental animal. This represents an important consideration when large cohorts of experimental animals are desirable as in many common behavioral assays. We describe the diverse strategies that have been implemented for developing transgenic mouse and rat lines and highlight recent advances that have led to dramatic improvements in achieving functional transgene expression of engineered opsins. Furthermore, we discuss considerations and caveats associated with implementing recently developed transgenic lines for optogenetics-based experimentation. Lastly, we propose strategies that can be implemented to develop and refine the next generation of genetically modified animals for behaviorally-focused optogenetics-based applications.

  11. Dynamics and ethics of comprehensive preimplantation genetic testing: a review of the challenges.

    PubMed

    Hens, Kristien; Dondorp, Wybo; Handyside, Alan H; Harper, Joyce; Newson, Ainsley J; Pennings, Guido; Rehmann-Sutter, Christoph; de Wert, Guido

    2013-01-01

    Genetic testing of preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Microarray technology is being introduced in both these contexts, and whole genome sequencing of blastomeres is also expeted to become possible soon. The amount of extra information such tests will yield may prove to be beneficial for embryo selection, will also raise various ethical issues. We present an overview of the developments and an agenda-setting exploration of the ethical issues. The paper is a joint endeavour by the presenters at an explorative 'campus meeting' organized by the European Society of Human Reproduction and Embryology in cooperation with the department of Health, Ethics & Society of the Maastricht University (The Netherlands). The increasing amount and detail of information that new screening techniques such as microarrays and whole genome sequencing offer does not automatically coincide with an increasing understanding of the prospects of an embryo. From a technical point of view, the future of comprehensive embryo testing may go together with developments in preconception carrier screening. From an ethical point of view, the increasing complexity and amount of information yielded by comprehensive testing techniques will lead to challenges to the principle of reproductive autonomy and the right of the child to an open future, and may imply a possible larger responsibility of the clinician towards the welfare of the future child. Combinations of preconception carrier testing and embryo testing may solve some of these ethical questions but could introduce others. As comprehensive testing techniques are entering the IVF clinic, there is a need for a thorough rethinking of traditional ethical paradigms regarding medically assisted reproduction.

  12. Melatonin inhibits paraquat-induced cell death in bovine preimplantation embryos.

    PubMed

    Pang, Yun-Wei; Sun, Ye-Qing; Sun, Wei-Jun; Du, Wei-Hua; Hao, Hai-Sheng; Zhao, Shan-Jiang; Zhu, Hua-Bin

    2016-03-01

    Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Is the zona pellucida an intrinsic source of signals activating maternal recognition of the developing mammalian embryo?

    PubMed

    Fujiwara, Hiroshi; Araki, Yoshihiko; Toshimori, Kiyotaka

    2009-07-01

    Mammalian mothers undergoing embryo implantation must specifically recognize the developing embryo in a species-restricted manner. We previously observed that immune cells derived from early pregnant mice could promote endometrial differentiation and embryo implantation in blastocyst-transferred pseudopregnant mice. Although the precise mechanism remains unknown, it is suggested that the maternal immune system undergoes functional changes after recognizing developing embryos from the very early stages of pregnancy. Since it is physically impossible for immune cells to directly interact with the developing embryo while it is surrounded by the zona pellucida (ZP), it is speculated that the embryo produces certain embryo- and species-specific soluble factor(s) in the oviduct before hatching. As a candidate for this factor, we have paid attention to the ZP that is normally protected from immunological attack during oogenesis in the ovarian follicle. ZP-specific glycoproteins are known to play important roles in the species- and oocyte-specific binding of sperm, and the ZP can also be considered an abundant store of oocyte- and species-specific glycoproteins. In contrast to unfertilized oocytes, developing embryos may degrade the ZP starting just after fertilization and proceeding until hatching using enzymes that are released from cortical granules or produced by the developing embryo. Accordingly, the developing embryo might provide ZP-degradation products including oligosaccharide chains to the immune system from the very early stages. Taken together, we propose here a novel hypothesis that these ZP-derivatives can act as an intrinsic signal from the developing embryo for maternal recognition by the immune system.

  14. Timing of mammalian peripheral trigeminal system development relative to body size: A comparison of metatherians with rodents and monotremes.

    PubMed

    Ashwell, Ken W S

    2015-01-01

    Specializations of the trigeminal sensory system are present in all three infraclasses of mammals (metatheria, eutheria, prototheria or monotremata). The trigeminal sensory system has been suggested as a critically important modality for sampling the path to the pouch and detecting the nipple or milk patch, but the degree to which that system may be required to function at birth varies significantly. Archived sections of the snout and brainstem of embryonic and postnatal mammals were used to test the relationship between structural maturity of the two ends of the trigeminal nerve pathway and the body size of mammalian young in metatherians, rodents and monotremes. A system for staging different levels of structural maturity of the vibrissae and trigeminal sensory was applied to embryos, pouch young and hatchlings and correlated with body length. Dasyurids are born at the most immature state with respect to vibrissal and trigeminal sensory nucleus development of any available metatherian, but these components of the trigeminal system are also developmentally advanced relative to body size when dasyurids are compared to other metatherians. Vibrissal and trigeminal sensory nucleus development is at a similar stage of development at birth and for a given body size in non-dasyurid metatherians; and trigeminal sensory nucleus development in monotremes is at a similar stage at birth to metatherians. Rodents reach a far more advanced stage of vibrissal and trigeminal sensory nucleus development at birth than do metatherians, and in the case of the mouse have a more developmentally advanced trigeminal system than all available metatherians at any given body length. Precocious development of the trigeminal sensory pathway relative to body size is evident in dasyurids, as might be expected given the small birth size of those metatherians. Nevertheless, the trigeminal sensory system in metatherians in general is not precocious relative to body size when these species are

  15. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells

    PubMed Central

    Grow, Edward J.; Flynn, Ryan A.; Chavez, Shawn L.; Bayless, Nicholas L.; Wossidlo, Mark; Wesche, Daniel; Martin, Lance; Ware, Carol; Blish, Catherine A.; Chang, Howard Y.; Reijo Pera, Renee A.; Wysocka, Joanna

    2015-01-01

    Summary Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, which comprise nearly 8% of the human genome1. The most recently acquired human ERV is HERV-K (HML-2), which repeatedly infected the primate lineage both before and after the divergence of humans and chimpanzees2,3. Unlike most other human ERVs, HERV-K retained multiple copies of intact open reading frames (ORFs) encoding retroviral proteins4. However, HERV-K is transcriptionally silenced by the host with exception of certain pathological contexts, such as germ cell tumors, melanoma, or HIV infection5–7. Here we demonstrate that DNA hypomethylation at LTR elements representing the most recent genomic integrations, together with transactivation by OCT4, synergistically facilitate HERV-K expression. Consequently, HERV-K is transcribed during normal human embryogenesis beginning with embryonic genome activation (EGA) at the 8-cell stage, continuing through the emergence of epiblast cells in pre-implantation blastocysts, and ceasing during hESC derivation from blastocyst outgrowths. Remarkably, HERV-K viral-like particles and Gag proteins are detected in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, HERV-K accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERV-K can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, arguing that complex interactions between retroviral proteins and host factors can fine-tune regulatory properties of early human development. PMID:25896322

  16. The biology of mammalian parenting and its effect on offspring social development

    PubMed Central

    Rilling, James K.; Young, Larry J.

    2015-01-01

    Parents know the transformative nature of having and caring for a child. Among many mammals, giving birth leads from an aversion to infant stimuli to irresistible attraction. Here, we review the biological mechanisms governing this shift in parental motivation in mammals. Estrogen and progesterone prepare the uterus for embryo implantation and placental development. Prolactin stimulates milk production, whereas oxytocin initiates labor and triggers milk ejection during nursing. These same molecules, interacting with dopamine, also activate specific neural pathways to motivate parents to nurture, bond with, and protect their offspring. Parenting in turn shapes the neural development of the infant social brain. Recent work suggests that many of the principles governing parental behavior and its effect on infant development are conserved from rodent to humans. PMID:25124431

  17. The biology of mammalian parenting and its effect on offspring social development.

    PubMed

    Rilling, James K; Young, Larry J

    2014-08-15

    Parents know the transformative nature of having and caring for a child. Among many mammals, giving birth leads from an aversion to infant stimuli to irresistible attraction. Here, we review the biological mechanisms governing this shift in parental motivation in mammals. Estrogen and progesterone prepare the uterus for embryo implantation and placental development. Prolactin stimulates milk production, whereas oxytocin initiates labor and triggers milk ejection during nursing. These same molecules, interacting with dopamine, also activate specific neural pathways to motivate parents to nurture, bond with, and protect their offspring. Parenting in turn shapes the neural development of the infant social brain. Recent work suggests that many of the principles governing parental behavior and its effect on infant development are conserved from rodent to humans.

  18. Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

    NASA Technical Reports Server (NTRS)

    Spooner, B. S.; Hardman, P.; Paulsen, A.

    1994-01-01

    Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

  19. Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight

    NASA Technical Reports Server (NTRS)

    Spooner, B. S.; Hardman, P.; Paulsen, A.

    1994-01-01

    Organ culture of embryonic mouse lung and pancreas rudiments has been used to investigate development and differentiation, and to assess the effects of microgravity on culture differentiation, during orbital spaceflight of the shuttle Endeavour (mission STS-54). Lung rudiments continue to grow and branch during spaceflight, an initial result that should allow future detailed study of lung morphogenesis in microgravity. Cultured embryonic pancreas undergoes characteristic exocrine acinar tissue and endocrine islet tissue differentiation during spaceflight, and in ground controls. The rudiments developing in the microgravity environment of spaceflight appear to grow larger than their ground counterparts, and they may have differentiated more rapidly than controls, as judged by exocrine zymogen granule presence.

  20. The functional diversity of essential genes required for mammalian cardiac development.

    PubMed

    Clowes, Christopher; Boylan, Michael G S; Ridge, Liam A; Barnes, Emma; Wright, Jayne A; Hentges, Kathryn E

    2014-08-01

    Genes required for an organism to develop to maturity (for which no other gene can compensate) are considered essential. The continuing functional annotation of the mouse genome has enabled the identification of many essential genes required for specific developmental processes including cardiac development. Patterns are now emerging regarding the functional nature of genes required at specific points throughout gestation. Essential genes required for development beyond cardiac progenitor cell migration and induction include a small and functionally homogenous group encoding transcription factors, ligands and receptors. Actions of core cardiogenic transcription factors from the Gata, Nkx, Mef, Hand, and Tbx families trigger a marked expansion in the functional diversity of essential genes from midgestation onwards. As the embryo grows in size and complexity, genes required to maintain a functional heartbeat and to provide muscular strength and regulate blood flow are well represented. These essential genes regulate further specialization and polarization of cell types along with proliferative, migratory, adhesive, contractile, and structural processes. The identification of patterns regarding the functional nature of essential genes across numerous developmental systems may aid prediction of further essential genes and those important to development and/or progression of disease.

  1. A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

    PubMed Central

    Bruun, Tim-Henrik; Mühlbauer, Katharina; Benen, Thomas; Kliche, Alexander; Wagner, Ralf

    2014-01-01

    An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates. PMID:25279768

  2. A mammalian cell based FACS-panning platform for the selection of HIV-1 envelopes for vaccine development.

    PubMed

    Bruun, Tim-Henrik; Mühlbauer, Katharina; Benen, Thomas; Kliche, Alexander; Wagner, Ralf

    2014-01-01

    An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.

  3. Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development

    PubMed Central

    Mao, Yaopan; Mulvaney, Joanna; Zakaria, Sana; Yu, Tian; Morgan, Katherine Malanga; Allen, Steve; Basson, M. Albert; Francis-West, Philippa; Irvine, Kenneth D.

    2011-01-01

    The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs. PMID:21303848

  4. The evidence base regarding the experiences of and attitudes to preimplantation genetic diagnosis in prospective parents.

    PubMed

    Cunningham, Jenny; Goldsmith, Lesley; Skirton, Heather

    2015-02-01

    Preimplantation genetic diagnosis was developed as an alternative to prenatal diagnosis for couples with a family history of genetic disease. After in vitro fertilization, the embryos can be analysed to ensure that only healthy embryos are transferred to the uterus. Past studies have suggested that couples who wish to avoid having a child with an inherited genetic condition look favourably on preimplantation genetic diagnosis as it prevents the need for termination of pregnancy following prenatal diagnosis of an affected fetus. However, it is important to understand the experiences of couples who have used or consider using this technique. To ascertain the current evidence base on this topic, we conducted a mixed methods systematic review. Four databases were searched for relevant peer-reviewed papers published between 2000 and 2013. Of 453 papers, nine satisfied the inclusion criteria and were assessed for quality. Results of nine papers were analysed and synthesised using a narrative approach. Three main themes emerged: (1) motivating factors; (2) emotional labour; (3) choices and uncertainty. The review has identified an emotional and difficult journey for couples pursuing preimplantation genetic diagnosis. While use of the technique gives hope to families who wish to prevent transmission of a genetic disease this is not without hard decision-making and periods of uncertainty. Lack of information was perceived as a barrier to access this reproductive option. Recommendations include: training and education in genetics for midwives who are the first point of contact for pregnant women; clinics to use a decision-making tool to emphasise the uncertainty involved in PGD and improved communication and psychological support to couples. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme.

    PubMed

    Feng, Weijun; Kawauchi, Daisuke; Körkel-Qu, Huiqin; Deng, Huan; Serger, Elisabeth; Sieber, Laura; Lieberman, Jenna Ariel; Jimeno-González, Silvia; Lambo, Sander; Hanna, Bola S; Harim, Yassin; Jansen, Malin; Neuerburg, Anna; Friesen, Olga; Zuckermann, Marc; Rajendran, Vijayanad; Gronych, Jan; Ayrault, Olivier; Korshunov, Andrey; Jones, David T W; Kool, Marcel; Northcott, Paul A; Lichter, Peter; Cortés-Ledesma, Felipe; Pfister, Stefan M; Liu, Hai-Kun

    2017-03-20

    Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.

  6. Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme

    PubMed Central

    Feng, Weijun; Kawauchi, Daisuke; Körkel-Qu, Huiqin; Deng, Huan; Serger, Elisabeth; Sieber, Laura; Lieberman, Jenna Ariel; Jimeno-González, Silvia; Lambo, Sander; Hanna, Bola S.; Harim, Yassin; Jansen, Malin; Neuerburg, Anna; Friesen, Olga; Zuckermann, Marc; Rajendran, Vijayanad; Gronych, Jan; Ayrault, Olivier; Korshunov, Andrey; Jones, David T. W.; Kool, Marcel; Northcott, Paul A.; Lichter, Peter; Cortés-Ledesma, Felipe; Pfister, Stefan M.; Liu, Hai-Kun

    2017-01-01

    Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation. PMID:28317875

  7. The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome.

    PubMed

    Bachiller, Daniel; Klingensmith, John; Shneyder, Natalya; Tran, Uyen; Anderson, Ryan; Rossant, Janet; De Robertis, E M

    2003-08-01

    The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. We present the phenotype produced by the targeted inactivation of the chordin gene in mouse. Chordin homozygous mutant mice show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally, displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis.

  8. Local apoptosis modulates early mammalian brain development through the elimination of morphogen-producing cells.

    PubMed

    Nonomura, Keiko; Yamaguchi, Yoshifumi; Hamachi, Misato; Koike, Masato; Uchiyama, Yasuo; Nakazato, Kenichi; Mochizuki, Atsushi; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Yoshida, Hiroki; Kuida, Keisuke; Miura, Masayuki

    2013-12-23

    Apoptotic cells are observed in the early developing brain. Apoptosis deficiency is proposed to cause brain overgrowth, but here we show that brain malformations in apoptosis-deficient mutants are due to insufficient brain ventricle expansion as a result of uncompleted cranial neural tube closure. Apoptosis eliminates Fgf8-expressing cells in the anterior neural ridge (ANR), which acts as an organizing center of the forebrain by producing FGF8 morphogen. Deficiency of apoptosis leads to the accumulation of undead and nonproliferative cells in the ventral part of the ANR. The undead cells in apoptosis-deficient mutants express Fgf8 continuously, which perturbs gene expression in the ventral forebrain. Thus, apoptosis within a specific subdomain of the ANR is required for correct temporal elimination of an FGF8-producing region within a limited developmental time window, thereby ensuring proper forebrain development. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Development of coherent neuronal activity patterns in mammalian cortical networks: common principles and local hetereogeneity.

    PubMed

    Egorov, Alexei V; Draguhn, Andreas

    2013-01-01

    Many mammals are born in a very immature state and develop their rich repertoire of behavioral and cognitive functions postnatally. This development goes in parallel with changes in the anatomical and functional organization of cortical structures which are involved in most complex activities. The emerging spatiotemporal activity patterns in multi-neuronal cortical networks may indeed form a direct neuronal correlate of systemic functions like perception, sensorimotor integration, decision making or memory formation. During recent years, several studies--mostly in rodents--have shed light on the ontogenesis of such highly organized patterns of network activity. While each local network has its own peculiar properties, some general rules can be derived. We therefore review and compare data from the developing hippocampus, neocortex and--as an intermediate region--entorhinal cortex. All cortices seem to follow a characteristic sequence starting with uncorrelated activity in uncoupled single neurons where transient activity seems to have mostly trophic effects. In rodents, before and shortly after birth, cortical networks develop weakly coordinated multineuronal discharges which have been termed synchronous plateau assemblies (SPAs). While these patterns rely mostly on electrical coupling by gap junctions, the subsequent increase in number and maturation of chemical synapses leads to the generation of large-scale coherent discharges. These patterns have been termed giant depolarizing potentials (GDPs) for predominantly GABA-induced events or early network oscillations (ENOs) for mostly glutamatergic bursts, respectively. During the third to fourth postnatal week, cortical areas reach their final activity patterns with distinct network oscillations and highly specific neuronal discharge sequences which support adult behavior. While some of the mechanisms underlying maturation of network activity have been elucidated much work remains to be done in order to fully

  10. [Molecular Karyotyping of Cell-Free DNA from Blastocoele Fluid as a Basis for Noninvasive Preimplantation Genetic Screening of Aneuploidy].

    PubMed

    Skryabin, N A; Lebedev, I N; Artukhova, V G; Zhigalina, D I; Stepanov, I A; Krivoschekova, G V; Svetlakov, A V

    2015-11-01

    The discovery of DNA fragments in the blastocoele fluid is promising for the development of new noninvasive methods for the preimplantation genetic diagnosis of chromosomal diseases. However, to date there are no data confirming the concordance between the molecular karyotype of cell-free DNA from blastocoele fluid and the blastocyst cells per se. This paper reports on this concordance according to the results of molecular-cytogenetic analysis of the chromosomal set with the use of comparative genomic hybridization.

  11. Results of a survey of the legal status and attitudes towards preimplantation genetic diagnosis conducted in 13 different countries.

    PubMed

    Viville, S; Pergament, D

    1998-12-01

    Considerable differences in the regulatory oversight of preimplantation genetic diagnostic (PGD) services exist among countries, ranging from total bans on any embryo manipulation to the almost complete absence of any regulations or authority. A survey of centres offering PGD services revealed common concerns about the creation of embryos for research, altering the pattern of normal embryo development and the potential for misuse in the clinical application of PGD.

  12. The experience of 3 years of external quality assessment of preimplantation genetic diagnosis for cystic fibrosis

    PubMed Central

    Deans, Zandra; Fiorentino, Francesco; Biricik, Anil; Traeger-Synodinos, Joanne; Moutou, Céline; De Rycke, Martine; Renwick, Pamela; SenGupta, Sioban; Goossens, Veerle; Harton, Gary

    2013-01-01

    Preimplantation genetic diagnosis (PGD) was first performed over 20 years ago and has become an accepted part of genetic testing and assisted reproduction worldwide. The techniques and protocols necessary to carry out genetic testing at the single-cell level can be difficult to master and have been developed independently by the laboratories worldwide offering preimplantation testing. These factors indicated the need for an external quality assessment (EQA) scheme for monogenic disease PGD. Toward this end, the European Society for Human Reproduction and Embryology came together with United Kingdom National External Quality Assessment Services for Molecular Genetics, to create a pilot EQA scheme followed by practical EQA schemes for all interested parties. Here, we detail the development of the pilot scheme as well as development and findings from the practical (clinical) schemes that have followed. Results were generally acceptable and there was marked improvement in results and laboratory scores for those labs that participated in multiple schemes. Data from the first three schemes indicate that the EQA scheme is working as planned and has helped laboratories improve their techniques and result reporting. The EQA scheme for monogenic PGD will continue to be developed to offer assessment for other monogenic disorders. PMID:23150080

  13. Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione.

    PubMed

    Moss, James I; Pontes, Eduardo; Hansen, Peter James

    2009-11-01

    Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 microM) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 muM can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

  14. The role of the innate immune response regulatory gene ABCF1 in mammalian embryogenesis and development.

    PubMed

    Wilcox, Sara M; Arora, Hitesh; Munro, Lonna; Xin, Jian; Fenninger, Franz; Johnson, Laura A; Pfeifer, Cheryl G; Choi, Kyung Bok; Hou, Juan; Hoodless, Pamela A; Jefferies, Wilfred A

    2017-01-01

    ABCF1 is an ABC transporter family protein that has been shown to regulate innate immune response and is a risk gene for autoimmune pancreatitis and arthritis. Unlike other members of ABC transporter family, ABCF1 lacks trans-membrane domains and is thought to function in translation initiation through an interaction with eukaryotic translation initiation factor 2 (eIF2). To study ABCF1 expression and function in development and disease, we used a single gene trap insertion in the Abcf1 gene in murine embryonic stem cells (ES cells) that allowed lineage tracing of the endogenous Abcf1 promoter by following the expression of a β-galactosidase reporter gene. From the ES cells, heterozygous mice (Abcf1+/-) were produced. No live born Abcf1-/- progeny were ever generated, and the lethality was not mouse strain-specific. Thus, we have determined that Abcf1 is an essential gene in development. Abcf1-/- mice were found to be embryonic lethal at 3.5 days post coitum (dpc), while Abcf1+/- mice appeared developmentally normal. Abcf1+/- mice were fertile and showed no significant differences in their anatomy when compared with their wild type littermates. The Abcf1 promoter was found to be active in all organs in adult mice, but varies in levels of expression in specific cell types within tissues. Furthermore, we observed high promoter activity in the blastocysts and embryos. Overall, Abcf1 expression in embryos is required for development and its expression in adults was highly correlated with actively proliferating and differentiating cell types.

  15. Expression and function of FGF10 in mammalian inner ear development

    NASA Technical Reports Server (NTRS)

    Pauley, Sarah; Wright, Tracy J.; Pirvola, Ulla; Ornitz, David; Beisel, Kirk; Fritzsch, Bernd

    2003-01-01

    We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors. Copyright 2003 Wiley-Liss, Inc.

  16. Development of a 3D matrix for modeling mammalian spinal cord injury in vitro.

    PubMed

    Diaz Quiroz, Juan Felipe; Li, Yuping; Aparicio, Conrado; Echeverri, Karen

    2016-11-01

    Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel therapies, this is in part due to the complexity of the injury and the difficulty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrix in vitro and when injured, the cells respond as they do in vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells after exposure to drugs.

  17. Expression and function of FGF10 in mammalian inner ear development

    NASA Technical Reports Server (NTRS)

    Pauley, Sarah; Wright, Tracy J.; Pirvola, Ulla; Ornitz, David; Beisel, Kirk; Fritzsch, Bernd

    2003-01-01

    We have investigated the expression of FGF10 during ear development and the effect of an FGF10 null mutation on ear development. Our in situ hybridization data reveal expression of FGF10 in all three canal crista sensory epithelia and the cochlea anlage as well as all sensory neurons at embryonic day 11.5 (E11.5). Older embryos (E18.5) displayed strong graded expression in all sensory epithelia. FGF10 null mutants show complete agenesis of the posterior canal crista and the posterior canal. The posterior canal sensory neurons form initially and project rather normally by E11.5, but they disappear within 2 days. FGF10 null mutants have no posterior canal system at E18.5. In addition, these mutants have deformations of the anterior and horizontal cristae, reduced formation of the anterior and horizontal canals, as well as altered position of the remaining sensory epithelia with respect to the utricle. Hair cells form but some have defects in their cilia formation. No defects were detected in the organ of Corti at the cellular level. Together these data suggest that FGF10 plays a major role in ear morphogenesis. Most of these data are consistent with earlier findings on a null mutation in FGFR2b, one of FGF10's main receptors. Copyright 2003 Wiley-Liss, Inc.

  18. Development of a 3D matrix for modeling mammalian spinal cord injury in vitro

    PubMed Central

    Diaz Quiroz, Juan Felipe; Li, Yuping; Aparicio, Conrado; Echeverri, Karen

    2016-01-01

    Spinal cord injury affects millions of people around the world, however, limited therapies are available to improve the quality of life of these patients. Spinal cord injury is usually modeled in rats and mice using contusion or complete transection models and this has led to a deeper understanding of the molecular and cellular complexities of the injury. However, it has not to date led to development of successful novel therapies, this is in part due to the complexity of the injury and the difficulty of deciphering the exact roles and interactions of different cells within this complex environment. Here we developed a collagen matrix that can be molded into the 3D tubular shape with a lumen and can hence support cell interactions in a similar architecture to a spinal cord. We show that astrocytes can be successfully grown on this matrix in vitro and when injured, the cells respond as they do in vivo and undergo reactive gliosis, one of the steps that lead to formation of a glial scar, the main barrier to spinal cord regeneration. In the future, this system can be used to quickly assess the effect of drugs on glial scar protein activity or to perform live imaging of labeled cells after exposure to drugs. PMID:28123426

  19. Inhibitory effects of catechin derivatives on mammalian DNA polymerase and topoisomerase activities and mouse one-cell zygote development.

    PubMed

    Yoshida, Naoko; Kuriyama, Isoko; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2013-03-01

    In this study, the inhibitory activities against DNA polymerases (pols) and DNA topoisomerases (topos) by eight major green tea catechin derivatives (flavan-3-ols) were investigated. Some catechins inhibited mammalian pols (α and β) and human topos (I and II), with (-)-epigallocatechin gallate (EGCg) the strongest inhibitor of both enzyme types, showing IC(50) values of 3.8-21.5 and 2.0-20.0 μM, respectively. EGCg did not affect the activities of plant (cauliflower) pol α or prokaryotic pols and showed no effect on the activities of other DNA metabolic enzymes tested. Next, a method was established for assay of mouse one-cell zygote development inhibition, the catechin derivatives screened for bioactivity, and the inhibition was assessed and their effects ranked as: EGCg > GCg > Cg > others. In the mouse one-cell zygote assay, EGCg at 50 μM increased abnormal cells and 75 μM of EGCg-induced apoptosis. The observed ranking of catechin derivative inhibition effects against mouse one-cell zygote development in vivo was similar to their ranking by topo inhibition in vitro rather than by pol inhibition; therefore, topo inhibition might have been effecting zygote development inhibition. These results suggested that catechin derivatives indeed reached the nuclear DNA where topo inhibition can occur, thus causing the observed cellular effects. From these findings, this zygote development inhibition assay will be useful as an anti-pregnant agent screening. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Dynamic Expression of Sox2, Gata3, and Prox1 during Primary Auditory Neuron Development in the Mammalian Cochlea

    PubMed Central

    Dabdoub, Alain

    2017-01-01

    Primary auditory neurons (PANs) connect cochlear sensory hair cells in the mammalian inner ear to cochlear nucleus neurons in the brainstem. PANs develop from neuroblasts delaminated from the proneurosensory domain of the otocyst and keep maturing until the onset of hearing after birth. There are two types of PANs: type I, which innervate the inner hair cells (IHCs), and type II, which innervate the outer hair cells (OHCs). Glial cells surrounding these neurons originate from neural crest cells and migrate to the spiral ganglion. Several transcription factors are known to regulate the development and differentiation of PANs. Here we systematically examined the spatiotemporal expression of five transcription factors: Sox2, Sox10, Gata3, Mafb, and Prox1 from early delamination at embryonic day (E) 10.5 to adult. We found that Sox2 and Sox10 were initially expressed in the proneurosensory cells in the otocyst (E10.5). By E12.75 both Sox2 and Sox10 were downregulated in the developing PANs; however, Sox2 expression transiently increased in the neurons around birth. Furthermore, both Sox2 and Sox10 continued to be expressed in spiral ganglion glial cells. We also show that Gata3 and Prox1 were first expressed in all developing neurons, followed by a decrease in expression of Gata3 and Mafb in type I PANs and Prox1 in type II PANs as they matured. Moreover, we describe two subtypes of type II neurons based on Peripherin expression. These results suggest that Sox2, Gata3 and Prox1 play a role during neurogenesis as well as maturation of the PANs. PMID:28118374

  1. Secretagogin is a Ca2+-binding protein identifying prospective extended amygdala neurons in the developing mammalian telencephalon

    PubMed Central

    Mulder, Jan; Spence, Lauren; Tortoriello, Giuseppe; DiNieri, Jennifer A.; Uhlén, Mathias; Shui, Bo; Kotlikoff, Michael I.; Yanagawa, Yuchio; Aujard, Fabienne; Hökfelt, Tomas; Hurd, Yasmin L.; Harkany, Tibor

    2010-01-01

    The Ca2+-binding proteins (CBPs) calbindin D28k, calretinin and parvalbumin are phenotypic markers of functionally diverse subclasses of neurons in the adult brain. The developmental dynamics of CBP expression are precisely timed: calbindin and calretinin are present in prospective cortical interneurons from mid-gestation, while parvalbumin only becomes expressed during the early postnatal period in rodents. Secretagogin (scgn) is a CBP cloned from pancreatic β and neuroendocrine cells. We hypothesized that scgn may be expressed by particular neuronal contingents during prenatal development of the mammalian telencephalon. We find that scgn is expressed in neurons transiting in the subpallial differentiation zone by embryonic day (E) 11 in mouse. From E12, scgn+ cells commute towards the extended amygdala and colonize the bed nucleus of stria terminalis, interstitial nucleus of the posterior limb of the anterior commissure, dorsal substantia innominata (SI), and the central and medial amygdaloid nuclei. Scgn+ neurons can acquire a cholinergic phenotype in the SI or differentiate into GABA cells in the central amygdala. We also uncover phylogenetic differences in scgn expression since this CBP defines not only neurons destined to the extended amygdala but also cholinergic projection cells and cortical pyramidal cells in the fetal non-human primate and human brains, respectively. Overall, our findings emphasize the developmentally shared origins of neurons populating the extended amygdala, and suggest that secretagogin can be relevant to the generation of functional modalities in specific neuronal circuitries. PMID:20529129

  2. The mammalian Cos2 homolog Kif7 plays an essential role in modulating Hh signal transduction during development.

    PubMed

    Endoh-Yamagami, Setsu; Evangelista, Marie; Wilson, Deanna; Wen, Xiaohui; Theunissen, Jan-Willem; Phamluong, Khanhky; Davis, Matti; Scales, Suzie J; Solloway, Mark J; de Sauvage, Frederic J; Peterson, Andrew S

    2009-08-11

    The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, we demonstrate that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. We show that Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. We also demonstrate a requirement for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh.

  3. Gene-Chemical Interactions in the Developing Mammalian Nervous System: Effects on Proliferation, Neurogenesis and Differentiation

    PubMed Central

    Fox, Donald A.; Opanashuk, Lisa; Zharkovsky, Aleksander; Weiss, Bernie

    2010-01-01

    The orderly formation of the nervous system requires a multitude of complex, integrated and simultaneously occurring processes. Neural progenitor cells expand through proliferation, commit to different cell fates, exit the cell cycle, generate different neuronal and glial cell types, and new neurons migrate to specified areas and establish synaptic connections. Gestational and perinatal exposure to environmental toxicants, pharmacological agents and drugs of abuse produce immediate, persistent or late-onset alterations in behavioral, cognitive, sensory and/or motor functions. These alterations reflect the disruption of the underlying processes of CNS formation and development. To determine the neurotoxic mechanisms that underlie these deficits it is necessary to analyze and dissect the complex molecular processes that occur during the proliferation, neurogenesis and differentiation of cells. This symposium will provide a framework for understanding the orchestrated events of neurogenesis, the coordination of proliferation and cell fate specification by selected genes, and the effects of well-known neurotoxicants on neurogenesis in the retina, hippocampus and cerebellum. These three tissues share common developmental profiles, mediate diverse neuronal activities and function, and thus provide important substrates for analysis. This paper summarizes four invited talks that were presented at the 12th International Neurotoxicology Association meeting held in Jerusalem, Israel during the summer of 2009. Donald A. Fox described the structural and functional alterations following low-level gestational lead exposure in children and rodents that produced a supernormal electroretinogram and selective increases in neurogenesis and cell proliferation of late-born retinal neurons (rod photoreceptors and bipolar cells), but not Müller glia cells, in mice. Lisa Opanashuk discussed how dioxin [TCDD] binding to the arylhydrocarbon receptor [AhR], a transcription factor that

  4. Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

    PubMed

    Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

    2011-01-01

    Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

  5. Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series

    PubMed Central

    Bowman, Caitlyn E.; Hartung, Thomas

    2016-01-01

    Glucose and oxygen are two of the most important molecules transferred from mother to fetus during eutherian pregnancy, and the metabolic fates of these nutrients converge at the transport and metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex in the inner mitochondrial membrane that consists of two essential components, MPC1 and MPC2. Here, we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. Mpc1 deletion was homozygous lethal in midgestation, but Mpc1 hypomorphs and tissue-specific deletion of Mpc1 presented as early perinatal lethality. The allelic series demonstrated that graded suppression of MPC resulted in dose-dependent metabolic and transcriptional changes. Steady-state metabolomics analysis of brain and liver from Mpc1 hypomorphic embryos identified compensatory changes in amino acid and lipid metabolism. Flux assays in Mpc1-deficient embryonic fibroblasts also reflected these changes, including a dramatic increase in mitochondrial alanine utilization. The mitochondrial alanine transaminase GPT2 was found to be necessary and sufficient for increased alanine flux upon MPC inhibition. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be mitigated in part by alternative anaplerotic substrates in utero. PMID:27215380

  6. Skeletal development in sloths and the evolution of mammalian vertebral patterning.

    PubMed

    Hautier, Lionel; Weisbecker, Vera; Sánchez-Villagra, Marcelo R; Goswami, Anjali; Asher, Robert J

    2010-11-02

    Mammals show a very low level of variation in vertebral count, particularly in the neck. Phenotypes exhibited at various stages during the development of the axial skeleton may play a key role in testing mechanisms recently proposed to explain this conservatism. Here, we provide osteogenetic data that identify developmental criteria with which to recognize cervical vs. noncervical vertebrae in mammals. Except for sloths, all mammals show the late ossification of the caudal-most centra in the neck after other centra and neural arches. In sloths with 8-10 ribless neck vertebrae, the caudal-most neck centra ossify early, matching the pattern observed in cranial thoracic vertebrae of other mammals. Accordingly, we interpret the ribless neck vertebrae of three-toed sloths caudal to V7 as thoracic based on our developmental criterion. Applied to the unusual vertebral phenotype of long-necked sloths, these data support the interpretation that elements of the axial skeleton with origins from distinct mesodermal tissues have repatterned over the course of evolution.

  7. Golgb1 regulates protein glycosylation and is crucial for mammalian palate development.

    PubMed

    Lan, Yu; Zhang, Nian; Liu, Han; Xu, Jingyue; Jiang, Rulang

    2016-07-01

    Cleft palate is a common major birth defect for which currently known causes account for less than 30% of pathology in humans. In this study, we carried out mutagenesis screening in mice to identify new regulators of palatogenesis. Through genetic linkage mapping and whole-exome sequencing, we identified a loss-of-function mutation in the Golgb1 gene that co-segregated with cleft palate in a new mutant mouse line. Golgb1 is a ubiquitously expressed large coiled-coil protein, also known as giantin, that is localized at the Golgi membrane. Using CRISPR/Cas9-mediated genome editing, we generated and analyzed developmental defects in mice carrying additional Golgb1 loss-of-function mutations, which supported a crucial requirement for Golgb1 in palate development. Through maxillary explant culture assays, we demonstrate that the Golgb1 mutant embryos have intrinsic defects in palatal shelf elevation. Just prior to the developmental stage of palatal shelf elevation in wild-type littermates, Golgb1 mutant embryos exhibit increased cell density, reduced hyaluronan accumulation and impaired protein glycosylation in the palatal mesenchyme. Together, these results demonstrate that, although it is a ubiquitously expressed Golgi-associated protein, Golgb1 has specific functions in protein glycosylation and tissue morphogenesis. © 2016. Published by The Company of Biologists Ltd.

  8. Skeletal development in sloths and the evolution of mammalian vertebral patterning

    PubMed Central

    Hautier, Lionel; Weisbecker, Vera; Sánchez-Villagra, Marcelo R.; Goswami, Anjali; Asher, Robert J.

    2010-01-01

    Mammals show a very low level of variation in vertebral count, particularly in the neck. Phenotypes exhibited at various stages during the development of the axial skeleton may play a key role in testing mechanisms recently proposed to explain this conservatism. Here, we provide osteogenetic data that identify developmental criteria with which to recognize cervical vs. noncervical vertebrae in mammals. Except for sloths, all mammals show the late ossification of the caudal-most centra in the neck after other centra and neural arches. In sloths with 8–10 ribless neck vertebrae, the caudal-most neck centra ossify early, matching the pattern observed in cranial thoracic vertebrae of other mammals. Accordingly, we interpret the ribless neck vertebrae of three-toed sloths caudal to V7 as thoracic based on our developmental criterion. Applied to the unusual vertebral phenotype of long-necked sloths, these data support the interpretation that elements of the axial skeleton with origins from distinct mesodermal tissues have repatterned over the course of evolution. PMID:20956304

  9. Complex coacervate microcapsules for mammalian cell culture and artificial organ development.

    PubMed

    Matthew, H W; Salley, S O; Peterson, W D; Klein, M D

    1993-01-01

    A number of combinations of anionic and cationic polymers, the majority being polysaccharides, were screened to determine their suitability for the development of alternative microcapsule formulations capable of supporting cells. The capsules were taken through a limited optimization and then evaluated on the bases of rupture strength, permeability to albumin, and ability of their components to promote the attachment, aggregation, and function of encapsulated rabbit hepatocytes. The widely used alginate-polylysine capsules were employed as a comparative standard in all tests. A number of the new formulations compared favorably with the standard, and some exhibited superior performance in specific areas. Hepatocyte function, as evaluated by the rate of urea synthesis, showed no significant differences between formulations over a 24-h test period. One formulation, composed of the polysaccharides (carboxymethyl)cellulose, chondroitin sulfate A, chitosan, and polygalacturonate, was found to be superior to alginate-polylysine capsules in the areas investigated and supported the long-term survival and growth of liver endothelial cells.

  10. Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

    PubMed Central

    Beam, KG; Knudson, CM

    1988-01-01

    Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle. PMID:2458430

  11. Embryo genome profiling by single-cell sequencing for preimplantation genetic diagnosis in a β-thalassemia family.

    PubMed

    Xu, Yanwen; Chen, Shengpei; Yin, Xuyang; Shen, Xiaoting; Pan, Xiaoyu; Chen, Fang; Jiang, Hui; Liang, Yu; Wang, Wei; Xu, Xun; Wang, Jian; Zhang, Xiuqing; Zhou, Canquan; Wang, Jun

    2015-04-01

    The embryonic genome, including genotypes and haplotypes, contains all the information for preimplantation genetic diagnosis, representing great potential for mendelian disorder carriers to conceive healthy babies. We developed a strategy to obtain the full embryonic genome for a β-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome. The final accuracy for homozygous and heterozygous single-nucleotide polymorphisms reached 99.62% and 98.39%, respectively. The aneuploidies of embryos were detected as well. Based on the comprehensive embryonic genome, we effectively performed whole-genome mendelian disorder diagnosis and human leukocyte antigen matching tests. This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single-cell sequencing technology in preimplantation genetic diagnosis clinical practices. © 2015 American Association for Clinical Chemistry.

  12. Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation.

    PubMed

    Moreira, P N; Fernández-Gonzalez, R; Ramirez, M A; Pérez-Crespo, M; Rizos, D; Pintado, B; Gutiérrez-Adán, A

    2006-02-01

    It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMalpha, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMalpha, as well as on in vivo cultured and MEMalpha cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMalpha cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

  13. Developmental onset of mixed-function oxidase activity in preimplantation mouse embryos.

    PubMed Central

    Filler, R; Lew, K J

    1981-01-01

    Two-cell embryos, obtained from the C57BL/6N and DBA/2N strains, were cultured in media that supported in vitro differentiation and that contained [3H]benzo[a]pyrene. High-pressure liquid chromatography of the activated intermediates formed during in vitro early embryonic development indicated that the onset of polynuclear aromatic hydrocarbon activation coincided with blastocyst formation. Comparison of individual oxygenated intermediates metabolically formed from embryos genetically "responsive" or "nonresponsive" to aromatic hydrocarbons revealed significant quantitative differences in the production of dihydrodiol, quinone, and phenolic derivatives. In addition to exhibiting basal mixed-function oxidase activity, blastocysts were also responsive to enzymatic induction when exposed to 2,-3,7,8-tetrachlorodibenzo-p-dioxin. The presence of operative metabolite-detoxifying pathways was also assayed. Enzymatic treatment of water-soluble metabolites with beta-glucuronidase or arylsulfatase revealed that neither glucuronic acid conjugates nor sulfate ester derivatives were present. These data, therefore, provide direct evidence that late preimplantation mouse embryos (day 3 1/2 of gestation) are similar to later developmental stages in having the enzymatic capability for xenobiotic activation and enzyme induction but are dissimilar with respect to their detoxification mechanism(s). Moreover, the ability of preimplantation embryos to activate directly polynuclear aromatic hydrocarbon to bioreactive intermediates may be of importance in assessing the ontological susceptibility of the developing embryo to carcinogenic or teratogenic chemicals. PMID:6273901

  14. Development of an enzyme immunoassay for detection of antibodies against Coccidioides in dogs and other mammalian species.

    PubMed

    Chow, Nancy A; Lindsley, Mark D; McCotter, Orion Z; Kangiser, Dave; Wohrle, Ron D; Clifford, Wayne R; Yaglom, Hayley D; Adams, Laura E; Komatsu, Kenneth; Durkin, Michelle M; Baker, Rocky J; Shubitz, Lisa F; Derado, Gordana; Chiller, Tom M; Litvintseva, Anastasia P

    2017-01-01

    Coccidioides is a soil-dwelling fungus that causes coccidioidomycosis, a disease also known as Valley fever, which affects humans and a variety of animal species. Recent findings of Coccidioides in new, unexpected areas of the United States have demonstrated the need for a better understanding of its geographic distribution. Large serological studies on animals could provide important information on the geographic distribution of this pathogen. To facilitate such studies, we used protein A/G, a recombinant protein that binds IgG antibodies from a variety of mammalian species, to develop an enzyme immunoassay (EIA) that detects IgG antibodies against Coccidioides in a highly sensitive and high-throughput manner. We showed the potential of this assay to be adapted to multiple animal species by testing a collection of serum and/or plasma samples from dogs, mice, and humans with or without confirmed coccidioidomycosis. We then evaluated the performance of the assay in dogs, using sera from dogs residing in a highly endemic area, and found seropositivity rates significantly higher than those in dogs of non-endemic areas. We further evaluated the specificity of the assay in dogs infected with other fungal pathogens known to cross-react with Coccidioides. Finally, we used the assay to perform a cross-sectional serosurvey investigating dogs from Washington, a state in which infection with Coccidioides has recently been documented. In summary, we have developed a Coccidioides EIA for the detection of antibodies in canines that is more sensitive and has higher throughput than currently available methods, and by testing this assay in mice and humans, we have shown a proof of principle of its adaptability for other animal species.

  15. [Unaffected child born following preimplantation genetic diagnosis with karyomapping].

    PubMed

    Nánássy, László; Téglás, Gyöngyvér; Csenki, Marianna; Vereczkey, Attila

    2016-12-01

    Preimplantation genetic diagnosis for single gene defects is a well established method in assisted reproductive technologies. Karyomapping is a genome wide parental haplotyping using a high density single nucleotide polymorphism array that allows the diagnosis of any single gene defects. A couple with an affected child with primary congenital glaucoma attended at our clinic. Six oocyte-cumulus-complex was retrieved and all three mature oocytes were inseminated. One zygote showed the signs of normal fertilization and was cultured for five days. Trophectoderm biopsy and karyomapping analysis were carried out. Result showed a heterozygous carrier for primary congenital glaucoma. Embryo was thawed and transferred and a healthy girl was delivered at term. Here we report the first live birth following in vitro fertilization combined with preimplantation genetic diagnosis using karyomapping in Hungary. Karyomapping is able to accurately detect single gene disorders from a limited amount of samples without a significant preclinical workup. Orv. Hetil., 2016, 157(51), 2048-2050.

  16. Preimplantation genetic diagnosis of DiGeorge syndrome.

    PubMed

    Iwarsson, E; Ahrlund-Richter, L; Inzunza, J; Fridström, M; Rosenlund, B; Hillensjö, T; Sjöblom, P; Nordenskjöld, M; Blennow, E

    1998-09-01

    We report the first case of preimplantation genetic diagnosis used in order to avoid chromosomal imbalance in the progeny of a woman mildly affected by DiGeorge syndrome and carrier of a microdeletion of chromosome 22q11.2. In total, seven embryos were biopsied in three separate treatments and analysed by fluorescent in-situ hybridization (FISH). Of these, four were carrying the deletion, two were normal and in one the analysis was inconclusive. The diagnostic procedure was performed within 5 h. This allowed the biopsied embryos to be transferred the same day as the biopsy was taken (day 3). Two embryos were transferred in the third treatment, but no pregnancy was established. Patients with a 22q11 microdeletion, who have a 50% risk of transmitting the deletion to their offspring, can now be offered preimplantation genetic diagnosis using FISH for the detection of a 22q11 deletion.

  17. Preimplantation genetic diagnosis for cystic fibrosis: a case report

    PubMed Central

    Biazotti, Maria Cristina Santoro; Pinto, Walter; de Albuquerque, Maria Cecília Romano Maciel; Fujihara, Litsuko Shimabukuro; Suganuma, Cláudia Haru; Reigota, Renata Bednar; Bertuzzo, Carmen Sílvia

    2015-01-01

    Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby. PMID:25993078

  18. Successful application of preimplantation genetic diagnosis for Leigh syndrome.

    PubMed

    Unsal, Evrim; Aktaş, Yasemin; Uner, Ozge; BaltacI, Aysun; Ozcan, Sarp; Turhan, Feriba; Baltaci, Volkan

    2008-11-01

    To perform preimplantation genetic diagnosis (PGD) for a SURF1 gene mutation of the Leigh syndrome to transfer unaffected or carrier embryo/embryos. Case report. Clinical IVF laboratory. A couple carrying an nt769 G/A mutation that is associated with Leigh syndrome. Oocytes were fertilized by means of intracytoplasmic sperm injection. The resulting embryos were biopsied 3 days after fertilization. One blastomere was taken and whole-genome amplification was performed. Amplification of the mutation site was achieved by polymerase chain reaction (PCR) and restriction digestion was completed. Gel Imager was used to measure the digests of normal and mutant load. Embryo testing by means of PGD-PCR and pregnancy. Successful preimplantation genetic diagnosis for a SURF1 gene mutation and transfer of healthy or carrier embryos. Successful singleton pregnancy resulting in the delivery of healthy baby girl. We report the first case of successful PGD for Leigh syndrome resulting in delivery of a healthy newborn.

  19. New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2

    PubMed Central

    Botquin, Valérie; Hess, Heike; Fuhrmann, Guy; Anastassiadis, Constantinos; Gross, Michael K.; Vriend, Gerrit; Schöler, Hans R.

    1998-01-01

    The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development. PMID:9649510

  20. Early preimplantation cells expressing Cdx2 exhibit plasticity of specification to TE and ICM lineages through positional changes.

    PubMed

    Toyooka, Yayoi; Oka, Sanae; Fujimori, Toshihiko

    2016-03-01

    The establishment of the trophectoderm (TE) and the inner cell mass (ICM) is the first cell lineage segregation to occur in mouse preimplantation development. These two cell lineages arise in a position-dependent manner at the blastocyst stage: the outer cells form TE, which will generate the future placenta, while the inner cells give rise to the ICM, from which the epiblast (EPI) and primitive endoderm (PrE) arise. Previous studies have shown that a portion of cells relocate from the outside position to the inside during this preimplantation stage, but few studies have investigated the correlation between cell relocation and the expression of key transcription factors critical for cell differentiation. To monitor cell movement and the status of the TE-specification pathway in living embryos, we established Cdx2-GFP reporter mice allowing us to visualize the expression of Caudal-type transcriptional factor (Cdx2), a key regulator of the initiation of TE differentiation. Observation of Cdx2-GFP preimplantation embryos by live cell imaging revealed that all cells localized in an initial outer position initiated the expression of Cdx2. Subsequently, cells that changed their position from an outer to an inner position downregulated Cdx2 expression and contributed to the ICM. Finally we showed that internalized cells likely contribute to both the EPI and PrE. Our datas indicate that cells expressing even high levels of Cdx2 can internalize, deactivate an activated TE-specification molecular pathway and integrate into the pluripotent cell population.

  1. [First experiences with preimplantation genetic screening of chromosomal aberrations using oligonucleotide-based array comparative genomic hybridization].

    PubMed

    Kuglík, Petr; Smetana, Jan; Němcová, Darja; Vallová, Vladimíra; Mikulášová, Aneta; Gaillyová, Renata; Hubinka, Vít; Koudelka, Marek

    2015-01-01

    Preimplantation genetic diagnosis (PGD) is a complex approach for detecting genetic abnormalities in early-stage embryos using genetic or molecular cytogenetic methods. Recently, single cell genomic methods based on DNA microarrays have been used for PGD. In the presented paper, we discuss and demonstrate the possibility to detect copy number variation (CNVs) in trophectoderm cells biopsied from 5-day embryos using 60-mer oligonucleotide-based array-CGH with CytoSure 8 × 15K Aneuploidy Array. Whereas this microarray platform was originally designed for analysis of unamplified DNA derived from many cells, the new methods, developed for single-cell genomics, allow the application of oligo arrays technology in preimplanation genetic diagnosis. Preclinical validation of single cell array-CGH was made by analysis of 30 positive and negative controls. Validation process included whole genome amplification of DNA from 5-10 cells with normal karyotype and from samples with known aneuploidies and structural aberrations. Subsequently, we analyzed the whole genome profiles in 118 embryos; aneuploidies of chromosomes were observed in 26.7%; segmental imbalances were proved in 6.8% of embryos. Our first experience confirmed that this oligonucleotide-based array technique enables high-resolution preimplantation aneuploidy screening of all the 23 chromosome pairs and sensitive preimplantation diagnosis of segmental imbalances such as deletions, duplications and amplifications.

  2. Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media.

    PubMed

    Kleijkers, Sander H M; Eijssen, Lars M T; Coonen, Edith; Derhaag, Josien G; Mantikou, Eleni; Jonker, Martijs J; Mastenbroek, Sebastiaan; Repping, Sjoerd; Evers, Johannes L H; Dumoulin, John C M; van Montfoort, Aafke P A

    2015-10-01

    Is gene expression in human preimplantation embryos affected by the medium used for embryo culture in vitro during an IVF treatment? Six days of in vitro culture of human preimplantation embryos resulted in medium-dependent differences in expression level of genes involved in apoptosis, protein degradation, metabolism and cell-cycle regulation. Several human studies have shown an effect of culture medium on embryo development, pregnancy outcome and birthweight. However, the underlying mechanisms in human embryos are still unknown. In animal models of human development, it has been demonstrated that culture of preimplantation embryos in vitro affects gene expression. In humans, it has been found that culture medium affects gene expression of cryopreserved embryos that, after thawing, were cultured in two different media for 2 more days. In a multicenter trial, women were randomly assigned to two culture medium groups [G5 and human tubal fluid (HTF)]. Data on embryonic development were collected for all embryos. In one center, embryos originating from two pronuclei (2PN) zygotes that were not selected for transfer or cryopreservation on Day 2 or 3 because of lower morphological quality, were cultured until Day 6 and used in this study, if couples consented. Ten blastocysts each from the G5 and HTF study groups, matched for fertilization method, maternal age and blastocyst quality, were selected and their mRNA was isolated and amplified. Embryos were examined individually for genome-wide gene expression using Agilent microarrays and PathVisio was used to identify the pathways that showed a culture medium-dependent activity. Expression of 951 genes differed significantly (P < 0.01) between the G5 and HTF groups. Eighteen pathways, involved in apoptosis, metabolism, protein processing and cell-cycle regulation, showed a significant overrepresentation of differentially expressed genes. The DNA replication, G1 to S cell-cycle control and oxidative phosphorylation pathways

  3. Preimplantation Genetic Diagnosis: Prenatal Testing for Embryos Finally Achieving Its Potential.

    PubMed

    Stern, Harvey J

    2014-03-17

    Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology.

  4. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

    PubMed Central

    Cimadomo, Danilo; Capalbo, Antonio; Ubaldi, Filippo Maria; Scarica, Catello; Palagiano, Antonio; Canipari, Rita; Rienzi, Laura

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential. PMID:26942198

  5. [Assisted Reproduction and Preimplantation Genetic Diagnosis in Patients Susceptible to Breast Cancer].

    PubMed

    Veselá, K; Kocur, T; Horák, J; Horňák, M; Oráčová, E; Hromadová, L; Veselý, J; Trávník, P

    2016-01-01

    Assisted reproduction, as well as pregnancy itself, in patients with breast cancer or other hereditary type of cancer, is a widely discussed topic. In the past, patients treated for breast cancer were rarely involved in the discussion about reproductive possibilities or infertility treatment. However, current knowledge suggests, that breast cancer is neither a contraindication to pregnancy, nor to assisted reproduction techniques. On the contrary, assisted reproduction and preimplantation genetic diagnosis methods might prevent the transmission of genetic risks to the fetus. In this review we summarize data concerning pregnancy risks in patients with increased risk of breast cancer. In addition, we introduce current possibilities and approaches to fertility preservation prior to assisted reproduction treatment as well as novel methods improving the safety of fertility treatment. In the second part of this review, we focus on karyomapping--an advanced molecular genetic tool for elimination of germinal mutations in patients with predisposition to cancer. Moreover, the rapid development of preimplantation genetic diagnosis methods contributes to detection of both chromosomal aneuploidy and causal mutations in a relatively short time-span.

  6. Determination of the reactivity of cytotoxic immune cells with preimplantation mouse embryos

    SciTech Connect

    Ewoldsen, M.A.

    1987-01-01

    Cytotoxic immune cells were used in an assay, MELIA (mixed embryo leukocyte interaction assay) to test the ability of the cells to kill blastocyst stage embryos. The cytotoxic immune cells generated for use in this study, cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and lymphokine activated killer (LAK) cells were shown to have phenotypic and cytolytic characteristics similar to those reported by other investigators. The lysis of the blastocysts in the MELIA was determined by measuring the inhibition of blastocoel retention and/or by the inhibition of incorporation of tritiated thymidine (/sup 3/H-TdR) into embryonic DNA. Blastocysts which possess or lack their zonae pellucidae were tested to determine whether the zona pellucida plays an immunoprotective role in preimplantation development. The results indicated that CTLs only lysed embryonic cells when the zona pellucida was absent, but NK and LAK cells lysed embryonic cells whether the zona pellucida was present or absent. The results suggest that the zona pellucida may protect the preimplantation mouse embryo from lysis by CTLs but what protects the embryo from lysis by NK and LAK cells is unclear.

  7. Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

    PubMed

    Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

    2004-07-01

    Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

  8. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.

    PubMed

    Cimadomo, Danilo; Capalbo, Antonio; Ubaldi, Filippo Maria; Scarica, Catello; Palagiano, Antonio; Canipari, Rita; Rienzi, Laura

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

  9. What do French patients and geneticists think about prenatal and preimplantation diagnoses in Marfan syndrome?

    PubMed

    Coron, F; Rousseau, T; Jondeau, G; Gautier, E; Binquet, C; Gouya, L; Cusin, V; Odent, S; Dulac, Y; Plauchu, H; Collignon, P; Delrue, M-A; Leheup, B; Joly, L; Huet, F; Thevenon, J; Mace, G; Cassini, C; Thauvin-Robinet, C; Wolf, J E; Hanna, N; Sagot, P; Boileau, C; Faivre, L

    2012-12-01

    Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with manifestations mainly involving the skeletal, ocular, and cardiovascular systems. The phenotypic variability observed in MFS makes genetic counselling difficult. Prenatal diagnosis (PND) and preimplantation genetic diagnosis are technically feasible when a causal mutation is identified, but both raise many ethical questions in this condition. Little is known about opinions and practices in such reproductive issues in MFS. The goal of this study was to report on patients' points of view and geneticists' standard practices. Two different questionnaires were produced. Fifty geneticists filled in the questionnaire. Twenty-two per cent thought that PND was acceptable, 72% debatable and 6% not acceptable. Preimplantation genetic diagnosis was more often reported acceptable (34% of answers). Results varied according to the physician's experience with the disease. Fifty-four answers were collected for patients' questionnaires. Most of them (74%) were favourable to the development of prenatal testing, and believed that the choice should be given to parents. However, only a minority would opt for prenatal diagnosis for themselves. This study showed that the majority of patients were in favour of PND and that opinions among practitioners varied widely, but that overall, practitioners favoured a systematic multidisciplinary evaluation of the couple's request. © 2012 John Wiley & Sons, Ltd.

  10. Preimplantation Genetic Diagnosis: Prenatal Testing for Embryos Finally Achieving Its Potential

    PubMed Central

    Stern, Harvey J.

    2014-01-01

    Preimplantation genetic diagnosis was developed nearly a quarter-century ago as an alternative form of prenatal diagnosis that is carried out on embryos. Initially offered for diagnosis in couples at-risk for single gene genetic disorders, such as cystic fibrosis, spinal muscular atrophy and Huntington disease, preimplantation genetic diagnosis (PGD) has most frequently been employed in assisted reproduction for detection of chromosome aneuploidy from advancing maternal age or structural chromosome rearrangements. Major improvements have been seen in PGD analysis with movement away from older, less effective technologies, such as fluorescence in situ hybridization (FISH), to newer molecular tools, such as DNA microarrays and next generation sequencing. Improved results have also started to be seen with decreasing use of Day 3 blastomere biopsy in favor of polar body or Day 5 trophectoderm biopsy. Discussions regarding the scientific, ethical, legal and social issues surrounding the use of sequence data from embryo biopsy have begun and must continue to avoid concern regarding eugenic or inappropriate use of this technology. PMID:26237262

  11. Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.

    PubMed

    Kleinschmidt, K; Wilkens, E; Glaeser, S P; Kaempfer, P; Staerk, A; Roesti, D

    2017-04-01

    The aim of this study was to develop and evaluate a real-time PCR technology for microbiological control methods to examine individualized cell therapeutics, an emerging class of pharmaceutical formulations. Oligonucleotide primers and hybridization probe for bacterial detection targeting the 16SrRNA gene were adapted based on Nadkarni et al. [Microbiology148 (2002) 257]. For detection of yeast and moulds, primers and probe were designed from conserved sequences of the 18SrRNA gene in this study. The real-time PCR assays were tested on genomic DNA of Escherichia coli and Candida albicans to assess efficiency and linear dynamic range. After successful establishment of robust real-time PCRs, applicability of the assays was evaluated by extracting microbial target DNA from cell-based preparations. Different commercial DNA extraction methods were compared identifying the MagNA Pure DNA Isolation Kit III as the method of choice. Sensitivity was examined for different strains and a detection limit of 10(2) -10(3) CFU per ml in a sample containing ~10(6) mammalian cells per ml was achieved. This study reports the successful establishment of two qualitative real-time PCR assays, enabling in general the broad-range detection of microbial contaminants in a cell-based sample matrix. Individualized cell therapeutics tend to have a short shelf life. Due to lengthy incubation periods, compendial testing according to current pharmacopoeial guidelines may not be applicable. We report a suitable alternative method upon which future microbiological quality control methods for such products could be based on. However, to implement valid rapid microbiological testing methods using real-time PCR technology, further challenges need to be addressed. © 2016 The Society for Applied Microbiology.

  12. Pax9 is required for filiform papilla development and suppresses skin-specific differentiation of the mammalian tongue epithelium.

    PubMed

    Jonker, Leon; Kist, Ralf; Aw, Andrew; Wappler, Ilka; Peters, Heiko

    2004-11-01

    The epidermis is a derivative of the surface ectoderm. It forms a protective barrier and specific appendages including hair, nails, and different eccrine glands. The surface ectoderm also forms the epithelium of the oral cavity and tongue, which develop a slightly different barrier and form different appendages such as teeth, filiform papillae, taste papillae, and salivary glands. How this region-specific differentiation is genetically controlled is largely unknown. We show here that Pax9, which is expressed in the epithelium of the tongue but not in skin, regulates several aspects of tongue-specific epithelial differentiation. In Pax9-deficient mice filiform papillae lack the anterior-posterior polarity, a defect that is associated with temporal-spatial changes in Hoxc13 expression. Barrier formation is disturbed in the mutant tongue and genome-wide expression profiling revealed that the expression of specific keratins (Krt), keratin-associated proteins, and members of the epidermal differentiation complex is significantly down-regulated. In situ hybridization demonstrated that several 'hard' keratins, Krt1-5, Krt1-24, and Krt2-16, are not expressed in the absence of Pax9. Notably, specific 'soft' keratins, Krt2-1 and Krt2-17, normally weakly expressed in the tongue but present at high levels in skin and in orthokeratinized oral dysplasia are up-regulated in the mutant tongue epithelium. This result indicates a partial trans-differentiation to an epithelium with skin-specific characteristics. Together, our findings show that Pax9 regulates appendage formation in the mammalian tongue and identify Pax9 as an important factor for the region-specific differentiation of the surface ectoderm.

  13. Distinct and cooperative roles of mammalian Vg1 homologs GDF1 and GDF3 during early embryonic development.

    PubMed

    Andersson, Olov; Bertolino, Philippe; Ibáñez, Carlos F

    2007-11-15

    Vg1, a member of the TGF-beta superfamily of ligands, has been implicated in the induction of mesoderm, formation of primitive streak, and left-right patterning in Xenopus and chick embryos. In mice, GDF1 and GDF3 - two TGF-beta superfamily ligands that share high sequence identity with Vg1 - have been shown to independently mimic distinct aspects of Vg1's functions. However, the extent to which the developmental processes controlled by GDF1 and GDF3 and the underlying signaling mechanisms are evolutionarily conserved remains unclear. Here we show that phylogenetic and genomic analyses indicate that Gdf1 is the true Vg1 ortholog in mammals. In addition, and similar to GDF1, we find that GDF3 signaling can be mediated by the type I receptor ALK4, type II receptors ActRIIA and ActRIIB, and the co-receptor Cripto to activate Smad-dependent reporter genes. When expressed in heterologous cells, the native forms of either GDF1 or GDF3 were incapable of inducing downstream signaling. This could be circumvented by using chimeric constructs carrying heterologous prodomains, or by co-expression with the Furin pro-protein convertase, indicating poor processing of the native GDF1 and GDF3 precursors. Unexpectedly, co-expression with Nodal - another TGF-beta superfamily ligand involved in mesoderm formation - could also expose the activities of native GDF1 and GDF3, suggesting a potentially novel mode of cooperation between these ligands. Functional complementarity between GDF1 and GDF3 during embryonic development was investigated by analyzing genetic interactions between their corresponding genes. This analysis showed that Gdf1(-/-);Gdf3(-/-) compound mutants are more severely affected than either Gdf1(-/-) or Gdf3(-/-) single mutants, with defects in the formation of anterior visceral endoderm and mesoderm that recapitulate Vg1 loss of function, suggesting that GDF1 and GDF3 together represent the functional mammalian homologs of Vg1.

  14. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome.

    PubMed

    Harlow, Philippa H; Perry, Simon J; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A; Flemming, Anthony J

    2016-03-18

    To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals.

  15. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome

    PubMed Central

    Harlow, Philippa H.; Perry, Simon J.; Widdison, Stephanie; Daniels, Shannon; Bondo, Eddie; Lamberth, Clemens; Currie, Richard A.; Flemming, Anthony J.

    2016-01-01

    To determine whether a C. elegans bioassay could predict mammalian developmental activity, we selected diverse compounds known and known not to elicit such activity and measured their effect on C. elegans egg viability. 89% of compounds that reduced C. elegans egg viability also had mammalian developmental activity. Conversely only 25% of compounds found not to reduce egg viability in C. elegans were also inactive in mammals. We conclude that the C. elegans egg viability assay is an accurate positive predictor, but an inaccurate negative predictor, of mammalian developmental activity. We then evaluated C. elegans as a tool to identify mechanisms affecting toxicological outcomes among related compounds. The difference in developmental activity of structurally related fungicides in C. elegans correlated with their rate of metabolism. Knockdown of the cytochrome P450s cyp-35A3 and cyp-35A4 increased the toxicity to C. elegans of the least developmentally active compounds to the level of the most developmentally active. This indicated that these P450s were involved in the greater rate of metabolism of the less toxic of these compounds. We conclude that C. elegans based approaches can predict mammalian developmental activity and can yield plausible hypotheses for factors affecting the biological potency of compounds in mammals. PMID:26987796

  16. The Development of a Two-Dimensional Multielectrode Array for Visual Perception Research in the Mammalian Brain.

    DTIC Science & Technology

    1980-12-01

    26 Physiological Research of the Visual System .......... 28 Chemical Neuroresearch . .. .. .. .. .. .. .. .. 28 Bioelectric...supported by physiological research where points on the surface of the primary visual cortex are stimulated and the resulting response is observed. Such...Introd-c-t ion Study of the mammalian visual perception process requires knowledge of the cnatomical, physiological , and psychological aspects of the

  17. Preimplantation embryo-secreted factors modulate maternal gene expression in rat uterus.

    PubMed

    Yamagami, Kazuki; Islam, M Rashedul; Yoshii, Yuka; Mori, Kazuki; Tashiro, Kosuke; Yamauchi, Nobuhiko

    2016-05-01

    In mammalian reproduction, embryo implantation into the uterus is spatiotemporally regulated by a complex process triggered by a number of factors. Although previous studies have suggested that uterine receptivity is mediated by blastocyst-derived factors, specific functions of embryos remain to be defined during preimplantation. Therefore, the present study was conducted to identify the maternal genes regulated by embryo-secreted factors in the rat uterus. RNA-sequencing (RNA-seq) data revealed that 10 genes are up-regulated in the delayed implantation uterus compared with the pseudopregnancy uterus. The RNA-seq results were further verified by real-time quantitative polymerase chain reaction. Sulf1 expression is significantly (P < 0.05) induced in the delayed implantation uterus, although Areg, Calca, Fxyd4 and Lamc3 show a definite but non-statistically significant increase in their expression levels. During early pregnancy, the levels of Areg, Calca, Fxyd4, Lamc3 and Sulf1 expression at 3.5 days post coitus (dpc) are significantly (P < 0.05) higher than those at 1.5 dpc. Treatment with embryo-conditioned media revealed that Lamc3 and Sulf1 are up-regulated compared with the other genes studied. Thus, embryo-derived factors regulate maternal gene expression, with Lamc3 and Sulf1 possibly being suitable markers for a response study of embryo-secreted factors to improve our understanding of embryo-maternal communication.

  18. Estimation of chromosomal imbalances in preimplantation embryos from preimplantation genetic diagnosis cycles of reciprocal translocations with or without acrocentric chromosomes.

    PubMed

    Lim, Chun Kyu; Cho, Jae Won; Song, In Ok; Kang, Inn Soo; Yoon, Yong-Dal; Jun, Jin Hyun

    2008-12-01

    To estimate chromosomal imbalances in preimplantation embryos from reciprocal translocation carriers with or without acrocentric chromosomes (Acro-Ch) 13, 14, 15, 21, and 22 in preimplantation genetic diagnosis (PGD) cycles. Fluorescence in situ hybridization was applied to PGD cycles for reciprocal translocation carriers. University-based centers for reproductive medicine. Ten and 24 patients of reciprocal translocation with and without Acro-Ch, respectively. Fluorescence in situ hybridization in biopsied blastomeres. Estimation of meiotic segregation mode in embryos from translocation carriers. The proportion of alternative segregation for normal or balanced chromosome contents in preimplantation embryos from PGD cycles in reciprocal translocations without Acro-Ch was significantly higher than that with Acro-Ch (26.0% vs. 14.6%). The proportion of interchange trisomy in 3:1 segregation was significantly lower in reciprocal translocations without Acro-Ch than that with Acro-Ch (4.3% vs. 9.5%). This is the first report that the incidence of alternative segregation producing normal or balanced embryos was relatively low in reciprocal translocations associated with Acro-Ch. Our data may be useful to predict the possibility of normal or balanced embryos and to counsel with reciprocal translocation carriers for PGD-fluorescence in situ hybridization cycles.

  19. Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

    SciTech Connect

    Kumari, S. Sindhu; Varadaraj, Kulandaiappan

    2014-10-03

    Highlights: • Intact AQP0 functions as fiber cell-to-fiber cell adhesion protein. • AQP0 facilitates reduction in extracellular space and lens water content. • AQP0 adhesion function aids in lens refractive index gradient (RING) formation. • AQP0 prevents lens spherical aberration by establishing RING. • AQP0 is critical for lens transparency and homeostasis. - Abstract: Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0{sup +/−}) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0{sup +/−}/AQP1{sup +/−}) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS–PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28 kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26–24 kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (P{sub f}) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA

  20. Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration.

    PubMed

    Kumari, S Sindhu; Varadaraj, Kulandaiappan

    2014-10-03

    Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0(+/-)) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0(+/)(-)/AQP1(+/)(-)) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS-PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26-24kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (Pf) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA

  1. Relationship between pre-implant interleukin-6 levels, inflammatory response, and early outcome in patients supported by left ventricular assist device: a prospective study.

    PubMed

    Caruso, Raffaele; Botta, Luca; Verde, Alessandro; Milazzo, Filippo; Vecchi, Irene; Trivella, Maria Giovanna; Martinelli, Luigi; Paino, Roberto; Frigerio, Maria; Parodi, Oberdan

    2014-01-01

    The immune response is crucial in the development of multi-organ failure (MOF) and complications in end-stage heart failure patients supported by left ventricular assist device (LVAD). However, at pre-implant, the association between inflammatory state and post-LVAD outcome is not yet clarified. Aim of the study was to assess the relationship among pre-implant levels of immune-related cytokines, postoperative inflammatory response and 3-month outcome in LVAD-patients. In 41 patients undergoing LVAD implantation, plasma levels of interleukin (IL)-6, IL-8, crucial for monocyte modulation, and urine neopterin/creatinine ratio (Neo/Cr), marker of monocyte activation, were assessed preoperatively, at 3 days, 1 and 4 weeks post-LVAD. MOF was evaluated by total sequential organ failure assessment (tSOFA) score. Intensive care unit (ICU)-death and/or post-LVAD tSOFA ≥11 was considered as main adverse outcome. Length of ICU-stay, 1 week-tSOFA score, hospitalisation and 3-month survival were considered additional end-points. During ICU-stay, 8 patients died of MOF, while 8 of the survivors experienced severe MOF with postoperative tSOFA score ≥11. Pre-implant level of IL-6 ≥ 8.3 pg/mL was identified as significant marker of discrimination between patients with or without adverse outcome (OR 6.642, 95% CI 1.201-36.509, p = 0.030). Patients were divided according to pre-implant IL-6 cutoff of 8.3 pg/ml in A [3.5 (1.2-6.1) pg/mL] and B [24.6 (16.4-38.0) pg/mL] groups. Among pre-implant variables, only white blood cells count was independently associated with pre-implant IL-6 levels higher than 8.3 pg/ml (OR 1.491, 95% CI 1.004-2.217, p = 0.048). The ICU-stay and hospitalisation resulted longer in B-group (p = 0.001 and p = 0.030, respectively). Postoperatively, 1 week-tSOFA score, IL-8 and Neo/Cr levels were higher in B-group. LVAD-candidates with elevated pre-implant levels of IL-6 are associated, after intervention, to higher release of monocyte

  2. The preimplantation mouse embryo is a target for cannabinoid ligand-receptor signaling.

    PubMed Central

    Paria, B C; Das, S K; Dey, S K

    1995-01-01

    Using a reverse transcription-coupled PCR, we demonstrated that both brain and spleen type cannabinoid receptor (CB1-R and CB2-R, respectively) mRNAs are expressed in the preimplantation mouse embryo. The CB1-R mRNA expression was coincident with the activation of the embryonic genome late in the two-cell stage, whereas the CB2-R mRNA was present from the one-cell through the blastocyst stages. The major psychoactive component of marijuana (-)-delta-9-tetrahydrocannabinol [(-)-THC] inhibited forskolin-stimulated cAMP generation in the blastocyst, and this inhibition was prevented by pertussis toxin. However, the inactive cannabinoid cannabidiol (CBD) failed to influence this response. These results suggest that cannabinoid receptors in the embryo are coupled to inhibitory guanine nucleotide binding proteins. Further, the oviduct and uterus exhibited the enzymatic capacity to synthesize the putative endogenous cannabinoid ligand arachidonylethanolamide (anandamide). Synthetic and natural cannabinoid agonists [WIN 55,212-2, CP 55,940, (-)-THC, and anandamide], but not CBD or arachidonic acid, arrested the development of two-cell embryos primarily between the four-cell and eight-cell stages in vitro in a dose-dependent manner. Anandamide also interfered with the development of eight-cell embryos to blastocysts in culture. The autoradiographic studies readily detected binding of [3H]anandamide in embryos at all stages of development. Positive signals were present in one-cell embryos and all blastomeres of two-cell through four-cell embryos. However, most of the binding sites in eight-cell embryos and morulae were present in the outer cells. In the blastocyst, these signals were primarily localized in the mural trophectoderm with low levels of signals in the polar trophectoderm, while little or no signals were noted in inner cell mass cells.These results establish that the preimplantation mouse embryo is a target for cannabinoid ligands. Consequently, many of the

  3. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos

    PubMed Central

    Petropoulos, Sophie; Edsgärd, Daniel; Reinius, Björn; Deng, Qiaolin; Panula, Sarita Pauliina; Codeluppi, Simone; Plaza Reyes, Alvaro; Linnarsson, Sten; Sandberg, Rickard; Lanner, Fredrik

    2016-01-01

    Summary Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research. PMID:27062923

  4. Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening.

    PubMed

    Montag, M; van der Ven, K; Rösing, B; van der Ven, H

    2009-01-01

    Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle, which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. However, the paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected, in contrast to preimplantation genetic diagnosis (PGD) by blastomere biopsy. The rapid pace of developments in the field of molecular diagnostics will also influence the advantages of PBD, and probably allow more general diagnostic applications in the future.

  5. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.

    PubMed

    Petropoulos, Sophie; Edsgärd, Daniel; Reinius, Björn; Deng, Qiaolin; Panula, Sarita Pauliina; Codeluppi, Simone; Plaza Reyes, Alvaro; Linnarsson, Sten; Sandberg, Rickard; Lanner, Fredrik

    2016-05-05

    Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.

  6. Transcriptional profiling of mouse uterus at pre-implantation stage under VEGF repression.

    PubMed

    Ji, Yan; Lu, Xiaodan; Zhong, Qingping; Liu, Peng; An, Yao; Zhang, Yuntao; Zhang, Shujie; Jia, Ruirui; Tesfamariam, Isaias G; Kahsay, Abraha G; Zhang, Luqing; Zhu, Wensheng; Zheng, Yaowu

    2013-01-01

    Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina's digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated were associated with RNA polymerase III activity while down-regulated were strongly related with muscle development. Comparable numbers of antisense transcripts were identified. Expression levels of the antisense transcripts were found tightly correlated with their sense expression levels, an indication of possibly non-specific transcripts generated around the active promoters and enhancers. The antisense transcripts with exceptionally high or low expression levels and the antisense transcripts under VEGF regulation were also identified. These transcripts may be important candidates in regulation of uterus development. This study provides a global survey on genes and antisense transcripts regulated by VEGF in

  7. Transcriptional Profiling of Mouse Uterus at Pre-Implantation Stage under VEGF Repression

    PubMed Central

    Ji, Yan; Lu, Xiaodan; Zhong, Qingping; Liu, Peng; An, Yao; Zhang, Yuntao; Zhang, Shujie; Jia, Ruirui; Tesfamariam, Isaias G.; Kahsay, Abraha G.; Zhang, Luqing; Zhu, Wensheng; Zheng, Yaowu

    2013-01-01

    Uterus development during pre-implantation stage affects implantation process and embryo growth. Aberrant uterus development is associated with many human reproductive diseases. Among the factors regulating uterus development, vascular remodeling promoters are critical for uterus function and fertility. Vascular endothelial growth factor (VEGF), as one of the major members, has been found to be important in endothelial cell growth and blood vessel development, as well as in non-endothelial cells. VEGF mediation in reproduction has been broadly studied, but VEGF-induced transcriptional machinery during implantation window has not been systematically studied. In this study, a genetically repressed VEGF mouse model was used to analyze uterus transcriptome at gestation 2.5 (G2.5) by Solexa/Illumina’s digital gene expression (DGE) system. A number of 831 uterus-specific and 2398 VEGF-regulated genes were identified. Gene ontology (GO) analysis indicated that genes actively involved in uterus development were members of collagen biosynthesis, cell proliferation and cell apoptosis. Uterus-specific genes were enriched in activities of phosphatidyl inositol phosphate kinase, histone H3-K36 demethylation and protein acetylation. Among VEGF-regulated genes, up-regulated wer