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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. PMID:27475848

  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. PMID:11750739

  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. PMID:25498236

  4. 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.

  5. Dynamic transcriptional symmetry-breaking in pre-implantation mammalian embryo development revealed by single-cell RNA-seq.

    PubMed

    Shi, Junchao; Chen, Qi; Li, Xin; Zheng, Xiudeng; Zhang, Ying; Qiao, Jie; Tang, Fuchou; Tao, Yi; Zhou, Qi; Duan, Enkui

    2015-10-15

    During mammalian pre-implantation embryo development, when the first asymmetry emerges and how it develops to direct distinct cell fates remain longstanding questions. Here, by analyzing single-blastomere transcriptome data from mouse and human pre-implantation embryos, we revealed that the initial blastomere-to-blastomere biases emerge as early as the first embryonic cleavage division, following a binomial distribution pattern. The subsequent zygotic transcriptional activation further elevated overall blastomere-to-blastomere biases during the two- to 16-cell embryo stages. The trends of transcriptional asymmetry fell into two distinct patterns: for some genes, the extent of asymmetry was minimized between blastomeres (monostable pattern), whereas other genes, including those known to be lineage specifiers, showed ever-increasing asymmetry between blastomeres (bistable pattern), supposedly controlled by negative or positive feedbacks. Moreover, our analysis supports a scenario in which opposing lineage specifiers within an early blastomere constantly compete with each other based on their relative ratio, forming an inclined 'lineage strength' that pushes the blastomere onto a predisposed, yet flexible, lineage track before morphological distinction.

  6. 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

  7. The landscape of accessible chromatin in mammalian preimplantation embryos.

    PubMed

    Wu, Jingyi; Huang, Bo; Chen, He; Yin, Qiangzong; Liu, Yang; Xiang, Yunlong; Zhang, Bingjie; Liu, Bofeng; Wang, Qiujun; Xia, Weikun; Li, Wenzhi; Li, Yuanyuan; Ma, Jing; Peng, Xu; Zheng, Hui; Ming, Jia; Zhang, Wenhao; Zhang, Jing; Tian, Geng; Xu, Feng; Chang, Zai; Na, Jie; Yang, Xuerui; Xie, Wei

    2016-06-30

    In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development. PMID:27309802

  8. 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.

  9. Human pre-implantation embryo development

    PubMed Central

    Niakan, Kathy K.; Han, Jinnuo; Pedersen, Roger A.; Simon, Carlos; Pera, Renee A. Reijo

    2012-01-01

    Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals. PMID:22318624

  10. The inorganic anatomy of the mammalian preimplantation embryo and the requirement of zinc during the first mitotic divisions

    PubMed Central

    Kong, Betty Y.; Duncan, Francesca E.; Que, Emily L.; Xu, Yuanming; Vogt, Stefan; O’Halloran, Thomas V.; Woodruff, Teresa K.

    2015-01-01

    Background Zinc is the most abundant transition metal in the mammalian oocyte, and dynamic fluxes in intracellular concentration are essential for regulating both meiotic progression and fertilization. Whether the defined pathways of zinc utilization in female meiosis directly translate to mitotic cells, including the mammalian preimplantation embryo, have not been studied previously. Results We determined that zinc is the most abundant transition metal in the preimplantation embryo, with levels an order of magnitude higher than those of iron or copper. Using a zinc-specific fluorescent probe, we demonstrated that labile zinc is distributed in vesicle-like structures in the cortex of cells at all stages of preimplantation embryo development. To test the importance of zinc during this period, we induced zinc insufficiency using the heavy metal chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). Incubation of embryos in media containing TPEN resulted in a developmental arrest that was specific to zinc chelation and associated with compromised mitotic parameters. The developmental arrest due to zinc insufficiency was associated with altered chromatin structure in the blastomere nuclei and decreased global transcription. Conclusions These results demonstrate for the first time that the preimplantation embryo requires tight zinc regulation and homeostasis for the initial mitotic divisions of life. PMID:25903945

  11. 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

  12. 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.

  13. 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.

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

    PubMed Central

    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 [1-13C8] 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. PMID:23226596

  15. 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

  16. Mitochondria and human preimplantation embryo development.

    PubMed

    Wilding, Martin; Coppola, Gianfranco; Dale, Brian; Di Matteo, Loredana

    2009-04-01

    Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

  17. Intracellular Ca2+ signaling and preimplantation development.

    PubMed

    Armant, D Randall

    2015-01-01

    The key, versatile role of intracellular Ca2+ signaling during egg activation after fertilization has been appreciated for several decades. More recently, evidence has accumulated supporting the concept that cytoplasmic Ca2+ is also a major signaling nexus during subsequent development of the fertilized ovum. This chapter will review the molecular reactions that regulate intracellular Ca2+ levels and cell function, the role of Ca2+ signaling during egg activation and specific examples of repetitive Ca2+ signaling found throughout pre- and peri-implantation development. Many of the upstream and downstream pathways utilized during egg activation are also critical for specific processes that take place during embryonic development. Much remains to be done to elucidate the full complexity of Ca2+ signaling mechanisms in preimplantation embryos to the level of detail accomplished for egg activation. However, an emerging concept is that because this second messenger can be modulated downstream of numerous receptors and is able to bind and activate multiple cytoplasmic signaling proteins, it can help the coordination of development through up- and downstream pathways that change with each embryonic stage.

  18. 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

  19. 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.

  20. The Genetic Regulation of Cell Fate During Preimplantation Mouse Development.

    PubMed

    Lokken, A A; Ralston, A

    2016-01-01

    The adult body is estimated to contain several hundred distinct cell types, each with a specialized physiological function. Failure to maintain cell fate can lead to devastating diseases and cancer, but understanding how cell fates are assigned and maintained during animal development provides new opportunities for human health intervention. The mouse is a premier model for evaluating the genetic regulation of cell fate during development because of the wide variety of tools for measuring and manipulating gene expression levels, the ability to access embryos at desired developmental stages, and the similarities between mouse and human development, particularly during the early stages of development. During the first 3 days of mouse development, the preimplantation embryo sets aside cells that will contribute to the extraembryonic tissues. The extraembryonic tissues are essential for establishing pregnancy and ensuring normal fetal development in both mice and humans. Genetic analyses of mouse preimplantation development have permitted identification of genes that are essential for specification of the extraembryonic lineages. In this chapter, we review the tools and concepts of mouse preimplantation development. We describe genes that are essential for cell fate specification during preimplantation stages, and we describe diverse models proposed to account for the mechanisms of cell fate specification during early development. PMID:27475852

  1. Metabolism of Preimplantation Embryo Development: A Bystander or an Active Participant?

    PubMed

    Kaneko, K J

    2016-01-01

    Unicellular organisms are exquisitely sensitive to nutrient availability in the environment and have evolved elaborate mechanisms to sense the levels and types of nutrients, altering gene expression patterns accordingly to adjust the metabolic activities required to survive. Thus, environmental cues induce adaptive metabolic differentiation through transcriptional and posttranscriptional changes. Similarly, preimplantation embryos are exposed to various environmental cues within the maternal reproductive tract prior to implantation. Because only "simple" culture conditions are required, it is assumed that these embryos are genetically preprogrammed to develop with little influence from the environment, with the exception of few "necessities" provided by the environment. However, a wealth of literature now suggests that the developing embryos are greatly influenced by the maternal environment. Even though the developing embryos have the capacity and plasticity to deal with nutritional imbalance posed by an altered maternal environment, there is often a trade-off to the overall fitness of those embryos later in life. Despite these studies that underline the general importance of the reproductive environment during development, it is thought that the primary driver of mammalian development is strictly genetic and that metabolic adaptation by the preimplantation embryo is secondary to genetic control. In this review, I propose that not only does the maternal environment of developing preimplantation embryos influence developmental potential, pregnancy outcomes, and postnatal disease states, but that it has an active role in induction and potentiation of the first differentiation event, the production of trophectoderm and inner cell mass lineages. PMID:27475855

  2. 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.

  3. Functional Analysis of Lysosomes During Mouse Preimplantation Embryo Development

    PubMed Central

    TSUKAMOTO, Satoshi; HARA, Taichi; YAMAMOTO, Atsushi; OHTA, Yuki; WADA, Ayako; ISHIDA, Yuka; KITO, Seiji; NISHIKAWA, Tetsu; MINAMI, Naojiro; SATO, Ken; KOKUBO, Toshiaki

    2012-01-01

    Abstract Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos. PMID:23080372

  4. 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

  5. Preimplantation genetic diagnosis: current status and new developments.

    PubMed

    Lissens, W; Sermon, K

    1997-08-01

    Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis aimed at eliminating embryos carrying serious genetic diseases before implantation. To this end, two major technologies are in use: the polymerase chain reaction (PCR) for monogenic diseases and fluorescent in-situ hybridization (FISH) for chromosomal aberrations. In this review, a number of problems arising from the use of these technologies, as well as their possible solutions and new developments, are discussed. Concerning PCR, the phenomenon of allelic drop-out, as well as methods to reduce this problem, such as fluorescent PCR, are described. The advantages and disadvantages of sperm separation by flow cytometry as an adjunct to sex determination for the avoidance of X-linked disease are discussed. The application of FISH for aneuploidy detection is commented upon and the advances in cell recycling, in which PCR and FISH are combined, are analysed. Finally, diseases for which PGD is currently possible are summarized.

  6. 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

  7. Effects of WT1 down-regulation on oocyte maturation and preimplantation embryo development in pigs.

    PubMed

    Gao, Fei; Guan, Jiyu; Liu, Limei; Zhang, Sheng; An, Peipei; Fan, Anran; Song, Guangqi; Zhang, Peng; Zhao, Tianchuang; Tang, Bo; Zhang, Xueming; Li, Ziyi

    2014-10-01

    The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function of WT1 during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions of WT1 during oocyte maturation and preimplantation embryonic development in pigs. We show that WT1 is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulating WT1 expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation of WT1 did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate that WT1 plays important roles in the development of porcine preimplantation embryos. PMID:25030893

  8. The Roles of Parathyroid Hormone-Like Hormone during Mouse Preimplantation Embryonic Development

    PubMed Central

    Guo, Lei; Qi, Shu-Tao; Miao, De-Qiang; Liang, Xing-Wei; Li, Hui; Ou, Xiang-Hong; Huang, Xin; Yang, Cai-Rong; Ouyang, Ying-Chun; Hou, Yi; Sun, Qing-Yuan; Han, Zhiming

    2012-01-01

    Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development. PMID:22808183

  9. Expression of LINE-1 retroposons is essential for murine preimplantation development.

    PubMed

    Beraldi, Rosanna; Pittoggi, Carmine; Sciamanna, Ilaria; Mattei, Elisabetta; Spadafora, Corrado

    2006-03-01

    In higher eukaryotes, reverse transcriptase (RT) activities are encoded by a variety of endogenous retroviruses and retrotransposable elements. We previously found that mouse preimplantation embryos are endowed with an endogenous RT activity. Inhibition of that activity by the non nucleosidic inhibitor nevirapine or by microinjection of anti-RT antibody caused early embryonic developmental arrest. Those experiments indicated that RT is required for early development, but did not identify the responsible coding elements. We now show that microinjection of morpholino-modified antisense oligonucleotides targeting the 5' end region of active LINE-1 retrotransposons in murine zygotes irreversibly arrests preimplantation development at the two- and four-cell stages; the overall level of functional RT is concomitantly downregulated in arrested embryos. Furthermore, we show that the induction of embryo developmental arrest is associated with a substantial reprogramming of gene expression. Together, these results support the conclusion that expression of LINE-1 retrotransposons is required for early embryo preimplantation development.

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

    PubMed

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

    2016-02-15

    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.

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

    PubMed

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

    2016-02-15

    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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development.

    PubMed Central

    Hou, Q; Gorski, J

    1993-01-01

    Estrogen and progesterone play an important role in the development and implantation of preimplantation embryos. However, it is controversial whether these hormones act directly on the embryos. The effects of these hormones depend on the existence of their specific receptors. To determine whether estrogen receptor (ER) and progesterone receptor genes are expressed in mouse preimplantation embryos, we examined RNA from embryos at different stages of preimplantation development by reverse transcription-polymerase chain reaction techniques. ER mRNA was found in oocytes and fertilized eggs. The message level began to decline at the two-cell stage and reached its lowest level at the five- to eight-cell stage. ER mRNA was not detectable at the morula stage but reappeared at the blastocyst stage. Progesterone receptor mRNA was not detectable until the blastocyst stage. The embryonic expression of ER and progesterone receptor genes in the blastocyst suggests a possible functional requirement for ER and progesterone receptor at this stage of development. These results provide a basis for determining the direct role of estrogen and progesterone in preimplantation embryos. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8415723

  14. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs

    PubMed Central

    KIM, Jin-Woo; PARK, Hyo-Jin; CHAE, Sung-Kyu; AHN, Jae-Hyun; DO, Geon-Yeop; CHOO, Young-Kug; PARK, Joung Jun; JUNG, Bae Dong; KIM, Sun-Uk; CHANG, Kyu-Tae; KOO, Deog-Bon

    2016-01-01

    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

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

    PubMed

    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.

  16. 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

  17. 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

  18. CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

    PubMed Central

    Lu, Yong Chao; Chen, Hui; Fok, Kin Lam; Tsang, Lai Ling; Yu, Mei Kuen; Zhang, Xiao Hu; Chen, Jing; Jiang, Xiaohua; Chung, Yiu Wa; Ma, Alvin Chun Hang; Leung, Anskar Yu Hung; Huang, He Feng; Chan, Hsiao Chang

    2012-01-01

    Although HCO3− is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO3− acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO3− on preimplantation embryo development can be suppressed by interfering the function of a HCO3−-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO3− or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO3− removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO3− to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs. PMID:22664907

  19. 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. PMID:6711631

  20. 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.

  1. Dietary Sugar in Healthy Female Primates Perturbs Oocyte Maturation and In Vitro Preimplantation Embryo Development

    PubMed Central

    Latham, Keith E.; Mtango, Namdori R.; Midic, Uros; VandeVoort, Catherine A.

    2014-01-01

    The consumption of refined sugars continues to pose a significant health risk. However, nearly nothing is known about the effects of sugar intake by healthy women on the oocyte or embryo. Using rhesus monkeys, we show that low-dose sucrose intake over a 6-month period has an impact on the oocyte with subsequent effects on the early embryo. The ability of oocytes to resume meiosis was significantly impaired, although the differentiation of the somatic component of the ovarian follicle into progesterone-producing cells was not altered. Although the small subset of oocytes that did mature were able to be fertilized in vitro and develop into preimplantation blastocysts, there were >1100 changes in blastocyst gene expression. Because sucrose treatment ended before fertilization, the effects of sugar intake by healthy primates are concluded to be epigenetic modifications to the immature oocyte that are manifest in the preimplantation embryo. PMID:24731100

  2. Dietary sugar in healthy female primates perturbs oocyte maturation and in vitro preimplantation embryo development.

    PubMed

    Chaffin, Charles L; Latham, Keith E; Mtango, Namdori R; Midic, Uros; VandeVoort, Catherine A

    2014-07-01

    The consumption of refined sugars continues to pose a significant health risk. However, nearly nothing is known about the effects of sugar intake by healthy women on the oocyte or embryo. Using rhesus monkeys, we show that low-dose sucrose intake over a 6-month period has an impact on the oocyte with subsequent effects on the early embryo. The ability of oocytes to resume meiosis was significantly impaired, although the differentiation of the somatic component of the ovarian follicle into progesterone-producing cells was not altered. Although the small subset of oocytes that did mature were able to be fertilized in vitro and develop into preimplantation blastocysts, there were >1100 changes in blastocyst gene expression. Because sucrose treatment ended before fertilization, the effects of sugar intake by healthy primates are concluded to be epigenetic modifications to the immature oocyte that are manifest in the preimplantation embryo. PMID:24731100

  3. Dietary sugar in healthy female primates perturbs oocyte maturation and in vitro preimplantation embryo development.

    PubMed

    Chaffin, Charles L; Latham, Keith E; Mtango, Namdori R; Midic, Uros; VandeVoort, Catherine A

    2014-07-01

    The consumption of refined sugars continues to pose a significant health risk. However, nearly nothing is known about the effects of sugar intake by healthy women on the oocyte or embryo. Using rhesus monkeys, we show that low-dose sucrose intake over a 6-month period has an impact on the oocyte with subsequent effects on the early embryo. The ability of oocytes to resume meiosis was significantly impaired, although the differentiation of the somatic component of the ovarian follicle into progesterone-producing cells was not altered. Although the small subset of oocytes that did mature were able to be fertilized in vitro and develop into preimplantation blastocysts, there were >1100 changes in blastocyst gene expression. Because sucrose treatment ended before fertilization, the effects of sugar intake by healthy primates are concluded to be epigenetic modifications to the immature oocyte that are manifest in the preimplantation embryo.

  4. CTR9/PAF1c regulates molecular lineage identity, histone H3K36 trimethylation and genomic imprinting during preimplantation development.

    PubMed

    Zhang, Kun; Haversat, Jocelyn M; Mager, Jesse

    2013-11-01

    Genome-wide epigenetic reprogramming is required for successful preimplantation development. Inappropriate or deficient chromatin regulation can result in defective lineage specification and loss of genomic imprinting, compromising normal development. Here we report that two members of the RNA polymerase II associated factor, homolog (Saccharomyces cerevisiae) complex (PAF1 complex) components, Ctr9 and Rtf1, are required during mammalian preimplantation development. We demonstrate that Ctr9-deficient embryos fail to correctly specify lineages at the blastocyst stage. Expression of some lineage specific factors is markedly reduced in Ctr9 knockdown embryos, including Eomes, Elf5 and Sox2, while others are inappropriately expressed (Oct4, Nanog, Gata6, Fgf4 and Sox17). We also show that several imprinted genes (Mest, Peg3, Snrpn and Meg3) are aberrantly expressed although allele specific DNA methylation is not altered. We document a loss of histone H3 lysine 36 trimethylation (H3K36me3) in Ctr9-deficient embryos and confirm that knockdown of either Setd2 or Rtf1 results in similar phenotypes. These findings show that the PAF1 complex is required for mammalian development, likely through regulation of H3K36me3, and indicate functional conservation of the PAF1 complex from yeast to mammals in vivo.

  5. Osteopontin is expressed in the oviduct and promotes fertilization and preimplantation embryo development of mouse.

    PubMed

    Liu, Qian; Xie, Qing-zhen; Zhou, Yun; Yang, Jing

    2015-08-01

    Osteopontin (OPN) is a multifunctional phosphoprotein that is detected in various tissues, including male and female reproductive tracts. In this study, we evaluated OPN expression in mouse oviducts during the estrus cycle, and at days 1-5 of pregnancy and pseudopregnancy by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The mice oocytes, sperm and embryos were treated with different concentrations of anti-OPN antibody in vitro to detect the function of OPN in fertilization and preimplantation embryo development. OPN mRNA and protein expression in mouse oviducts were cyclic dependent throughout the estrous cycle, which was highest at estrous and lowest at diestrous. Such a phenomenon was consistent with the change in estrogen level in mice. The expression levels of OPN in mice oviduct of normal pregnancy and pseudopregnancy were significantly different, which indicated that OPN expression in mouse oviducts was depend on estrogen and preimplantation embryo. Furthermore, anti-OPN antibody treatment could reduce the rates of fertilization, cleavage and blastocyst formation in vitro in a dose-dependent way. Overall, our results indicated that the expression of OPN in mouse oviducts during the estrous cycle and early pregnancy is likely regulated by estrogen and the embryo, and OPN may play a vital role in oocyte fertilization and preimplantation embryo development.

  6. Injection of ligand-free gold and silver nanoparticles into murine embryos does not impact pre-implantation development

    PubMed Central

    Taylor, Ulrike; Garrels, Wiebke; Barchanski, Annette; Peterson, Svea; Sajti, Laszlo; Lucas-Hahn, Andrea; Gamrad, Lisa; Baulain, Ulrich; Klein, Sabine; Kues, Wilfried A

    2014-01-01

    Summary Intended exposure to gold and silver nanoparticles has increased exponentially over the last decade and will continue to rise due to their use in biomedical applications. In particular, reprotoxicological aspects of these particles still need to be addressed so that the potential impacts of this development on human health can be reliably estimated. Therefore, in this study the toxicity of gold and silver nanoparticles on mammalian preimplantation development was assessed by injecting nanoparticles into one blastomere of murine 2 cell-embryos, while the sister blastomere served as an internal control. After treatment, embryos were cultured and embryo development up to the blastocyst stage was assessed. Development rates did not differ between microinjected and control groups (gold nanoparticles: 67.3%, silver nanoparticles: 61.5%, sham: 66.2%, handling control: 79.4%). Real-time PCR analysis of six developmentally important genes (BAX, BCL2L2, TP53, OCT4, NANOG, DNMT3A) did not reveal an influence on gene expression in blastocysts. Contrary to silver nanoparticles, exposure to comparable Ag+-ion concentrations resulted in an immediate arrest of embryo development. In conclusion, the results do not indicate any detrimental effect of colloidal gold or silver nanoparticles on the development of murine embryos. PMID:24991505

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

    PubMed

    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; Reijo Pera, Renee A

    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.

  8. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

    PubMed

    Petropoulos, S; Panula, S P; Schell, J P; Lanner, F

    2016-09-01

    Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line.

  9. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

    PubMed

    Petropoulos, S; Panula, S P; Schell, J P; Lanner, F

    2016-09-01

    Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line. PMID:27046137

  10. [Effect of maternal genotype on the rate of preimplantation development in mice].

    PubMed

    Osipov, V V; Vakhrusheva, M P

    1981-01-01

    The genetic control of the rate of preimplantation development was studied in the mouse embryos. The number of cells in the embryo and the percentage of embryos at the blastocyst stage were determined on the 3.5 day of pregnancy. The experiments were carried out with CBA, A/He, C57Bl/Mib mice and mice homozygous by the mutant genes white (Miwh), fidget (fi) and ocular retardation (or), congenic with the inbred C57Bl/Mib mice. Contrasting differences were found between C57Bl/Mib and fi/fi mice. The rate of development of the morphologically normal C57Bl/Mib and fi/fi and F1 embryo was shown to depend on the maternal genotype, rather than on the paternal one. The effect of maternal genotype of the rate of preimplantation development was related to differences in the time of beginning of the cleavage. The rate of cleavage is similar for the C57Bl/Mib, fi/fi and F1 embryos.

  11. 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

  12. Preimplantation mouse embryo development as a target of the pesticide methoxychlor.

    PubMed

    Amstislavsky, Sergei Y; Kizilova, Elena A; Eroschenko, Victor P; Amstislavksy, Sergei Y

    2003-01-01

    Effects of methoxychlor (MXC) and estradiol-17beta (E) were studied in mouse preimplantation embryos. Pregnant mice received s.c. injections of sesame oil only, 10 microg E, or 0.5 mg purified (95%) MXC on Days 2-4 of pregnancy (plug = Day 1). Another group received a single dose of 2.5 microg E on Day 2 only. Based on the average weight of pregnant females, 10 microg of estradiol was equivalent to 0.33 mg/kg of bw, 2.5 microg of estradiol was equivalent to 0.082 mg/kg of bw, and the 0.5-mg dose of MXC was equivalent to 16.5 mg/kg of bw. All embryos were collected for analyses on Day 4. MXC and both estradiol-17beta doses suppressed embryonic development to blastocyst, decreased embryo cell numbers, and caused abnormal blastocyst formation. The high estradiol-17beta dose significantly increased the percent degenerating embryos and caused a tube-locking effect, with retention of embryos in the oviduct. In contrast to estradiol-17beta, MXC at the dose used in this study did not alter tubal transport of embryos. Also in contrast to estradiol-17beta, MXC increased the percentage of nuclear fragmentation and micronuclei. In preimplantation embryos, MXC and estradiol-17beta both suppressed embryo development. MXC effects were, however, different from those of estradiol-17beta, indicating a difference in mechanism of action, possibly due to cytotoxicity and induction of apoptosis. PMID:12507662

  13. 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.

  14. Microwells support high-resolution time-lapse imaging and development of preimplanted mouse embryos

    PubMed Central

    Chung, Yu-Hsiang; Hsiao, Yi-Hsing; Kao, Wei-Lun; Hsu, Chia-Hsien; Chen, Chihchen

    2015-01-01

    A vital aspect affecting the success rate of in vitro fertilization is the culture environment of the embryo. However, what is not yet comprehensively understood is the affect the biochemical, physical, and genetic requirements have over the dynamic development of human or mouse preimplantation embryos. The conventional microdrop technique often cultures embryos in groups, which limits the investigation of the microenvironment of embryos. We report an open microwell platform, which enables micropipette manipulation and culture of embryos in defined sub-microliter volumes without valves. The fluidic environment of each microwell is secluded from others by layering oil on top, allowing for non-invasive, high-resolution time-lapse microscopy, and data collection from each individual embryo without confounding factors. We have successfully cultured mouse embryos from the two-cell stage to completely hatched blastocysts inside microwells with an 89% success rate (n = 64), which is comparable to the success rate of the contemporary practice. Development timings of mouse embryos that developed into blastocysts are statistically different to those of embryos that failed to form blastocysts (p–value < 10−10, two-tailed Student's t-test) and are robust indicators of the competence of the embryo to form a blastocyst in vitro with 94% sensitivity and 100% specificity. Embryos at the cleavage- or blastocyst-stage following the normal development timings were selected and transferred to the uteri of surrogate female mice. Fifteen of twenty-two (68%) blastocysts and four of ten (40%) embryos successfully developed into normal baby mice following embryo transfer. This microwell platform, which supports the development of preimplanted embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology. PMID:26015830

  15. Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development.

    PubMed

    Shao, Gen-Bao; Chen, Jun-Chao; Zhang, Liu-Ping; Huang, Pan; Lu, Hong-Yan; Jin, Jie; Gong, Ai-Hua; Sang, Jian-Rong

    2014-08-01

    Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P < 0.05), corresponding to the period of embryonic genome activation (EGA). Subsequently, the H3K4me2 level dramatically decreased at the four-cell stage and remained at low level until the blastocyst stage (P < 0.05), whereas the H3K4me3 level transiently decreased in the four-cell embryos but steadily increased to the peak in the blastocysts (P < 0.05). The high level of H3K4me2 during the EGA was coinciding with a peak expression of its methyltransferase, ASH2L, which may stabilize this methylation level during this period. Correspondingly, a concomitant decrease in levels of its demethylases, KDM5B and KDM1A, was observed. H3K4me3 was correlated to the expression of its methyltransferase (KMT2B) and demethylase (KDM5A). Thus, these enzymes may function for the EGA and the first lineage segregation in preimplantation mouse embryos. PMID:24619213

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

    PubMed

    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.

  17. Genetic Analysis of Human Preimplantation Embryos.

    PubMed

    Garcia-Herrero, S; Cervero, A; Mateu, E; Mir, P; Póo, M E; Rodrigo, L; Vera, M; Rubio, C

    2016-01-01

    Preimplantation development comprises the initial stages of mammalian development, before the embryo implants into the mother's uterus. In normal conditions, after fertilization the embryo grows until reaching blastocyst stage. The blastocyst grows as the cells divide and the cavity expands, until it arrives at the uterus, where it "hatches" from the zona pellucida to implant into the uterine wall. Nevertheless, embryo quality and viability can be affected by chromosomal abnormalities, most of which occur during gametogenesis and early embryo development; human embryos produced in vitro are especially vulnerable. Therefore, the selection of chromosomally normal embryos for transfer in assisted reproduction can improve outcomes in poor-prognosis patients. Additionally, in couples with an inherited disorder, early diagnosis could prevent pregnancy with an affected child and would, thereby, avoid the therapeutic interruption of pregnancy. These concerns have prompted advancements in the use of preimplantation genetic diagnosis (PGD). Genetic testing is applied in two different scenarios: in couples with an inherited genetic disorder or carriers of a structural chromosomal abnormality, it is termed PGD; in infertile couples with increased risk of generating embryos with de novo chromosome abnormalities, it is termed preimplantation genetic screening, or PGS. PMID:27475859

  18. 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

  19. Long-term, six-dimensional live-cell imaging for the mouse preimplantation embryo that does not affect full-term development.

    PubMed

    Yamagata, Kazuo; Suetsugu, Rinako; Wakayama, Teruhiko

    2009-06-01

    Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which ;out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research.

  20. Impact of cytokine expression in the pre-implanted donor lung on the development of chronic lung allograft dysfunction subtypes.

    PubMed

    Saito, T; Takahashi, H; Kaneda, H; Binnie, M; Azad, S; Sato, M; Waddell, T K; Cypel, M; Liu, M; Keshavjee, S

    2013-12-01

    The long-term success of lung transplantation continues to be challenged by the development of chronic lung allograft dysfunction (CLAD). The purpose of this study was to investigate the relationship between cytokine expression levels in pre-implanted donor lungs and the posttransplant development of CLAD and its subtypes, bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS). Of 109 patients who underwent bilateral lung or heart-lung transplantation and survived for more than 3 months, 50 BOS, 21 RAS and 38 patients with No CLAD were identified by pulmonary function test results. Using donor lung tissue biopsies sampled from each patient, expression levels of IL-6, IL-1β, IL-8, IL-10, interferon-γ and tumor necrosis factor-α mRNA were measured. IL-6 expression levels were significantly higher in pre-implanted lungs of patients that ultimately developed BOS compared to RAS and No CLAD (p = 0.025 and 0.011, respectively). Cox regression analysis demonstrated an association between high IL-6 expression levels and BOS development (hazard ratio = 4.98; 95% confidence interval = 2.42-10.2, p < 0.001). In conclusion, high IL-6 mRNA expression levels in pre-implanted donor lungs were associated with the development of BOS, not RAS. This association further supports the contention that early graft injury impacts on both late graft function and early graft function. PMID:24164971

  1. Altering Intracellular pH Disrupts Development and Cellular Organization in Preimplantation Hamster Embryos

    PubMed Central

    Squirrell, Jayne M.; Lane, Michelle; Bavister, Barry D.

    2016-01-01

    In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pHi and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos. PMID:11369617

  2. [Dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos.].

    PubMed

    Zhang, Qing-Hua; Shan, Zhi-Yan; Guan, Na; Xu, Yan-Ning; Shen, Jing-Ling; Zhong, Shu-Qi; Lei, Lei

    2008-12-25

    Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and

  3. 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. PMID:27070551

  4. Development of a real-time PCR method for rapid sexing of human preimplantation embryos.

    PubMed

    Martinhago, Cd; Vagnini, Ld; Petersen, Cg; Mauri, Al; Baruffi, Rl; de Oliveira, Rm; Franco, Jg

    2010-01-01

    Genes on the X chromosome are known to be responsible for more than 200 hereditary diseases. After IVF, the simple selection of embryo sex before uterine transfer can prevent the occurrence of affected offspring among couples at risk for these genetic disorders. The aim of this investigation was to develop a rapid method of preimplantation genetic diagnosis (PGD) using real-time polymerase chain reaction (PCR) for the sexing of human embryos, and to compare it to the fluorescence in-situ hybridization technique, considered to be the gold standard. After biopsies were obtained from 40 surplus non-viable embryos for transfer, a total of 98 blastomeres were analysed. It was possible to analyse 24 embryos (60%) by both techniques, generating a total of 70 blastomeres (35 per technique), while 28 blastomeres from 16 embryos (40%) were analysed only by real-time PCR. A rapid and safe method was developed in the present study for the sexual diagnosis of a single human cell (blastomere and buccal cell) using the emerging technology of real-time PCR.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. The effect of extracellular matrix molecules on mouse preimplantation embryo development in vitro.

    PubMed

    Figueiredo, F; Jones, G M; Thouas, G A; Trounson, A O

    2002-01-01

    The extracellular matrix (ECM) molecules, laminin (LN), chondroitin sulfate (CS), fibronectin (FN), hyaluronic acid (HA), mucin (MUC) and heparan sulfate proteoglycan (HS), were investigated as supplements to culture medium to improve the in vitro development of mouse 1-cell zygotes to blastocysts. Development was also compared with that in medium supplemented with bovine serum albumin (BSA) to determine the potential for ECM molecules as suitable alternatives to serum albumin in culture medium. Supplementation of sequential culture media with LN at all concentrations examined failed to result in more than 70% of zygotes developing to blastocysts; therefore, LN was considered unsuitable as a replacement for BSA and was not examined further. The optimal concentration of the remaining ECM molecules was used to supplement sequential culture media and the effect on blastocyst quality was assessed by determining the differential cell numbers of blastocysts grown in BSA-supplemented medium. Development to blastocyst was similar, regardless of the macromolecule used. The number of inner cell mass cells was significantly higher in HS-supplemented medium compared with controls. Trophectoderm cell numbers were similar to control values for all ECM molecules examined except CS for which there were fewer trophectoderm cells. It is concluded that ECM molecules, FN, HA, MUC and HS may be used as substitutes for serum protein supplementation of culture media EG0/G2 for mouse preimplantation embryo development. Heparan sulfate proteoglycan increases inner cell mass numbers and this may be due to interactions with the growth factors fibroblast growth factor 4 (FGF-4) and granulocyte-macrophage colony-stimulating factor. PMID:12617788

  10. 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.

  11. Expression of adrenergic receptors in bovine and rabbit oocytes and preimplantation embryos.

    PubMed

    Čikoš, Š; Czikková, S; Chrenek, P; Makarevich, A V; Burkuš, J; Janštová, Ž; Fabian, D; Koppel, J

    2014-02-01

    Catecholamines play an important role in embryogenesis, and data obtained in the rodent model indicate that they can act even during the preimplantation period of development. Using RT-PCR with specific oligonucleotide primers distinguishing among all members of the adrenergic receptor family, we examined expression of adrenergic receptors in bovine and rabbit oocytes, morulas and blastocysts. We found several profiles of adrenoceptor mRNA expression. Transcripts for some receptor subtypes (bovine alpha 2 receptors, rabbit α2A, α2C, β1 and β2 receptors) were detected at all examined stages, which suggests receptor expression throughout (or at most stages) the preimplantation developmental period. Expression in oocytes but not at later stages was found in only one adrenoceptor subtype (rabbit α1B). In contrast, mRNA for several adrenoceptors was found in embryos but not in oocytes (bovine beta adrenoceptors and rabbit α1A). Nucleotide sequences of our PCR products amplified in rabbit oocytes, and preimplantation embryos represent the first published mRNA sequences (partial sequences coding at least one transmembrane region) of rabbit α2C, β1 and β2 adrenoceptors. Our results suggest that the expression of adrenergic receptors can be a general feature of mammalian oocytes and preimplantation embryos. On the other hand, comparison of three mammalian species (cattle, rabbit and mouse) revealed possible interspecies differences in the expression of particular adrenoceptor subtypes. Our results support the opinion that stress mediators can act directly in cells of preimplantation embryos.

  12. Potential role for PADI-mediated histone citrullination in preimplantation development

    PubMed Central

    2012-01-01

    Background The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. Results In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. Conclusions Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation

  13. 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. PMID:27587271

  14. 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.

  15. The Maternal Effect Genes UTX and JMJD3 Play Contrasting Roles in Mus musculus Preimplantation Embryo Development

    PubMed Central

    Yang, Lei; Song, Li-Shuang; Liu, Xue-Fei; Xia, Qing; Bai, Li-Ge; Gao, Li; Gao, Guang-Qi; Wang, Yu; Wei, Zhu-Ying; Bai, Chun-Ling; Li, Guang-Peng

    2016-01-01

    During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility. PMID:27384759

  16. Preimplantation development following in vitro fertilization of mouse oocytes: effects of timing of superovulation and preincubation in vitro.

    PubMed

    Edgar, D H; Whalley, K M; Mills, J A

    1987-04-01

    The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F1 hybrid (C57BL/6 X CBA/Ca) mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.

  17. 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.

  18. Conceptus development and transcriptome at preimplantation stages in lactating dairy cows of distinct genetic groups and estrous cyclic statuses.

    PubMed

    Ribeiro, E S; Monteiro, A P A; Bisinotto, R S; Lima, F S; Greco, L F; Ealy, A D; Thatcher, W W; Santos, J E P

    2016-06-01

    The objectives were to compare development and transcriptome of preimplantation conceptuses 15 d after synchronized ovulation and artificial insemination (AI) according to the genetic background of the cow and estrous cyclicity at the initiation of the synchronization program. On d 39±3 postpartum, Holstein cows that were anovular (HA; n=10), Holstein cows that were estrous cyclic (HC; n=25), and Jersey/Holstein crossbred cows that were estrous cyclic (CC; n=25) were randomly selected in a grazing herd and subjected to the Ovsynch protocol. All cows were inseminated on d 49±3 postpartum, which was considered study d 0. Blood was sampled and analyzed for concentrations of progesterone, estradiol, insulin, and insulin-like growth factor 1 (IGF-1) on study d -10, -3, -1, 7, and 15 relative to AI. On study d 15, uteri were flushed and recovered fluid had IFN-τ concentrations measured and subjected to metabolomic analysis. Morphology of the recovered conceptuses was evaluated, and mRNA was extracted and subjected to transcriptome microarray analysis. Compared with HC, CC presented greater concentrations of progesterone and estradiol in plasma, with corpora lutea and preovulatory follicles of similar size. Conceptuses from CC were larger, tended to secrete greater amounts of IFN-τ, and had greater transcript expression of peroxisome proliferator-activated receptor gamma (PPARγ), an important transcription factor that coordinates lipid metabolism and elongation at preimplantation development. In addition, pregnant CC had greater concentrations of anandamide in the uterine flush, which might be important for elongation of the conceptus and early implantation. Conceptuses from HA were also longer and secreted greater amounts of IFN-τ than conceptuses from HC, likely because of the distinct progesterone profiles before and after AI. Nonetheless, anovular cows had reduced concentrations of IGF-1 in plasma, and their conceptuses presented remarkable transcriptomic

  19. An evolutionary link for developing mammalian lungs.

    PubMed

    Maloney, J E; Darian-Smith, C; Russell, B; Varghese, M; Cooper, J; Limpus, C J

    1989-09-01

    Lungs of the human infant and those of other mammals are filled with fluid immediately prior to birth. Studies of the ionic composition of this fluid indicate that active ionic transport processes occur in the epithelial cells of the potential airspaces. The purpose of this study was to see if these active ion pumps were present in developing species other than mammals thus providing a possible evolutionary link to mammals. A series of samples of lung liquid, amniotic fluid, and plasma were taken from embryonic marine turtles gathered from clutches incubating in the beach at Mon Repos, Queensland, Australia during the summer of 1986-87. The concentrations of sodium, potassium and chloride ions and protein measured in these liquids indicated that active pumping processes similar to that seen in the mammalian lung were present in the developing lungs of these marine reptiles and further, circumstantial evidence was gathered to suggest that this liquid was partially reabsorbed prior to hatching. The results support the notion that processes responsible for the normal development of the human lung and lungs of other mammals are also present in the hollow lungs of marine turtles. Thus there is an evolutionary counterpart controlling lung development in more ancient species. It may be possible to generalize this observation to the development of hollow lungs of other species.

  20. 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development

    PubMed Central

    2012-01-01

    Background Embryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. Cells within this embryo will then differentiate and give rise to all the tissues of a new individual. Early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. When the organization of higher-order chromatin structures, such as pericentromeric heterochromatin, was first analyzed in mouse embryos, specific nuclear rearrangements were observed that correlated with embryonic genome activation at the 2-cell stage. However, most existing analyses have been conducted by visual observation of fluorescent images, in two dimensions or on z-stack sections/projections, but only rarely in three dimensions (3D). Results In the present study, we used DNA fluorescent in situ hybridization (FISH) to localize centromeric (minor satellites), pericentromeric (major satellites), and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos (from the 1-cell to blastocyst stage). Their distribution was then analyzed in 3D on confocal image stacks, focusing on the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. We used computational imaging to quantify various nuclear parameters in the 3D-FISH images, to analyze the organization of compartments of interest, and to measure physical distances between these compartments. Conclusions The results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. just after fertilization. We also found that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes other remarkable changes during preimplantation development, particularly at the 4-cell stage. PMID:23095683

  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.

  3. Functions of miRNAs during Mammalian Heart Development

    PubMed Central

    Yan, Shun; Jiao, Kai

    2016-01-01

    MicroRNAs (miRNAs) play essential roles during mammalian heart development and have emerged as attractive therapeutic targets for cardiovascular diseases. The mammalian embryonic heart is mainly derived from four major cell types during development. These include cardiomyocytes, endocardial cells, epicardial cells, and neural crest cells. Recent data have identified various miRNAs as critical regulators of the proper differentiation, proliferation, and survival of these cell types. In this review, we briefly introduce the contemporary understanding of mammalian cardiac development. We also focus on recent developments in the field of cardiac miRNAs and their functions during the development of different cell types. PMID:27213371

  4. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix.

    PubMed

    Sargus-Patino, Catherine N; Wright, Elane C; Plautz, Sarah A; Miles, Jeremy R; Vallet, Jeff L; Pannier, Angela K

    2014-08-01

    Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17

  5. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix.

    PubMed

    Sargus-Patino, Catherine N; Wright, Elane C; Plautz, Sarah A; Miles, Jeremy R; Vallet, Jeff L; Pannier, Angela K

    2014-08-01

    Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17

  6. Effects of high-dose and multiple-dose gonadotropin stimulation on mouse oocyte quality as assessed by preimplantation development following in vitro fertilization.

    PubMed

    Edgar, D H; Whalley, K M; Mills, J A

    1987-10-01

    The effects of increasing the level of ovarian stimulation on preimplantation embryonic development were assessed using a mouse in vitro fertilization system. When F1 hybrid (C57BL/6 X CBA/Ca) mice received a single injection of 5 IU pregnant mare's serum gonadotropin (PMSG) followed 60 hr later by 5 IU human chorionic gonadotropin (hCG) approximately 50% of the resultant postovulatory oocytes developed to the blastocyst stage following in vitro fertilization. Increasing the single dose of PMSG to 10 or 15 IU resulted in significant reductions in the frequency of development to the blastocyst stage. When one or two additional doses of 5 IU PMSG were administered 24 and 48 hr after an initial injection of 5 IU, lower frequencies of oocytes with the potential for full preimplantation development were again observed. This reduction in gamete quality was significantly greater when the final dose of PMSG was administered only 12 hr prior to hCG. The results suggest that excessive gonadotropin stimulation may compromise the quality of the preimplantation embryos obtained following in vitro fertilization and that the timing of gonadotropin administration may also be critical.

  7. Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development.

    PubMed

    Mihajlović, Aleksandar I; Bruce, Alexander W

    2016-09-01

    The differential activity of the Hippo-signalling pathway between the outer- and inner-cell populations of the developing preimplantation mouse embryo directs appropriate formation of trophectoderm and inner cell mass (ICM) lineages. Such distinct signalling activity is under control of intracellular polarization, whereby Hippo-signalling is either supressed in polarized outer cells or activated in apolar inner cells. The central role of apical-basolateral polarization to such differential Hippo-signalling regulation prompted us to reinvestigate the role of potential upstream molecular regulators affecting apical-basolateral polarity. This study reports that the chemical inhibition of Rho-associated kinase (Rock) is associated with failure to form morphologically distinct blastocysts, indicative of compromised trophectoderm differentiation, and defects in the localization of both apical and basolateral polarity factors associated with malformation of tight junctions. Moreover, Rock-inhibition mediates mislocalization of the Hippo-signalling activator Angiomotin (Amot), to the basolateral regions of outer cells and is concomitant with aberrant activation of the pathway. The Rock-inhibition phenotype is mediated by Amot, as RNAi-based Amot knockdown totally rescues the normal suppression of Hippo-signalling in outer cells. In conclusion, Rock, via regulating appropriate apical-basolateral polarization in outer cells, regulates the appropriate activity of the Hippo-signalling pathway, by ensuring correct subcellular localization of Amot protein in outer cells. PMID:27430121

  8. 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

  9. Axonal oscillations in developing mammalian nerve axons

    NASA Astrophysics Data System (ADS)

    Zeng, Shangyou; Jung, Peter

    2005-01-01

    We study neuronal spike propagation in a developing myelinated axon in various stages of its development through detailed computational modeling. Recently, a form of bursting (axonal bursting), has been reported in axons in developing nerves in the absence of potassium channels. We present a computational study using a detailed model for a myelinated nerve in development to explore under what circumstances such an effect can be expected. It is shown that axonal oscillation may be caused by backfiring between the nodes of Ranvier or through backfiring from internodal sodium channels or by reducing the thickness of the myelin wrapping the axon between the nodes of Ranvier.

  10. 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.

  11. 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

  12. The Role of Autophagy in Mammalian Development

    PubMed Central

    Cecconi, Francesco; Levine, Beth

    2009-01-01

    Autophagy is important for the degradation of bulk cytoplasm, long-lived proteins, and entire organelles. In lower eukaryotes, autophagy functions as a cell death mechanism or as a stress response during development. However, autophagy’s significance in vertebrate development, and the role (if any) of vertebrate-specific factors in its regulation, remains unexplained. Through careful analysis of the current autophagy gene mutant mouse models, we propose that in mammals, autophagy may be involved in specific cytosolic rearrangements needed for proliferation, death, and differentiation during embryogenesis and postnatal development. Thus, autophagy is a process of cytosolic “renovation,” crucial in cell fate decisions. PMID:18804433

  13. 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.

  14. 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.

  15. Genetic regulation of mammalian gonad development.

    PubMed

    Eggers, Stefanie; Ohnesorg, Thomas; Sinclair, Andrew

    2014-11-01

    Sex-specific gonadal development starts with formation of the bipotential gonad, which then differentiates into either a mature testis or an ovary. This process is dependent on activation of either the testis-specific or the ovary-specific pathway while the opposite pathway is continuously repressed. A network of transcription factors tightly regulates initiation and maintenance of these distinct pathways; disruption of these networks can lead to disorders of sex development in humans and male-to-female or female-to-male sex reversal in mice. Sry is the Y-linked master switch that is both required and sufficient to drive the testis-determining pathway. Another key component of the testis pathway is Sox9, which acts immediately downstream of Sry. In contrast to the testis pathway, no single sex-determining factor has been identified in the ovary pathway; however, multiple genes, such as Foxl2, Rspo1, Ctnnb1, and Wnt4, seem to work synergistically and in parallel to ensure proper ovary development. Our understanding of the regulatory networks that underpin testis and ovary development has grown substantially over the past two decades.

  16. 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

  17. The histone demethylase JMJD2C is stage-specifically expressed in preimplantation mouse embryos and is required for embryonic development.

    PubMed

    Wang, Jianle; Zhang, Miao; Zhang, Yu; Kou, Zhaohui; Han, Zhiming; Chen, Da-Yuan; Sun, Qing-Yuan; Gao, Shaorong

    2010-01-01

    Epigenetic modifications play a pivotal role in embryonic development by dynamically regulating DNA methylation and chromatin modifications. Although recent studies have shown that core histone methylation is reversible, very few studies have investigated the functions of the newly discovered histone demethylases during embryonic development. In the present study, we investigated the expression characteristics and function of JMJD2C, a histone demethylase that belongs to the JmjC-domain-containing histone demethylases, during preimplantation embryonic development of the mouse. We found that JMJD2C is stage-specifically expressed during preimplantation development, with the highest activity being observed from the two-cell to the eight-cell stage. Depletion of JMJD2C in metaphase II oocytes followed by parthenogenetic activation causes a developmental arrest before the blastocyst stage. Moreover, consistent with a previous finding in embryonic stem (ES) cells, depletion of JMJD2C causes a significant down-regulation of the pluripotency gene Nanog in embryos. However, contrary to a previous report in ES cells, we observed that other pluripotency genes, Pou5f1 and Sox2, are also significantly down-regulated in JMJD2C-depleted embryos. Furthermore, the depletion of JMJD2C in early embryos also caused significant down-regulation of the Myc and Klf4 genes, which are associated with cell proliferation. Our data suggest that the deregulation of these critical genes synergistically causes the developmental defects observed in JMJD2C-depleted embryos. PMID:19696013

  18. 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.

  19. Invited review: Genetic contributions underlying the development of preimplantation bovine embryos.

    PubMed

    Kropp, J; Peñagaricano, F; Salih, S M; Khatib, H

    2014-03-01

    In recent years, it has become evident that genetic selection to improve milk production has resulted in a decline in dairy cattle fertility. Growing evidence suggests that the greatest loss occurs early in pregnancy around the time of embryo implantation. As a means to make genetic improvements and to assist in reproductive performance, use of artificial reproductive technologies such as artificial insemination and in vitro production of embryos have been widely used. Both of these technologies rely on the competence and quality of gametes for successful development of embryos. Often, selection of animals is based on the genetic merit of the animal, although specific fertility markers are relatively underdeveloped compared with markers for production traits. Similarly, current in vitro fertilization systems could benefit from a uniform method for selection of the best quality embryos to transfer into recipients for successful implantation and delivery of healthy offspring. As genetics underlie biological processes such as fertility, the need exists to further identify and characterize genes that affect fertility and development within both the parental gametes and the embryo. Furthermore, the magnitude of the contribution of each parental genome to the success of embryo development and pregnancy is not clear. As such, the objective of this review is to provide an overview of studies relating to genetic markers at the DNA level, parental and embryonic gene expression, and the effects of epigenetics on embryonic development. Future studies should exploit advances in molecular technologies to identify and classify genes underlying fertility and development to establish biomarkers and predictors for improved genetic selection. PMID:24377798

  20. 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.

  1. Toxic effects of Hoechst staining and UV irradiation on preimplantation development of parthenogenetically activated mouse oocytes.

    PubMed

    Versieren, Karen; Heindryckx, Björn; Qian, Chen; Gerris, Jan; De Sutter, Petra

    2014-02-01

    Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.

  2. Intracytoplasmic morphologically selected sperm injection improves development and quality of preimplantation embryos in teratozoospermia patients.

    PubMed

    Knez, Katja; Tomazevic, Tomaz; Zorn, Branko; Vrtacnik-Bokal, Eda; Virant-Klun, Irma

    2012-08-01

    This prospective randomized study investigated whether intracytoplasmic sperm injection (ICSI) outcome can be improved with sperm preselection under ×6000 magnification and intracytoplasmic morphologically selected sperm injection (IMSI) in patients with teratozoospermia and characterized embryo development and quality regarding sperm morphology and presence of head vacuoles. Couples with isolated teratozoospermia were divided into two groups: IMSI group (n=52) and ICSI group (n=70) and fertilization, blastocyst and clinical pregnancy rates were compared. Oocytes from 30 randomly chosen patients from the IMSI group were injected with spermatozoa that had been previously classified under ×6000 magnification into four classes according to the number and size of vacuoles in the head and then cultured separately. Pronuclear morphology, embryo development and blastomere viability were estimated to investigate the influence of sperm morphology, especially vacuoles, on embryo developmental capacity. A significantly higher clinical pregnancy rate was achieved in the IMSI group compared with the ICSI group (48% versus 24%, P<0.05). Fertilization with spermatozoa without head vacuoles yielded higher number of morphologically normal zygotes, higher blastocyst rate and smaller proportion of arrested embryos than spermatozoa with vacuoles and other head defects. IMSI is a method of choice in patients with teratozoospermia.

  3. 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

  4. 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.

  5. Nepro is localized in the nucleolus and essential for preimplantation development in mice.

    PubMed

    Hashimoto, Masakazu; Sato, Tatsuya; Muroyama, Yuko; Fujimura, Lisa; Hatano, Masahiko; Saito, Tetsuichiro

    2015-09-01

    We generated knockout (KO) mice of Nepro, which has been shown to be necessary to maintain neural progenitor cells downstream of Notch in the mouse developing neocortex by using knockdown experiments, to explore its function in embryogenesis. Nepro KO embryos were morphologically indistinguishable from wild type (WT) embryos until the morula stage but failed in blastocyst formation, and many cells of the KO embryos resulted in apoptosis. We found that Nepro was localized in the nucleolus at the blastocyst stage. The number of nucleolus precursor bodies (NPBs) and nucleoli per nucleus was significantly higher in Nepro KO embryos compared with WT embryos later than the 2-cell stage. Furthermore, at the morula stage, whereas 18S rRNA and ribosomal protein S6 (rpS6), which are components of the ribosome, were distributed to the cytoplasm in WT embryos, they were mainly localized in the nucleoli in Nepro KO embryos. In addition, in Nepro KO embryos, the amount of the mitochondria-associated p53 protein increased, and Cytochrome c was distributed in the cytoplasm. These findings indicate that Nepro is a nucleolus-associated protein, and its loss leads to the apoptosis before blastocyst formation in mice.

  6. A systematic molecular genetic approach to study mammalian germline development

    PubMed Central

    Abe, Kuniya; Ko, Minoru S. H.; MacGregor, Grant R.

    2011-01-01

    It is difficult to study gene expression in mammalian embryonic germ cells as PGCs constitute only a minor proportion of the mouse embryo. We have overcome this problem by using a novel combination of established molecular and transgenic approaches. A line of mice has been generated in which the cells of the germ lineage express the β-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages during germline development by use of a stain for β-gal activity and a fluorescence activated cell sorter. Subsequently, cDNA libraries have been constructed from each germ cell population using a modified lone-linker PCR strategy. These combined cDNA libraries represent genes expressed in PGCs during mammalian germline development. To facilitate a molecular genetic approach to studying mammalian germline development, these cDNA libraries will be pooled to form an arrayed, addressed reference embryonic germ cell cDNA library. In parallel with large-scale cDNA sequencing efforts, genes that are differentially expressed in germ cells will be identified by screening the reference library with probes generated by subtractive hybridization. Complementary DNAs identified using this approach will be analyzed by sequencing, database comparison, genomic mapping and in situ hybridization to ascertain the potential functional importance of each gene to germline development. In addition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function of these genes in this process. PMID:9853837

  7. 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.

  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. 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.

  11. Preimplantation embryo programming: transcription, epigenetics, and culture environment.

    PubMed

    Duranthon, Veronique; Watson, Andrew J; Lonergan, Patrick

    2008-02-01

    Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryo's genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed can exceed its own adaptive capacity, resulting in aberrant embryonic development. Nuclear transfer or embryo culture-induced influences not only affect implantation and establishment of pregnancy but also can extend to fetal and postnatal development and affect susceptibility to disease in later life. It is therefore critical to define the basic program controlling preimplantation development, and also to utilize nuclear transfer and embryo culture models so that we may design healthier environments for preimplantation embryos to thrive in and also minimize the potential for negative consequences during pregnancy and post-gestational life. In addition, it is necessary to couple gene expression analysis with the investigation of gene function so that effects on gene expression can be fully understood. The purpose of this short review is to highlight our knowledge of the mechanisms controlling preimplantation development and report how those mechanisms may be influenced by nuclear transfer and embryo culture. PMID:18239045

  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. Engineering considerations for process development in mammalian cell cultivation.

    PubMed

    Zhang, Hu; Wang, Weixiang; Quan, Chunshan; Fan, Shengdi

    2010-01-01

    Mammalian cell cultivation plays a great role in producing protein therapeutics in the last decades. Many engineering parameters are considered for optimization during process development in mammalian cell cultivation, only shear and mixing are especially highlighted in this paper. It is believed that shear stress due to agitation has been over-estimated to damage cells, but shear may result in nonlethal physiological responses. There is no cell damage in the regions where bubbles form, break up and coalescence, but shear stress becomes significant in the wake of rising bubbles and causes great damage to cells in bubble burst regions. Mixing is not sufficient to provide homogeneous dissolved oxygen tension, pH, CO2 and nutrients in large-scale bioreactors, which can bring severe problems for cell growth, product formation and process control. Scale-down reactors have been developed to address mixing and shear problems for parallel operations. Engineering characterization in conventional and recently developed scale-down bioreactors has been briefly introduced. Process challenges for cultivation of industrial cell lines in high cell densities as well as cultivation of stem cells and other human cells for regenerative medicine, tissue engineering and gene therapy are prospected. Important techniques, such as micromanipulation and nanomanipulation (optical tweezers) for single cell analysis, computational fluid dynamics (CFD) for shear and mixing characterization, and miniaturized bioreactors, are being developed to address those challenges. PMID:19929819

  14. 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).

  15. Drosophila homolog of the mammalian jun oncogene is expressed during embryonic development and activates transcription in mammalian cells.

    PubMed Central

    Zhang, K; Chaillet, J R; Perkins, L A; Halazonetis, T D; Perrimon, N

    1990-01-01

    By means of low-stringency cross-species hybridization to Southern DNA blots, human c-jun sequences were used to identify a unique Drosophila melanogaster locus (Djun). The predicted DJun protein is highly homologous to members of the mammalian Jun family in both the DNA binding and leucine zipper regions. Djun was mapped by in situ hybridization to position 46E of the second chromosome. It encodes a 1.7-kilobase transcript constitutively expressed at all developmental stages. Functionally, Djun in cooperation with mouse c-fos can trans-activate activator protein 1 DNA binding site when introduced into mammalian cells. Taken together, these data suggest that Djun, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of Djun allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving Djun and thus c-jun. Images PMID:1696724

  16. 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.

  17. 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. PMID:26763693

  18. Understanding Mammalian Germ Line Development with In Vitro Models.

    PubMed

    Martínez-Arroyo, Ana M; Míguez-Forján, Jose M; Remohí, Jose; Pellicer, Antonio; Medrano, Jose V

    2015-09-15

    Germ line development is crucial in organisms with sexual reproduction to complete their life cycle. In mammals, knowledge about germ line development is based mainly on the mouse model, in which genetic and epigenetic events are well described. However, little is known about how germ line development is orchestrated in humans, especially in the earliest stages. New findings derived from human in vitro models to obtain germ cells can shed light on these questions. This comprehensive review summarizes the current knowledge about mammalian germ line development, emphasizing the state of the art obtained from in vitro models for germ cell-like cell derivation. Current knowledge of the pluripotency cycle and germ cell specification has allowed different in vitro strategies to obtain germ cells with proven functionality in mouse models. Several reports during the last 10 years show that in vitro germ cell derivation with proven functionality to generate a healthy offspring is possible in mice. However, differences in the embryo development and pluripotency potential between human and mouse make it difficult to extrapolate these results. Further efforts on both human and mouse in vitro models to obtain germ cells from pluripotent stem cells may help to elucidate how human physiological events take place; therefore, therapeutic strategies can also be considered.

  19. Expression patterns of FGF receptors in the developing mammalian cochlea

    PubMed Central

    Hayashi, Toshinori; Ray, Catherine A.; Younkins, Christa; Bermingham-McDonogh, Olivia

    2010-01-01

    Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1-4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from E13.5 through post-natal ages. We find that Fgfr1, 2 and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27kip1) and the early hair cells (math1). PMID:20131355

  20. Expression and distribution of forkhead activin signal transducer 2 (FAST2) during follicle development in mouse ovaries and pre-implantation embryos.

    PubMed

    Wang, Guiping; Liu, Linlin; Guo, Shujuan; Zhang, Cong

    2016-07-01

    Xenopus forkhead activin signal transducer 1 (xFAST 1) was first characterized in Xenopus as the transcriptional partner for Smad proteins. FAST2, which is the xFAST 1 homologues in mouse, is expressed during early developmental stages of the organism. However, the function of FAST2 in mouse ovaries and pre-implantation embryos is unclear. Therefore, we investigated its expression during these processes. In postnatal mice, FAST2 was expressed in oocytes and thecal cells from postnatal day (PD) 1 to PD 21. In gonadotropin-induced immature mice, FAST2 was expressed in oocytes, thecal cells and newly formed corpora lutea (CLs), but was expressed at a lower level in degenerated CLs. Similar results were observed upon western blot analyses. In meloxicam-treated immature mice, ovulation was inhibited and FAST2 was expressed in thecal cells, luteinized granulosa cells and entrapped oocytes. Immunofluorescence results showed that FAST2 was expressed in the cytoplasm and nucleus but not the nucleolus from the zygote to 8-cell embryo stage, after which it was localized to the cytoplasm of the morulae and inner cell mass of the blastocysts. Taken together, these observations suggest that FAST2 is expressed in a cell-specific manner during ovarian follicle development, ovulation, luteinization and early embryonic development, and that FAST2 might play important roles in these physiological processes. PMID:27432806

  1. 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. PMID:21521190

  2. The effects of delayed activation and MG132 treatment on nuclear remodeling and preimplantation development of embryos cloned by electrofusion are correlated with the age of recipient cytoplasts.

    PubMed

    Wu, Yan-Guang; Zhou, Ping; Lan, Guo-Cheng; Wang, Gang; Luo, Ming-Jiu; Tan, Jing-He

    2007-01-01

    The electrofusion method, used extensively in livestock cloning, cannot be used in mice, because it is believed that the mouse oocytes are more susceptible to detrimental effects of electrical stimulus than oocytes from other species. Reports on whether a delayed activation after electrofusion and a premature chromosome condensation (PCC) is essential for efficient cloning are inconclusive. To address these issues, effects of pulsing on activation and MPF activity of nonenucleated oocytes and effects of delayed activation and MG132 treatment on donor nuclear PCC and preimplantation development of embryos cloned by electrofusion or nuclear injection were compared between different cytoplast ages in mice and goats. The results indicated that the use of oocytes collected early after donor stimulation would make it possible to conduct somatic cell nuclear transfer in mice by electrofusion. Whether a delayed activation is essential was dependent upon the age, or rather, the level, of MPF activity of the cytoplasts at the time of electrofusion, as was the requirement for MG132 treatment. The competence for blastocyst formation of cloned embryos was highly correlated with the level of donor nuclear PCC in recipient cytoplasts. The nuclear injection technique was more adaptable to older cytoplast ages, and hence less dependent on drugs for inhibition of MPF inactivation, compared to electrofusion.

  3. The effects of delayed activation and MG132 treatment on nuclear remodeling and preimplantation development of embryos cloned by electrofusion are correlated with the age of recipient cytoplasts.

    PubMed

    Wu, Yan-Guang; Zhou, Ping; Lan, Guo-Cheng; Wang, Gang; Luo, Ming-Jiu; Tan, Jing-He

    2007-01-01

    The electrofusion method, used extensively in livestock cloning, cannot be used in mice, because it is believed that the mouse oocytes are more susceptible to detrimental effects of electrical stimulus than oocytes from other species. Reports on whether a delayed activation after electrofusion and a premature chromosome condensation (PCC) is essential for efficient cloning are inconclusive. To address these issues, effects of pulsing on activation and MPF activity of nonenucleated oocytes and effects of delayed activation and MG132 treatment on donor nuclear PCC and preimplantation development of embryos cloned by electrofusion or nuclear injection were compared between different cytoplast ages in mice and goats. The results indicated that the use of oocytes collected early after donor stimulation would make it possible to conduct somatic cell nuclear transfer in mice by electrofusion. Whether a delayed activation is essential was dependent upon the age, or rather, the level, of MPF activity of the cytoplasts at the time of electrofusion, as was the requirement for MG132 treatment. The competence for blastocyst formation of cloned embryos was highly correlated with the level of donor nuclear PCC in recipient cytoplasts. The nuclear injection technique was more adaptable to older cytoplast ages, and hence less dependent on drugs for inhibition of MPF inactivation, compared to electrofusion. PMID:17907952

  4. Polyamide Nanogels from Generally Recognized as Safe Components and Their Toxicity in Mouse Preimplantation Embryos.

    PubMed

    Prasad, Priyaa; Molla, Mijanur Rahaman; Cui, Wei; Canakci, Mine; Osborne, Barbara; Mager, Jesse; Thayumanavan, S

    2015-11-01

    Safe delivery systems that can not only encapsulate hydrophobic drug molecules, but also release them in response to specific triggers are important in several therapeutic and biomedical applications. In this paper, we have designed a nanogel based on molecules that are generally recognized as safe (GRAS). We have shown that the resultant polymeric nanogels exhibit responsive molecular release and also show high in vitro cellular viability on HEK 293T, HeLa, MCF 7, and A549 cell lines. The toxicity of these nanogels was further evaluated with a highly sensitive assay using mouse preimplantation embryo development, where blastocysts were formed after 4 days of in vitro culture, and live pups were born when morulae/early blastocysts were transferred to the uteri of surrogate recipients. Our results indicate that these nanogels are nontoxic during mammalian development and do not alter normal growth or early embryo success rate. PMID:26367020

  5. Polyamide Nanogels from GRAS Components and Their Toxicity in Mouse Pre-implantation Embryos

    PubMed Central

    Prasad, Priyaa; Molla, Mijanur Rahaman; Cui, Wei; Canakci, Mine; Osborne, Barbara; Mager, Jesse; Thayumanavan, S.

    2016-01-01

    Safe delivery systems that can not only encapsulate hydrophobic drug molecules, but also release them in response to specific triggers, are important in several therapeutic and biomedical applications. In this paper, we have designed a nanogel based on molecules that are generally recognized as safe (GRAS). We have shown that the resultant polymeric nanogels exhibit responsive molecular release, and also show high in vitro cellular viability on HEK 293T, HeLa, MCF 7 and A549 cell lines. The toxicity of these nanogels was further evaluated with a highly sensitive assay using mouse preimplantation embryo development, where blastocysts were formed after four days of in vitro culture and live pups were born when morulae/early blastocysts were transferred to the uteri of surrogate recipients. Our results indicate that these nanogels are non-toxic during mammalian development and do not alter normal growth or early embryo success rate. PMID:26367020

  6. 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.

  7. 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

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

    PubMed

    Thamodaran, Vasanth; Bruce, Alexander W

    2016-09-01

    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

  9. 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

  10. 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.

  11. 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

  12. [Pre-implantation genetic diagnosis: indications, techniques, and results].

    PubMed

    Veiga, A; Boada, M; Barri, P N

    1998-01-01

    The combination of the technique of In Vitro Fertilization (IVF) and molecular genetics has led to the development of Preimplantation Genetic Diagnosis (PGD). Oocyte and embryo biopsy, Fluorescent in situ Hybridization (FISH) and Polymerase Chain Reaction (PCR) allow diagnostic procedures in couples with high risk and also certain IVF couples. We present a review of PGD indications, techniques and results. PMID:9810133

  13. Dynamic regulation of DNA methylation during mammalian development.

    PubMed

    Guibert, Sylvain; Forné, Thierry; Weber, Michael

    2009-10-01

    DNA methylation occurs on cytosines, is catalyzed by DNA methyltransferases (DNMTs), and is present at high levels in all vertebrates. DNA methylation plays essential roles in maintaining genome integrity, but its implication in orchestrating gene-expression patterns remained a matter of debate for a long time. Recent efforts to map DNA methylation at the genome level helped to get a better picture of the distribution of this mark and revealed that DNA methylation is more dynamic between cell types than previously anticipated. In particular, these datasets showed that DNA methylation is targeted to important developmental genes and might act as a barrier to prevent accidental cellular reprogramming. In this review, we will discuss the distribution and function of DNA methylation in mammalian genomes, with particular emphasis on the waves of global DNA methylation reprogramming occurring in early embryos and primordial germ cells. PMID:22122638

  14. Intracellular protein degradation in mammalian cells: recent developments.

    PubMed

    Knecht, Erwin; Aguado, Carmen; Cárcel, Jaime; Esteban, Inmaculada; Esteve, Juan Miguel; Ghislat, Ghita; Moruno, José Félix; Vidal, José Manuel; Sáez, Rosana

    2009-08-01

    In higher organisms, dietary proteins are broken down into amino acids within the digestive tract but outside the cells, which incorporate the resulting amino acids into their metabolism. However, under certain conditions, an organism loses more nitrogen than is assimilated in the diet. This additional loss was found in the past century to come from intracellular proteins and started an intensive research that produced an enormous expansion of the field and a dispersed literature. Therefore, our purpose is to provide an updated summary of the current knowledge on the proteolytic machinery involved in intracellular protein degradation and its physiological and pathological relevance, especially addressed to newcomers in the field who may find further details in more specialized reviews. However, even providing a general overview, this is an extremely wide field and, therefore, we mainly focus on mammalian cells, while other cells will be mentioned only for comparison purposes.

  15. 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.

  16. Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

    PubMed

    White, Carlee R; MacDonald, William A; Mann, Mellissa R W

    2016-09-01

    In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

  17. 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.

  18. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    PubMed

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  19. Development of Orientation Preference in the Mammalian Visual Cortex

    PubMed Central

    Chapman, Barbara; Gödecke, Imke; Bonhoeffer, Tobias

    2008-01-01

    Recent experiments have studied the development of orientation selectivity in normal animals, visually deprived animals, and animals where patterns of neuronal activity have been altered. Results of these experiments indicate that orientation tuning appears very early in development, and that normal patterns of activity are necessary for its normal development. Visual experience is not needed for early development of orientation, but is crucial for maintaining orientation selectivity. Neuronal activity and vision thus seem to play similar roles in the development of orientation selectivity as they do in the development of eyespecific segregation in the visual system. PMID:10504188

  20. [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.

  1. Whole genome amplification in preimplantation genetic diagnosis.

    PubMed

    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.

  2. Preimplantation genetic diagnosis and screening.

    PubMed

    Kearns, W G; Pen, R; Graham, J; Han, T; Carter, J; Moyer, M; Richter, K S; Tucker, M; Hoegerman, S F; Widra, E

    2005-11-01

    Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer. PGD is an exciting technology that may improve the likelihood of a successful pregnancy and birth for five distinct patient groups: (1) those with infertility related to recurrent miscarriages or unsuccessful in vitro fertilization (IVF) cycles, (2) those with unexplained infertility, (3) advanced maternal age, (4) severe male factor infertility, and (5) couples at risk for transmitting a hereditary disease to their offspring. PGD is always performed following an IVF cycle where multiple oocytes are retrieved and fertilized. Sophisticated techniques such as multiprobe, multicolor fluorescence in situ hybridization are used to test single cells for structural or numerical chromosome abnormalities, whereas the polymerase chain reaction, linkage analysis, and DNA sequencing are used to analyze single cells for disease-specific DNA mutations. PGD allows one to transfer only those embryos identified as being free of genetic abnormalities, thus potentially increasing the implantation rate and decreasing the miscarriage rate. These technologies identify embryos free of specific genetic abnormalities and may increase the likelihood of achieving the patient's goal: the birth of a healthy infant.

  3. Use of short hairpin RNA expression vectors to study mammalian neural development.

    PubMed

    Yu, Jenn-Yah; Wang, Tsu-Wei; Vojtek, Anne B; Parent, Jack M; Turner, David L

    2005-01-01

    The use of RNA interference (RNAi) in mammalian cells has become a powerful tool for the analysis of gene function. Here we discuss the use of DNA vectors to produce short hairpin RNAs (shRNAs) and inhibit gene expression in mammalian neural progenitors and neurons. Protocols are presented for introducing shRNA vectors into mouse P19 cells differentiated as neurons in vitro and for electroporation of shRNA vectors into primary neural progenitors from the embryonic mouse dorsal telencephalon (prospective cerebral cortex). Transfected primary cortical progenitors can be differentiated in vitro either in dissociated culture or organotypic slice culture. The use of shRNA vectors for RNAi provides a versatile approach to understand gene function during mammalian neural development.

  4. 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.

  5. 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.

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

    PubMed Central

    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. PMID:25520627

  7. An Obligatory Role of Mind Bomb-1 in Notch Signaling of Mammalian Development

    PubMed Central

    Im, Sun-Kyoung; Kim, Yoon-Young; Kim, Cheol-Hee; Suh, Pann-Ghill; Jan, Yuh Nung; Kong, Young-Yun

    2007-01-01

    Background The Notch signaling pathway is an evolutionarily conserved intercellular signaling module essential for cell fate specification that requires endocytosis of Notch ligands. Structurally distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), cooperatively regulate the endocytosis of Notch ligands in Drosophila. However, the respective roles of the mammalian E3 ubiquitin ligases, Neur1, Neur2, Mib1, and Mib2, in mammalian development are poorly understood. Methodology/Principal Findings Through extensive use of mammalian genetics, here we show that Neur1 and Neur2 double mutants and Mib2−/− mice were viable and grossly normal. In contrast, conditional inactivation of Mib1 in various tissues revealed the representative Notch phenotypes: defects of arterial specification as deltalike4 mutants, abnormal cerebellum and skin development as jagged1 conditional mutants, and syndactylism as jagged2 mutants. Conclusions/Significance Our data provide the first evidence that Mib1 is essential for Jagged as well as Deltalike ligand-mediated Notch signaling in mammalian development, while Neur1, Neur2, and Mib2 are dispensable. PMID:18043734

  8. Functions of DNA methylation and hydroxymethylation in mammalian development.

    PubMed

    Guibert, Sylvain; Weber, Michael

    2013-01-01

    DNA methylation occurs at cytosines, predominantly in the CpG dinucleotide context and is a key epigenetic regulator of embryogenesis and stem-cell differentiation in mammals. The genomic patterns of 5-methylcytosine are extensively reprogrammed during early embryonic development as well as in the germ-cell lineage. Thanks to improvements in high-throughput mapping technologies, it is now possible to characterize the dynamics of this epigenetic mark at the genome scale. DNA methylation plays multiple roles during development and serves to establish long-term gene silencing. In 2009, it was revealed that 5-hydroxymethylcytosine (5hmC) is another prominent cytosine modification catalyzed by the enzymes of the TET family and abundant in certain cell types. 5hmC has been thought to serve as an intermediate in the reaction of DNA demethylation or act as a signal for chromatin factors. Here, we review the current knowledge on the roles of these DNA epigenetic marks in development, epigenetic reprogramming, and pluripotency. PMID:23587238

  9. 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

  10. [Preimplantation Genetic Diagnosis by Blastocentesis: Problems and Perspectives].

    PubMed

    Zhigalina, D I; Skryabin, N A; Artyukhova, V G; Svetlakov, A V; Lebedev, I N

    2016-01-01

    The discovery of cell-free DNA in blastocoele fluid opens new perspectives for the development of preimplantation genetic diagnosis of human chromosomal and genetic diseases. In this review we analyzed the results of the first studies, which made it possible to evaluate the effectiveness of the application of a new source of biological material and showed a high degree of agreement between the results of molecular karyotyping with cell-free DNA and blastocyst cells. The results suggest the possibility of developing a noninvasive method of preimplantation genetic diagnosis, which may open a new round of progress in the field of assisted reproductive technologies and the genetics of early stages of human ontogenesis.

  11. 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

  12. Identification of molecular compartments and genetic circuitry in the developing mammalian kidney

    PubMed Central

    Yu, Jing; Valerius, M. Todd; Duah, Mary; Staser, Karl; Hansard, Jennifer K.; Guo, Jin-jin; McMahon, Jill; Vaughan, Joe; Faria, Diane; Georgas, Kylie; Rumballe, Bree; Ren, Qun; Krautzberger, A. Michaela; Junker, Jan P.; Thiagarajan, Rathi D.; Machanick, Philip; Gray, Paul A.; van Oudenaarden, Alexander; Rowitch, David H.; Stiles, Charles D.; Ma, Qiufu; Grimmond, Sean M.; Bailey, Timothy L.; Little, Melissa H.; McMahon, Andrew P.

    2012-01-01

    Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations. PMID:22510988

  13. Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos

    PubMed Central

    Liu, Tiancheng; Yu, Lin; Ding, Guohui; Wang, Zhen; Liu, Lei; Li, Hong; Li, Yixue

    2015-01-01

    Evolutionary developmental biology (EVO-DEVO) tries to decode evolutionary constraints on the stages of embryonic development. Two models—the “funnel-like” model and the “hourglass” model—have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED) were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA). Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies. PMID:26273607

  14. Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos.

    PubMed

    Liu, Tiancheng; Yu, Lin; Ding, Guohui; Wang, Zhen; Liu, Lei; Li, Hong; Li, Yixue

    2015-01-01

    Evolutionary developmental biology (EVO-DEVO) tries to decode evolutionary constraints on the stages of embryonic development. Two models--the "funnel-like" model and the "hourglass" model--have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED) were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA). Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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 Central

    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 (XM) during early female development. In order to provide further insight into the XM 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 XM in some blastomeres of late 2-cell embryos concomitant with the general activation of the genome indicating that XM 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. PMID:26267271

  1. Conserved microRNA editing in mammalian evolution, development and disease

    PubMed Central

    2014-01-01

    Background Mammalian microRNAs (miRNAs) are sometimes subject to adenosine-to-inosine RNA editing, which can lead to dramatic changes in miRNA target specificity or expression levels. However, although a few miRNAs are known to be edited at identical positions in human and mouse, the evolution of miRNA editing has not been investigated in detail. In this study, we identify conserved miRNA editing events in a range of mammalian and non-mammalian species. Results We demonstrate deep conservation of several site-specific miRNA editing events, including two that date back to the common ancestor of mammals and bony fishes some 450 million years ago. We also find evidence of a recent expansion of an edited miRNA family in placental mammals and show that editing of these miRNAs is associated with changes in target mRNA expression during primate development and aging. While global patterns of miRNA editing tend to be conserved across species, we observe substantial variation in editing frequencies depending on tissue, age and disease state: editing is more frequent in neural tissues compared to heart, kidney and testis; in older compared to younger individuals; and in samples from healthy tissues compared to tumors, which together suggests that miRNA editing might be associated with a reduced rate of cell proliferation. Conclusions Our results show that site-specific miRNA editing is an evolutionarily conserved mechanism, which increases the functional diversity of mammalian miRNA transcriptomes. Furthermore, we find that although miRNA editing is rare compared to editing of long RNAs, miRNAs are greatly overrepresented among conserved editing targets. PMID:24964909

  2. Human oocytes and preimplantation embryos express mRNA for growth hormone receptor.

    PubMed

    Ménézo, Y J; el Mouatassim, S; Chavrier, M; Servy, E J; Nicolet, B

    2003-11-01

    Human genetic expression of growth hormone receptor (GHR) gene was qualitatively analysed using reverse transcription polymerase chain reaction (RT-PCR) in cumulus cells, immature germinal vesicle (GV) and mature metaphase II (MII) stage oocytes and preimplantation human embryos. The transcripts encoding GHR were detected in cumulus cells and also in naked oocytes, either mature or not. In this case, a nested PCR is needed, as for early embryo preimplantation stages, before genomic activation. The GHR gene is highly expressed from the 4-day morula onwards. This suggests that GHR transcription follows a classical scheme associated with genomic activation. It is probable that, in human, growth hormone plays a role in the final stages of oocyte maturation and early embryogenesis as it does for several other mammalian species. PMID:15085728

  3. Preimplantation genetic diagnosis--an overview.

    PubMed

    Ogilvie, Caroline Mackie; Braude, Peter R; Scriven, Paul N

    2005-03-01

    Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye. PMID:15749997

  4. 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

  5. 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

  6. Histone H3.3 maintains genome integrity during mammalian development

    PubMed Central

    Jang, Chuan-Wei; Shibata, Yoichiro; Starmer, Joshua; Yee, Della; Magnuson, Terry

    2015-01-01

    Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development. PMID:26159997

  7. Histone H3.3 maintains genome integrity during mammalian development.

    PubMed

    Jang, Chuan-Wei; Shibata, Yoichiro; Starmer, Joshua; Yee, Della; Magnuson, Terry

    2015-07-01

    Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.

  8. 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.

  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. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

    PubMed Central

    Lai, Tingfeng; Yang, Yuansheng; Ng, Say Kong

    2013-01-01

    From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics. PMID:24276168

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

    PubMed

    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

  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. 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

  14. 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.

  15. The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

    PubMed

    Cox, G A; Mahaffey, C L; Nystuen, A; Letts, V A; Frankel, W N

    2000-10-01

    The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

  16. Rapid and Pervasive Changes in Genome-Wide Enhancer Usage During Mammalian Development

    PubMed Central

    Nord, Alex S.; Blow, Matthew J.; Attanasio, Catia; Akiyama, Jennifer A.; Holt, Amy; Hosseini, Roya; Phouanenavong, Sengthavy; Plajzer-Frick, Ingrid; Shoukry, Malak; Afzal, Veena; Rubenstein, John L. R.; Rubin, Edward M.; Pennacchio, Len A.; Visel, Axel

    2014-01-01

    Summary Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. While substantial progress has been made towards genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ~90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following mid-gestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage underlying processes central to development and disease. PMID:24360275

  17. ttime: an R package for translating the timing of brain development across mammalian species.

    PubMed

    Nagarajan, Radhakrishnan; Darlington, Richard B; Finlay, Barbara L; Clancy, Barbara

    2010-10-01

    Understanding relationships between the sequence and timing of brain developmental events across a given set of mammalian species can provide information about both neural development and evolution. Yet neurodevelopmental event timing data available from the published literature are incomplete, particularly for humans. Experimental documentation of unknown event timings requires considerable effort that can be expensive, time consuming, and for humans, often impossible. Application of suitable statistical models for translating neurodevelopmental event timings across mammalian species is essential. The present study implements an established statistical model and related functions as an open-source R package (ttime, translating time). The model incorporated into ttime allows predictions of unknown neurodevelopmental timings and explorations of phylogenetic relationships. The open-source package will enable transparency and reproducibility while minimizing redundancy. Sustainability and widespread dissemination will be guaranteed by the active CRAN (Comprehensive R Archive Network) community. The package updates the web-service (Clancy et al. 2007b) www.translatingtime.net by permitting predictions based on curated event timing databases which may include species not yet incorporated in the current model. The R package can be integrated into complex workflows that use the event predictions in their analyses. The package ttime is publicly available and can be downloaded from http://cran.r-project.org/web/packages/ttime/index.html .

  18. Preimplantation death of xenomitochondrial mouse embryo harbouring bovine mitochondria

    PubMed Central

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

    2015-01-01

    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. PMID:26416548

  19. 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

    2015-01-01

    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. PMID:26416548

  20. 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

  1. From Meiosis to Mitosis: The Astonishing Flexibility of Cell Division Mechanisms in Early Mammalian Development.

    PubMed

    Bury, L; Coelho, P A; Glover, D M

    2016-01-01

    The execution of female meiosis and the establishment of the zygote is arguably the most critical stage of mammalian development. The egg can be arrested in the prophase of meiosis I for decades, and when it is activated, the spindle is assembled de novo. This spindle must function with the highest of fidelity and yet its assembly is unusually achieved in the absence of conventional centrosomes and with minimal influence of chromatin. Moreover, its dramatic asymmetric positioning is achieved through remarkable properties of the actin cytoskeleton to ensure elimination of the polar bodies. The second meiotic arrest marks a uniquely prolonged metaphase eventually interrupted by egg activation at fertilization to complete meiosis and mark a period of preparation of the male and female pronuclear genomes not only for their entry into the mitotic cleavage divisions but also for the imminent prospect of their zygotic expression. PMID:27475851

  2. 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

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

    PubMed

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

    2015-01-01

    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.

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

    PubMed

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

    2014-10-31

    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.

  5. Keeping things quiet: Roles of NuRD and Sin3 co-repressor complexes during mammalian development

    PubMed Central

    McDonel, Patrick; Costello, Ita; Hendrich, Brian

    2010-01-01

    Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic development. PMID:18775506

  6. 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

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

    PubMed

    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

  8. Regulation of mammalian epithelial differentiation and intestine development by class I histone deacetylases.

    PubMed

    Tou, Liqiang; Liu, Qiang; Shivdasani, Ramesh A

    2004-04-01

    The biochemical mechanisms underlying epigenetic control of gene expression are increasingly well known. In contrast, the contributions of individual modifications toward activation of lineage-specific genes during vertebrate development are poorly understood. Class II histone deacetylases (HDACs), which show restricted tissue distribution, regulate muscle-specific gene expression, in part through interactions with myogenic transcription factors. We have combined gene expression profiling with manipulation of fetal mouse intestinal tissue to define roles for other regulatory factors. We found that in the developing mouse intestine class I HDACs are confined to the prospective epithelium and that their levels decline coincidently with activation of differentiation genes, suggesting a functional relationship between these events. Overexpression of wild-type but not of mutant HDACs 1 and 2 in fetal intestine explants reverses expression of certain maturation markers. HDAC inhibitors, including the selective class I antagonist valproic acid, activate the same genes prematurely and accelerate cytodifferentiation. Chromatin immunoprecipitation of freshly isolated organs reveals early HDAC2 occupancy at differentiation gene promoters and corresponding histone hypoacetylation that reverses as HDAC levels fall. Thus, modulation of endogenous class I HDAC levels represents a previously unappreciated mechanism to enable onset of tissue-restricted gene expression in a developing mammalian organ.

  9. A major effect of simulated microgravity on several stages of preimplantation mouse development is lethality associated with elevated phosphorylated SAPK/JNK.

    PubMed

    Wang, Yingchun; Xie, Yufen; Wygle, Dana; Shen, Hayley H; Puscheck, Elizabeth E; Rappolee, Daniel A

    2009-10-01

    We tested whether microgravity affects mouse development during a period when gravity cues chick and frog embryo development. A rotating vessel developed approximately 0.1% simulated microgravity (MGS) for embryos. Microgravity simulation resulted in blocked cell accumulation in E2.5 embryos. E1.5 and E3.5 embryos showed lesser effects. For E1.5/2.5 embryos, cell accumulation block was followed by lethality at 48 hours after MGS. For E3.5 embryos, MGS blocked development without lethality but with apoptosis. E1.5-3.5 embryos from the rotational control developed lesser effects than MGS embryos. Embryonic stress-activated protein kinase (SAPK) was phosphorylated during MGS and mediated apoptosis. Increased pSAPK suggested that lethality is due to cellular stress induced by MGS, unlike the dysfunctional development after gravitational disorientation in frog and chick embryos. Thus, MGS causes lethality, a novel phenotype not often observed in microgravity or MGS. Embryonic lethality at E2.5 and apoptosis at E3.5 are associated with SAPK function, suggesting that MGS causes a general stress response that immediately affects many aspects of development. In addition, MGS and many aspects of In vitro fertilization/assisted reproductive technologies (IVF/ART) produce nonphysiological, nonevolutionary stresses that are mediated by SAPK, suggesting the primacy of this protein kinase in a wide range of mechanisms mediating negative reproductive outcomes in IVF/ART and potentially in spaceflight.

  10. 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

  11. Towards the Development of a Potent and Selective Organoruthenium Mammalian Sterile 20 Kinase Inhibitor

    PubMed Central

    Anand, Ruchi; Maksimoska, Jasna; Pagano, Nicholas; Wong, Eric Y.; Gimotty, Phyllis A.; Diamond, Scott L.; Meggers, Eric

    2009-01-01

    Mammalian sterile 20 (MST1) kinase, a member of the sterile 20 (Ste-20) family of proteins, is a proapoptotic cytosolic kinase that plays an important role in the cellular response to oxidative stress. In this study, we report on the development of a potent and selective MST1 kinase inhibitor based on a ruthenium half-sandwich scaffold. We show that the enantiopure organoruthenium inhibitor, 9E1, has an IC50 value of 45 nM for MST1 and a greater than 25-fold inhibitor selectivity over the related Ste-20 kinases, p21 activated kinase 1 (PAK1), and p21 activated kinase 4 (PAK4) and an almost 10-fold selectivity over the related Thousand and one amino acids kinase 2 (TAO2). Compound 9E1 also displays a promising selectivity profile against unrelated protein kinases, however, the proto-oncogene serine/threonine protein kinase PIM1 (PIM-1) and glycogen synthase kinase 3 (GSK-3β) are inhibited with IC50 values in the low nanomolar range. We also show that 9E1 can inhibit MST1 function in cells. A cocrystal structure of a related compound with PIM-1 and a homology model with MST1 reveals the binding mode of this scaffold to MST1 and provides a starting point for the development of improved MST1 kinase inhibitors for possible therapeutic application. PMID:19226137

  12. 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

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

    PubMed

    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

  14. How does Reelin control neuronal migration and layer formation in the developing mammalian neocortex?

    PubMed

    Sekine, Katsutoshi; Kubo, Ken-ichiro; Nakajima, Kazunori

    2014-09-01

    The mammalian neocortex has a laminar structure that develops in a birth-date-dependent "inside-out" pattern. Its layered structure is established by neuronal migration accompanied by sequential changes in migratory mode regulated by several signaling cascades. Although Reelin was discovered about two decades ago and is one of the best known molecules that is indispensable to the establishment of the "inside-out" neuron layers, the cellular and molecular functions of Reelin in layer formation are still largely unknown. In this review article, we summarize our recent understanding of Reelin's functions during neuronal migration. Reelin acts in at least two different steps of neuronal migration: the final step of neuronal migration (somal/terminal translocation) just beneath the marginal zone (MZ) and the regulation of cell polarity step when the neurons change their migratory mode from multipolar migration to locomotion. During the translocation mode, Reelin activates integrin α5β1 through an intracellular pathway that triggers the translocation and activates N-cadherin in concert with the nectin-afadin system. Reelin is also involved in the termination of neuronal migration by degrading Dab1 via the SOCS7-Cullin5-Rbx2 system, and Reelin has been found to induce the birth-date-dependent neuronal aggregation in vivo. Based on these findings, we hypothesize that the molecular function of Reelin during neuronal migration is to control cell-adhesiveness during development by regulating the expression/activation of cell adhesion molecules.

  15. 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. PMID:26476195

  16. Essential role of the zinc finger transcription factor Casz1 for mammalian cardiac morphogenesis and development.

    PubMed

    Liu, Zhihui; Li, Wenling; Ma, Xuefei; Ding, Nancy; Spallotta, Francesco; Southon, Eileen; Tessarollo, Lino; Gaetano, Carlo; Mukouyama, Yoh-Suke; Thiele, Carol J

    2014-10-24

    Chromosome 1p36 deletion syndrome is one of the most common terminal deletions observed in humans and is related to congenital heart disease (CHD). However, the 1p36 genes that contribute to heart disease have not been clearly delineated. Human CASZ1 gene localizes to 1p36 and encodes a zinc finger transcription factor. Casz1 is required for Xenopus heart ventral midline progenitor cell differentiation. Whether Casz1 plays a role during mammalian heart development is unknown. Our aim is to determine 1p36 gene CASZ1 function at regulating heart development in mammals. We generated a Casz1 knock-out mouse using Casz1-trapped embryonic stem cells. Casz1 deletion in mice resulted in abnormal heart development including hypoplasia of myocardium, ventricular septal defect, and disorganized morphology. Hypoplasia of myocardium was caused by decreased cardiomyocyte proliferation. Comparative genome-wide RNA transcriptome analysis of Casz1 depleted embryonic hearts identifies abnormal expression of genes that are critical for muscular system development and function, such as muscle contraction genes TNNI2, TNNT1, and CKM; contractile fiber gene ACTA1; and cardiac arrhythmia associated ion channel coding genes ABCC9 and CACNA1D. The transcriptional regulation of some of these genes by Casz1 was also found in cellular models. Our results showed that loss of Casz1 during mouse development led to heart defect including cardiac noncompaction and ventricular septal defect, which phenocopies 1p36 deletion syndrome related CHD. This suggests that CASZ1 is a novel 1p36 CHD gene and that the abnormal expression of cardiac morphogenesis and contraction genes induced by loss of Casz1 contributes to the heart defect.

  17. 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.

  18. 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. PMID:26241857

  19. 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.

  20. Thoughts on the development, structure and evolution of the mammalian and avian telencephalic pallium.

    PubMed Central

    Puelles, L

    2001-01-01

    Various lines of evidence suggest that the development and evolution of the mammalian isocortex cannot be easily explained without an understanding of correlative changes in surrounding areas of the telencephalic pallium and subpallium. These are close neighbours in a common morphogenetic field and are postulated as sources of some cortical neuron types (and even of whole cortical areas). There is equal need to explain relevant developmental evolutionary changes in the dorsal thalamus, the major source of afferent inputs to the telencephalon (to both the pallium and subpallium). The mammalian isocortex evolved within an initially small dorsal part of the pallium of vertebrates, surrounded by other pallial parts, including some with a non-cortical, nuclear structure. Nuclear pallial elements are markedly voluminous in reptiles and birds, where they build the dorsal ventricular ridge, or hypopallium, which has been recently divided molecularly and structurally into a lateral pallium and a ventral pallium. Afferent pallial connections are often simplified as consisting of thalamic fibres that project either to focal cell aggregates in the ventral pallium (predominant in reptiles and birds) or to corticoid areas in the dorsal pallium (predominant in mammals). Karten's hypothesis, put forward in 1969, on the formation of some isocortical areas postulates an embryonic translocation into the nascent isocortex of the ventropallial thalamorecipient foci and respective downstream ventropallial target populations, as specific layer IV, layers II- III, or layers V-VI neuron populations. This view is considered critically in the light of various recent data, contrasting with the alternative possibility of a parallel, separate evolution of the different pallial parts. The new scenario reveals as well a separately evolving tiered structure of the dorsal thalamus, some of whose parts receive input from midbrain sensory centres (collothalamic nuclei), whereas other parts receive

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

    PubMed

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

    2015-10-15

    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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.

    PubMed

    Zambrano, Fabiola; Aguila, Luis; Arias, María E; Sánchez, Raúl; Felmer, Ricardo

    2016-10-01

    In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique. PMID:27325573

  7. Somatic cell nuclear transfer in buffalos: effect of the fusion and activation protocols and embryo culture system on preimplantation embryo development.

    PubMed

    Simon, Liz; Veerapandian, C; Balasubramanian, S; Subramanian, A

    2006-01-01

    The present study was conducted primarily to evaluate several factors that affect the nuclear transfer programme in water buffalos, in which relatively few studies have been performed. Embryos reconstructed with quiescent fetal fibroblasts and metaphase II cytoplasts were matured for 24 h, and activation was found to be comparatively better than in those matured for 30 h. A significantly higher proportion of embryos fused (52.0 +/- 1.9) and cleaved (51.2 +/- 1.7) when the couplets were fused 4-6 h before activation than when fused and activated simultaneously (46.5 +/- 1.6 and 44.5 +/- 2.0, respectively). Development of nuclear transfer embryos to the blastocyst stage (4.8 +/- 2.2) was supported by a commercially available sequential medium, and cleavage (76.5 +/- 2.8) was significantly higher in this medium compared with cleavage in TCM-199 with oviduct epithelial cell coculture (45.6 +/- 1.5) and synthetic oviduct fluid (21.8 +/- 6.6). Of the 16 cloned embryos transferred, none resulted in pregnancy. The present study demonstrates that optimal numbers of cloned buffalo blastocysts can be obtained from oocytes matured for 24 h, fused 3-4 h before activation and cultured in a commercially available sequential media (G1/G2), thus providing further information to enable successful nuclear transfer in buffalos.

  8. 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

  9. 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. PMID:24453515

  10. 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. PMID:27272212

  11. 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. PMID:26022337

  12. 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.

  13. Expression of growth factor ligand and receptor genes in preimplantation stage water buffalo (Bubalus bubalis) embryos and oviduct epithelial cells.

    PubMed

    Daliri, M; Rao, K B; Kaur, G; Garg, S; Patil, S; Totey, S M

    1999-09-01

    The temporal pattern of expression of genes for several growth factor ligands and receptors was examined in preimplantation water buffalo embryos and oviduct epithelial cells using RT-PCR. The identity of the resulting PCR products was confirmed by their expected size, restriction analysis, Southern blot hybridization and nucleotide sequence analysis. Preimplantation stage embryos from the one-cell to the blastocyst stage were derived after maturation, fertilization and culture of oocytes in vitro. Expression of members of the insulin-like growth factor (IGF) family was observed predominantly in preimplantation stage embryos and oviduct epithelial cells. Similarly, transcripts encoding insulin and IGF-I receptors were detected at each stage of embryonic development. The mRNA transcript of the IGF-I receptor was not detected in oviduct epithelial cells, but a prominent band corresponding to the insulin receptor was observed. Insulin and IGF-II mRNA were expressed as maternal transcripts that were not detected at the two- to four-cell stage but were present as zygotic transcripts at the eight-cell stage. Transcripts encoding IGF-I were detected in oviduct epithelial cells, but were not observed in any of the preimplantation stage embryos. Transforming growth factor (TGF) alpha and beta and epidermal growth factor mRNA transcripts were not detected in any of the preimplantation stage embryos. These results indicate that IGF-I acts via a paracrine mechanism to promote growth and development of preimplantation water buffalo embryos. Similarly, IGF-II appears to act through a heterologous autocrine mechanism via the IGF-I or the insulin receptor. Furthermore, the presence of TGF-alpha in oviduct epithelial cells indicates that it may have a critical role during development.

  14. Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.

    PubMed

    Boroviak, Thorsten; Loos, Remco; Lombard, Patrick; Okahara, Junko; Behr, Rüdiger; Sasaki, Erika; Nichols, Jennifer; Smith, Austin; Bertone, Paul

    2015-11-01

    Naive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation. PMID:26555056

  15. Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis

    PubMed Central

    Boroviak, Thorsten; Loos, Remco; Lombard, Patrick; Okahara, Junko; Behr, Rüdiger; Sasaki, Erika; Nichols, Jennifer; Smith, Austin; Bertone, Paul

    2015-01-01

    Summary Naive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation. PMID:26555056

  16. DEVELOPMENT OF A REFINED DATABASE OF MAMMALIAN RELATIVE POTENCY ESTIMATES FOR DIOXIN-LIKE COMPOUNDS

    EPA Science Inventory

    The toxic equivalency factor (TEF) approach has been widely accepted as the most feasible method available at present for evaluating potential health risks associated with exposure to mixtures of dioxin-like compounds (DLCs). The current mammalian TEFs for the DLCs were establis...

  17. 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.

  18. Quantitative analysis of gene expression in preimplantation mouse embryos using green fluorescent protein reporter.

    PubMed

    Medvedev, Serguei Yuri; Tokunaga, Tomoyuki; Schultz, Richard M; Furukawa, Tsutomu; Nagai, Takashi; Yamaguchi, Manabu; Hosoe, Misa; Yakovlev, Alexander F; Takahashi, Seiya; Izaike, Yoshiaki

    2002-07-01

    We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available. PMID:12080029

  19. The parental non-equivalence of imprinting control regions during mammalian development and evolution.

    PubMed

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

    2010-11-01

    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

  20. 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.

  1. Dynamic expression of chromatin modifiers during developmental transitions in mouse preimplantation embryos

    PubMed Central

    Nestorov, Peter; Hotz, Hans-Rudolf; Liu, Zichuan; Peters, Antoine H.F.M.

    2015-01-01

    During mouse preimplantation development, major changes in cell fate are accompanied by extensive alterations of gene expression programs. Embryos first transition from a maternal to zygotic program and subsequently specify the pluripotent and the trophectodermal cell lineages. These processes are regulated by key transcription factors, likely in cooperation with chromatin modifiers that control histone and DNA methylation. To characterize the spatiotemporal expression of chromatin modifiers in relation to developmental transitions, we performed gene expression profiling of 156 genes in individual oocytes and single blastomeres of developing mouse embryos until the blastocyst stage. More than half of the chromatin modifiers displayed either maternal or zygotic expression. We also detected lineage-specific expression of several modifiers, including Ezh1, Prdm14, Scmh1 and Tet1 underscoring possible roles in cell fate decisions. Members of the SET-domain containing SMYD family showed differential gene expression during preimplantation development. We further observed co-expression of genes with opposing biochemical activities, such as histone methyltransferases and demethylases, suggesting the existence of a dynamic chromatin steady-state during preimplantation development. PMID:26403153

  2. 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.

  3. [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.

  4. 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. PMID:24743999

  5. 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.

  6. [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)'. PMID:27120827

  7. 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.

  8. 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

  9. 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. PMID:25104401

  10. Carbon dioxide starvation, the development of C4 ecosystems, and mammalian evolution.

    PubMed

    Cerling, T E; Ehleringer, J R; Harris, J M

    1998-01-29

    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

  11. 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

  12. 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

  13. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.

    PubMed

    Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko; Matsuzaki, Fumio

    2016-09-01

    Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. PMID:27578183

  14. Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain

    PubMed Central

    Tsunekawa, Yuji; Terhune, Raymond Kunikane; Fujita, Ikumi; Shitamukai, Atsunori; Suetsugu, Taeko

    2016-01-01

    Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues. PMID:27578183

  15. 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

  16. 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.

  17. Mammalian Target of Rapamycin: Its Role in Early Neural Development and in Adult and Aged Brain Function.

    PubMed

    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

  18. 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

  19. Collection and transfer of preimplantation mouse embryos.

    PubMed

    Watson, J G; Wright, R W; Chaykin, S

    1977-10-01

    An improved method of collection and transfer of preimplantation mouse embryos focusing on increasing the predictability of the mating process and utilizing artificial insemination and artifical induction of pseudopregnancy is presented. Donor females were superovulated by the injection of 10 IU pregnant mare's serum followed 60 hours later by 10 IU human chorionic gonadotropin. Immature C3HeB/J females were treated with 6 IU of the 2 hormones on exactly the same schedule. In some foster mothers the estrous cycle was phased with the administration of 1.5 or 3.0 IU of the gonadotropins. Artificial insemination and artificial inductions of pseudopregnancy were performed 12 hours after the 2nd injection. The appropriate time of artificial insemination or induction was predicted in animals untreated with hormones, on the basis of estrous smears. A modification of artificial insemination techniques, refraining from use of the artificial penis and vaginal tampon, was used. Embroys were flushed from the oviducts of females 36 hours after insemination. Attainment of the 2-celled stage was evidence of fertilization. Embroys were maintained in vitro and then transferred to foster mothers at the early blastula stage. 5 embroys were transferred to each uterine horn. Implantation was evaluated. The yield of embroys was doubled through the use of artificial insemination. 1 male was used to inseminate up to 20 females. It was found that selection of recipients from normally cycling females was preferable to hormone priming.

  20. 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

  1. 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

  2. Differential localization of SNARE complex proteins SNAP-25, syntaxin, and VAMP during development of the mammalian retina.

    PubMed

    Greenlee, M H; Roosevelt, C B; Sakaguchi, D S

    2001-02-12

    SNARE complex proteins have critical functions during regulated vesicular release of neurotransmitter. In addition, they play critical roles during neurite outgrowth and synaptogenesis. Although it is clear that the function of any one SNARE complex protein during release of neurotransmitter is dependent on its association with other members of the complex, it is less certain whether their function during development and differentiation is dependent on interaction with one another. Previously, we have observed transient high levels of SNARE complex protein SNAP-25 in developing cholinergic amacrine cells (West Greenlee et al. [1998] J Comp Neurol 394:374-385). In addition, we detected, high levels of SNAP-25 in developing and mature photoreceptors. To better understand the functional significance of these high levels of SNAP-25 expression, we used immunocytochemistry to examine the developmental expression of the three members of the SNARE complex, SNAP-25, Syntaxin, and vesicle associated membrane protein (VAMP/also Synaptobrevin). Our results demonstrate that the high levels of SNAP-25 in cholinergic amacrine cells and photoreceptors are not accompanied by the same relatively high levels of other SNARE complex proteins. These results suggest that high levels of SNAP-25 in specific cell types may function independently of association with Syntaxin and VAMP. In this analysis, we characterized the changing patterns of immunoreactivity for the three SNARE complex proteins during the development and differentiation of the mammalian retina. We have compared the pattern of expression of the core SNARE complex proteins in the Brazilian opossum, Monodelphis domestica, and in the rat and found common patterns of expression between these diverse mammalian species. We observed temporal differences in the onset of immunoreactivity between these three proteins, and differences in their localization within synaptic layers in the developing and mature mammalian retina. This

  3. The gyrification of mammalian cerebral cortex: quantitative evidence of anisomorphic surface expansion during phylogenetic and ontogenetic development.

    PubMed Central

    Mayhew, T M; Mwamengele, G L; Dantzer, V; Williams, S

    1996-01-01

    Describing the shapes of 3D objects has proved to be as problematical in biology as in other areas. In an attempt to tackle this problem, established stereological methods (the Cavalieri principle and vertical sectioning) have been used to estimate a 3D shape-dependent quantity which can detect anisomorphic changes and is related to the degree of cortical convolution or gyrification. This isomophy factor is employed to assess phylogenetic and ontogenetic changes in the mammalian cerebral cortex. Gross anatomical differences between cerebral hemispheres of adult domestic mammals (horses, oxen, pigs, goats, dogs, cats and rabbits) were tested by paying attention to species, laterality and sex differences. Human fetal brains were also studied. Mean body weights of domestic mammals varied from 4 kg to 460 kg and brain weights from 10 g to 636 g. Fetuses weighed 39-610 g (crown-rump lengths 85-185 mm) and brain volumes were 4-56 cm3. Isomorphy factors were derived from estimates of hemisphere volumes and cortical surface areas. Hemisphere shape varied between species but no lateral or sex differences were detected. It is concluded that these mammalian brains are, in terms of their gross anatomy, symmetric and not sexually dimorphic. Fetal brains became more convoluted during uterine development. The isomorphy factor offers a convenient measure of gyrification which demonstrates that brains become more convoluted as they enlarge. PMID:8655415

  4. Variation in mammalian proximal femoral development: comparative analysis of two distinct ossification patterns

    PubMed Central

    Serrat, Maria A; Reno, Philip L; McCollum, Melanie A; Meindl, Richard S; Lovejoy, C Owen

    2007-01-01

    The developmental anatomy of the proximal femur is complex. In some mammals, including humans, the femoral head and greater trochanter emerge as separate ossification centres within a common chondroepiphysis and remain separate throughout ontogeny. In other species, these secondary centres coalesce within the chondroepiphysis to form a single osseous epiphysis much like the proximal humerus. These differences in femoral ontogeny have not been previously addressed, yet are critical to an understanding of femoral mineralization and architecture across a wide range of mammals and may have key implications for understanding and treating hip abnormalities in humans. We evaluated femora from 70 mammalian species and categorized each according to the presence of a ‘separate’ or ‘coalesced’ proximal epiphysis based on visual assessment. We found that ossification type varies widely among mammals: taxa in the ‘coalesced’ group include marsupials, artiodactyls, perissodactyls, bats, carnivores and several primates, while the ‘separate’ group includes hominoids, many rodents, tree shrews and several marine species. There was no clear relationship to body size, phylogeny or locomotion, but qualitative and quantitative differences between the groups suggest that ossification type may be primarily an artefact of femoral shape and neck length. As some osseous abnormalities of the human hip appear to mimic the normal morphology of species with coalesced epiphyses, these results may provide insight into the aetiology and treatment of human hip disorders such as femoroacetabular impingement and early-onset osteoarthritis. PMID:17331175

  5. The inverted neurogenetic gradient of the mammalian isocortex: development and evolution.

    PubMed

    Aboitiz, F; Morales, D; Montiel, J

    2001-12-01

    In this paper we review recent evidence on the molecular control of cell migration in the isocortex, and present an hypothesis for the evolutionary origin of the inside-out neurogenetic gradient of this structure. We suggest that there are at least two key factors involved in the acquisition of the inside-out gradient: (i) the expression of the protein reelin, which arrests the migration of cortical plate cells by detaching them from the radial glial fiber. This permits younger neurons to use the same fiber to migrate past the previous neurons; and (ii) the second factor is an intracellular signaling pathway dependent on a cyclin-dependent protein kinase (Cdk5). Cdk5 may work by inhibiting N-cadherin mediated cell aggregation as young cells cross the cortical plate, permitting them to move to the more superficial layers. Interestingly, the mutation in Cdk5 affects the migration of only those cells belonging to superficial layers, which are considered to be an evolutionary acquisition of the mammalian isocortex.

  6. 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

  7. [Preimplantation genetic diagnosis: update of the Parisian group].

    PubMed

    Frydman, René; Tachdjian, Gérard; Achour-Frydman, Nelly; Ray, Pierre; Romana, Serge; Hamamah, Samir; Marcadet-Fredet, Sabine; Kerbrat, Violaine; Fanchin, Renato; Kadoch, Jacques; Attie, Tania; Lelorc'h, M; Vekemans, Michel; Munnich, Arnold

    2002-01-01

    To report the birth of the first fourteen infants conceived after preimplantation genetic diagnosis (PGD) in our unit. Fifty-nine couples were enrolled between January 2000 and July 2001. They had a total of 71 oocyte pick-up cycles. The collected oocytes were inseminated by intracytoplasmic sperm injection. The resulting embryos were biopsied on the third day of development and the genetic analysis was performed on the same day. Most of the embryo transfers were carried out on the fourth day. The 71 oocyte pick-up cycles yielded 872 oocytes of which 731 were suitable for intracytoplasmic sperm injection. Among the 505 embryos obtained, 421 embryos were biopsied and genetic diagnosis was performed for 312 (74%) of these 127 embryos were transferred during the course of 58 transfer procedures. There were 18 biochemical and 12 ongoing (7 singles, 4 twins and 1 triple) pregnancies. Sixteen infants have been born and 2 are expected. PGD has gained a place among the choices offered to couples at risk of transmission of a serious and incurable genetic disease.

  8. Provision and quality assurance of preimplantation genetic diagnosis in Europe.

    PubMed

    Corveleyn, Anniek; Morris, Michael A; Dequeker, Elisabeth; Sermon, Karen; Davies, James Lawford; Antiñolo, Guillermo; Schmutzler, Andreas; Vanecek, Jiri; Nagels, Nick; Zika, Eleni; Palau, Francesc; Ibarreta, Dolores

    2008-03-01

    Preimplantation genetic diagnosis (PGD) is now well established and provided in many European countries. However, regulations, professional standards and accreditation requirements can differ notably. Furthermore, no comprehensive independent data exist either about practice and provision in Europe or about the quality assurance practices and procedures designed to optimize the quality of the results. Consequently, a study was launched to obtain knowledge, currently lacking, of the provision and quality assurance of PGD services and cross-border activities in Europe. An online questionnaire was developed and sent to PGD providers, and expert opinions were obtained through interviews with professionals in specific countries. Information was gathered from 53 centres offering PGD in 17 European countries. There is a diverse array of tests available, with a trend for custom-made services. Although half of the centres have a designated quality manager, just 33% have achieved or are preparing for accreditation or certification. About 66% of the centres responded that they did not participate in external quality assessment, a problem exacerbated by the lack of existing PGD-specific schemes. Approximately 19% of the centres do not keep data on accuracy and 9% do not even follow up until birth. PGD is an expanding activity with an increasing international flow that accounts for approximately one-third of the activity reported. The survey highlights a significant need for improvement in quality assurance in PGD centres. On the positive side, important improvements in the quality management of these services are expected with the European Tissue Directive entering into force.

  9. Preimplantation genetic diagnosis for autosomal recessive polycystic kidney disease.

    PubMed

    Gigarel, N; Frydman, N; Burlet, P; Kerbrat, V; Tachdjian, G; Fanchin, R; Antignac, C; Frydman, R; Munnich, A; Steffann, J

    2008-01-01

    Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common hereditary renal cystic diseases, and is caused by mutations in the PKHD1 gene. Due to the poor prognosis, there is a strong demand for prenatal diagnosis. Preimplantation genetic diagnosis (PGD) represents an alternative because it avoids the physical and emotional trauma of a pregnancy termination in the case of an affected fetus. A standardized single-cell diagnostic procedure was developed, based on haplotype analysis, enabling PGD to be offered to couples at risk of transmitting ARPKD. Six linked markers within (D6S1714 and D6S243), or in close proximity to (D6S272, D6S436, KIAA0057, D6S1662) the PKHD1 gene were tested by multiplex nested-polymerase chain reaction (PCR), using a Qiagen multiplex PCR kit. PCR analyses were carried out on 50 single lymphocytes. The amplification rate was excellent (100%), with an allele drop-out (ADO) rate ranging from 0 to 8%. Five PGD cycles were performed and 23 embryos were biopsied and analysed using this test. Transferable embryos were obtained in 4 cycles, resulting in two pregnancies and the birth of a healthy boy. This standardized diagnostic procedure allowed the detection of recombination, contamination, and ADO events, providing high assay accuracy with wide applicability.

  10. AB011. Preimplantation genetic diagnosis—experience from Hong Kong

    PubMed Central

    Chung, Brian Hon-Yin

    2015-01-01

    Preimplantation genetic diagnosis (PGD) is a technology used to determine whether a genetic or chromosomal disorder is present in embryos during an in-vitro fertilization (IVF) cycle. PGD screens embryos prior to their transfer to the uterus. Initially, PGD was developed to detect early onset life threatening single gene disorders for couples who are aware of their hereditary risks through family history or based on carrier testing. Since then, the use of PGD has expanded to detect late onset disorders such as Huntington disease and hereditary cancer predisposition syndromes. PGD treatment has been available in Hong Kong since 2002. In Hong Kong, couples seeking PGD treatment are required to see two doctors, one of whom must have proper training in clinical genetics and/or genetic counselling. The purposes of genetic counselling include educating patients about the genetic condition and ensuring that patients are given informed choices on the available options. PGD can provide reassurance of having healthy children for couples at high risks passing the genetic change. Yet there are more ethical concerns when the requests become increasingly complex. Multidisciplinary PGD board meeting has a key role in cases with ethical concerns. We illustrate with case examples the practice of PGD-related genetic counselling in Hong Kong.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. The Niche Factor Syndecan-1 Regulates the Maintenance and Proliferation of Neural Progenitor Cells during Mammalian Cortical Development

    PubMed Central

    Wang, Qingjie; Yang, Landi; Alexander, Caroline; Temple, Sally

    2012-01-01

    Neural progenitor cells (NPCs) divide and differentiate in a precisely regulated manner over time to achieve the remarkable expansion and assembly of the layered mammalian cerebral cortex. Both intrinsic signaling pathways and environmental factors control the behavior of NPCs during cortical development. Heparan sulphate proteoglycans (HSPG) are critical environmental regulators that help modulate and integrate environmental cues and downstream intracellular signals. Syndecan-1 (Sdc1), a major transmembrane HSPG, is highly enriched in the early neural germinal zone, but its function in modulating NPC behavior and cortical development has not been explored. In this study we investigate the expression pattern and function of Sdc1 in the developing mouse cerebral cortex. We found that Sdc1 is highly expressed by cortical NPCs. Knockdown of Sdc1 in vivo by in utero electroporation reduces NPC proliferation and causes their premature differentiation, corroborated in isolated cells in vitro. We found that Sdc1 knockdown leads to reduced levels of β-catenin, indicating reduced canonical Wnt signaling. Consistent with this, GSK3β inhibition helps rescue the Sdc1 knockdown phenotype, partially restoring NPC number and proliferation. Moreover, exogenous Wnt protein promotes cortical NPC proliferation, but this is prevented by Sdc1 knockdown. Thus, Sdc1 in the germinal niche is a key HSPG regulating the maintenance and proliferation of NPCs during cortical neurogenesis, in part by modulating the ability of NPCs to respond to Wnt ligands. PMID:22936997

  20. 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.

  1. [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. PMID:26845860

  2. Activins and inhibins in mammalian testis development: new models, new insights.

    PubMed

    Barakat, B; Itman, C; Mendis, S H; Loveland, K L

    2012-08-15

    The discovery of activin and inhibins as modulators of the hypothalamic-pituitary-gonadal axis has set the foundation for understanding their central importance to many facets of development and disease. This review contains an overview of the processes and cell types that are central to testis development and spermatogenesis and then provides an update focussed on information gathered over the past five years to address new concepts about how these proteins function to control testis development in fetal and juvenile life. Current knowledge about the interactive nature of the transforming growth factor-β (TGFβ) superfamily signalling network is applied to recent findings about activins and inhibins in the testis. Information about the regulated synthesis of signalling components and signalling regulators in the testis is integrated with new concepts that demonstrate their functional significance. The importance of activin bioactivity levels or dosage in controlling balanced growth of spermatogonial cells and their niche at different stages of testis development is highlighted.

  3. Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease

    PubMed Central

    Lee, Hyoung-Song; Kim, Min Jee; Ko, Duck Sung; Jeon, Eun Jin; Kim, Jin Young

    2013-01-01

    Objective Preimplantation genetic diagnosis (PGD) is an assisted reproductive technique for couples carrying genetic risks. Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a prevalence rate of 1/2,500. In this study, we report on our experience with PGD cycles performed for CMT types 1A and 2F. Methods Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of multiplex fluorescent polymerase chain reaction (PCR) followed by fragment analysis or sequencing using single lymphocytes. We performed six cycles of PGD for CMT1A and one cycle for CMT2F. Results Two duplex and two triplex protocols were developed according to the available markers for each CMT1A couple. Depending on the PCR protocols, the amplification rates and ADO rates ranged from 90.0% to 98.3% and 0.0% to 11.1%, respectively. For CMT2F, the amplification rates and ADO rates were 93.3% and 4.8%, respectively. In case of CMT1A, 60 out of 63 embryos (95.2%) were diagnosed and 13 out of 21 unaffected embryos were transferred in five cycles. Two pregnancies were achieved and three babies were delivered without any complications. In the case of CMT2F, a total of eight embryos were analyzed and diagnosed. Seven embryos were diagnosed as unaffected and four embryos were transferred, resulting in a twin pregnancy. Two healthy babies were delivered. Conclusion This is the first report of successful pregnancy and delivery after specific PGD for CMT disease in Korea. Our PGD procedure could provide healthy babies to couples with a high risk of transmitting genetic diseases. PMID:24505562

  4. 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

  5. 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

  6. PROTEOMICS OF MINUTE AMOUNTS OF TISSUE SAMPLE IN AQUATIC SPECIES: APPLICATION TO DEVELOPMENT OF NON-MAMMALIAN SCREENING ASSAYS FOR ENDOCRINE DISRUPTERS

    EPA Science Inventory

    When combined with other biochemical/toxicology assays, this type of information will contribute to the classification of chemicals based upon their effects at different levels of biological organization, and to the development of a cost-effective, non-mammalian screening assay f...

  7. 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

  8. 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.

  9. 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

  10. Construction and development of a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma.

    PubMed

    Li, Feng; Liu, Yan-Hong; Li, Yan-Wen; Li, Yue-Hui; Xie, Ping-Li; Ju, Qiang; Chen, Lin; Li, Guan-Cheng

    2012-12-01

    We present a detailed method for constructing a mammalian cell-based full-length antibody display library for targeting hepatocellular carcinoma. Two novel mammalian library vectors pcDNA3-CHm and pcDNA3-CLm were constructed that contained restriction enzyme sites NheI, ClaI and antibody constant domain. Mammalian expression vector pcDNA3-CHm contains IgG heavy-chain (HC) constant region and glycosylphosphatidylinositol anchor (GPI) that could be anchored full-length antibodies on the surface of mammalian cells. GOLPH2 prokaryotic expression vector was carried out in Escherichia coli and purified by immobilized metal affinity chromatography. Variable domain of heavy-chain and variable domain of light-chain genes were respectively inserted into the vector pcDNA3-CHm and pcDNA3-CLm by ligation, and antibody libraries are displayed as whole IgG molecules on the cell surface by co-transfecting this HC-GPI with a light chain. By screening the cell library using magnetic beads and cell ELISA, the cell clone that displayed GOLPH2-specific antibodies on cell surfaces was identified. The mammalian cell-based antibody display library is a great potential application for displaying full-length functional antibodies of targeting hepatocellular carcinoma on the surface of mammalian cells. Anti-GOLPH2 display antibody was successfully isolated from the library.

  11. Development and evolution of the vestibular sensory apparatus of the mammalian ear

    PubMed Central

    Beisel, Kirk W.; Wang-Lundberg, Yesha; Maklad, Adel; Fritzsch, Bernd

    2014-01-01

    Herein, we will review molecular aspects of vestibular ear development and present them in the context of evolutionary changes and hair cell regeneration. Several genes guide the development of anterior and posterior canals. Although some of these genes are also important for horizontal canal development, this canal strongly depends on a single gene, Otx1. Otx1 also governs the segregation of saccule and utricle. Several genes are essential for otoconia and cupula formation, but protein interactions necessary to form and maintain otoconia or a cupula are not yet understood. Nerve fiber guidance to specific vestibular endorgans is predominantly mediated by diffusible neurotrophic factors that work even in the absence of differentiated hair cells. Neurotrophins, in particular Bdnf, are the most crucial attractive factor released by hair cells. If Bdnf is misexpressed, fibers can be redirected away from hair cells. Hair cell differentiation is mediated by Atoh1. However, Atoh1 may not initiate hair cell precursor formation. Resolving the role of Atoh1 in postmitotic hair cell precursors is crucial for future attempts in hair cell regeneration. Additional analyses are needed before gene therapy can help regenerate hair cells, restore otoconia, and reconnect sensory epithelia to the brain. PMID:16614470

  12. microRNA-dependent Temporal Gene Expression in the Ureteric Bud Epithelium during Mammalian Kidney Development

    PubMed Central

    Nagalakshmi, Vidya K.; Lindner, Volkhard; Wessels, Andy; Yu, Jing

    2014-01-01

    Background Our previous study on mouse mutants with the ureteric bud (UB) epithelium-specific Dicer deletion (Dicer UB mutants) demonstrated the significance of UB epithelium-derived miRNAs in UB development. Results Our whole-genome transcriptional profiling showed that the Dicer mutant UB epithelium abnormally retained transcriptional features of the early UB epithelium and failed to express many genes associated with collecting duct differentiation. Further, we identified a temporal expression pattern of early UB genes during UB epithelium development in which gene expression was detected at early developmental stages and became undetectable by E14.5. In contrast, expression of early UB genes persisted at later stages in the Dicer mutant UB epithelium and increased at early stages. Our bioinformatics analysis of the abnormally persistently expressed early genes in the Dicer mutant UB epithelium showed significant enrichment of the let-7 family miRNA targets. We further identified a temporal expression pattern of let-7 miRNAs in the UB epithelium that is anti-parallel to that of some early UB genes during kidney development. Conclusions We propose a model in which the let-7 family miRNAs silence the expression of a subset of early genes in the UB epithelium at later developmental stages in order to promote collecting duct differentiation. PMID:25369991

  13. Notes on the role of dynamic DNA methylation in mammalian development.

    PubMed

    Bestor, Timothy H; Edwards, John R; Boulard, Mathieu

    2015-06-01

    It has been nearly 40 y since it was suggested that genomic methylation patterns could be transmitted via maintenance methylation during S phase and might play a role in the dynamic regulation of gene expression during development [Holliday R, Pugh JE (1975) Science 187(4173):226-232; Riggs AD (1975) Cytogenet Cell Genet 14(1):9-25]. This revolutionary proposal was justified by "... our almost complete ignorance of the mechanism for the unfolding of the genetic program during development" that prevailed at the time. Many correlations between transcriptional activation and demethylation have since been reported, but causation has not been demonstrated and to date there is no reasonable proof of the existence of a complex biochemical system that activates and represses genes via reversible DNA methylation. Such a system would supplement or replace the conserved web of transcription factors that regulate cellular differentiation in organisms that have unmethylated genomes (such as Caenorhaditis elegans and the Dipteran insects) and those that methylate their genomes. DNA methylation does have essential roles in irreversible promoter silencing, as in the monoallelic expression of imprinted genes, in the silencing of transposons, and in X chromosome inactivation in female mammals. Rather than reinforcing or replacing regulatory pathways that are conserved between organisms that have either methylated or unmethylated genomes, DNA methylation endows genomes with the ability to subject specific sequences to irreversible transcriptional silencing even in the presence of all of the factors required for their expression, an ability that is generally unavailable to organisms that have unmethylated genomes.

  14. 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.

  15. Role of lipids on elongation of the preimplantation conceptus in ruminants.

    PubMed

    Ribeiro, Eduardo S; Santos, José E P; Thatcher, William W

    2016-10-01

    Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants.

  16. Role of lipids on elongation of the preimplantation conceptus in ruminants.

    PubMed

    Ribeiro, Eduardo S; Santos, José E P; Thatcher, William W

    2016-10-01

    Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants. PMID:27335133

  17. 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

    2010-11-01

    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.

  18. Requirement for the Mitochondrial Pyruvate Carrier in Mammalian Development Revealed by a Hypomorphic Allelic Series.

    PubMed

    Bowman, Caitlyn E; Zhao, Liang; Hartung, Thomas; Wolfgang, Michael J

    2016-08-01

    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

  19. 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. PMID:27226319

  20. 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

    2010-11-01

    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

  1. 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.

  2. Development, reliability, and validation of an infant mammalian penetration-aspiration scale.

    PubMed

    Holman, Shaina Devi; Campbell-Malone, Regina; Ding, Peng; Gierbolini-Norat, Estela M; Griffioen, Anne M; Inokuchi, Haruhi; Lukasik, Stacey L; German, Rebecca Z

    2013-06-01

    A penetration-aspiration scale exists for assessing airway protection in adult videofluoroscopy and fiberoptic endoscopic swallowing studies; however, no such scale exists for animal models. The aim of this study was threefold: (1) develop a penetration-aspiration scale (PAS) for infant mammals, (2) test the scale's intra- and interrater reliabilities, and (3) validate the use of the scale for distinguishing between abnormal and normal animals. After discussion and reviewing many videos, the result was a 7-point infant mammal PAS. Reliability was tested by having five judges score 90 swallows recorded with videofluoroscopy across two time points. In these videos, the frame rate was either 30 or 60 frames per second and the animals were either normal, had a unilateral superior laryngeal nerve (SLN) lesion, or had hard palate local anesthesia. The scale was validated by having one judge score videos of both normal and SLN lesioned pigs and testing the difference using a t test. Raters had a high intrarater reliability [average κ = 0.82, intraclass correlation coefficient (ICC) = 0.92] and high interrater reliability (average κ = 0.68, ICC = 0.66). There was a significant difference in reliability for videos captured at 30 and 60 frames per second for scores of 3 and 7 (P < 0.001). The scale was also validated for distinguishing between normal and abnormal pigs (P < 0.001). Given the increasing number of animal studies using videofluoroscopy to study dysphagia, this scale provides a valid and reliable measure of airway protection during swallowing in infant pigs that will give these animal models increased translational significance.

  3. Development, reliability and validation of an infant mammalian penetration-aspiration scale

    PubMed Central

    Holman, Shaina Devi; Campbell-Malone, Regina; Ding, Peng; Gierbolini-Norat, Estela M.; Griffioen, Anne M.; Inokuchi, Haruhi; Lukasik, Stacey L.; German, Rebecca Z.

    2012-01-01

    A penetration-aspiration scale exists for assessing airway protection in adult videofluoroscopy and fiberoptic endoscopic swallowing studies, however no such scale exists for animal models. The aim of this study was threefold to 1) develop a Penetration-Aspiration Scale (PAS) for infant mammals, 2) test the scale’s intra- and inter-rater reliability, and 3) to validate the use of the scale for distinguishing between abnormal and normal animals. After discussion and reviewing many videos, the result was a 7-Point Infant Mammal PAS. Reliability was tested by having 5 judges score 90 swallows recorded with videofluoroscopy across two time points. In these videos, the frame rate was either 30 or 60 frames per second and the animals were either normal, had a unilateral superior laryngeal nerve (SLN) lesion, or had hard palate local anesthesia. The scale was validated by having one judge score videos of both normal and SLN lesioned pigs and testing the difference using a t-test. Raters had a high intra-rater (average kappa of 0.82, intraclass correlation coefficient (ICC)= 0.92) and high inter-rater reliability (average kappa of 0.68, ICC= 0.66). There was a significant difference in reliability for videos captured at 30 and 60 frames per second for scores of 3 and 7 (p<0.001). The scale was also validated for distinguishing between normal and abnormal pigs (p<0.001). Given the increasing number of animal studies using videofluoroscopy to study dysphagia, this scale provides a valid and reliable measure of airway protection during swallowing in infant pigs that will give these animal models increased translational significance. PMID:23129423

  4. Notes on the role of dynamic DNA methylation in mammalian development

    PubMed Central

    Bestor, Timothy H.; Edwards, John R.; Boulard, Mathieu

    2015-01-01

    It has been nearly 40 y since it was suggested that genomic methylation patterns could be transmitted via maintenance methylation during S phase and might play a role in the dynamic regulation of gene expression during development [Holliday R, Pugh JE (1975) Science 187(4173):226–232; Riggs AD (1975) Cytogenet Cell Genet 14(1):9–25]. This revolutionary proposal was justified by “... our almost complete ignorance of the mechanism for the unfolding of the genetic program during development” that prevailed at the time. Many correlations between transcriptional activation and demethylation have since been reported, but causation has not been demonstrated and to date there is no reasonable proof of the existence of a complex biochemical system that activates and represses genes via reversible DNA methylation. Such a system would supplement or replace the conserved web of transcription factors that regulate cellular differentiation in organisms that have unmethylated genomes (such as Caenorhaditis elegans and the Dipteran insects) and those that methylate their genomes. DNA methylation does have essential roles in irreversible promoter silencing, as in the monoallelic expression of imprinted genes, in the silencing of transposons, and in X chromosome inactivation in female mammals. Rather than reinforcing or replacing regulatory pathways that are conserved between organisms that have either methylated or unmethylated genomes, DNA methylation endows genomes with the ability to subject specific sequences to irreversible transcriptional silencing even in the presence of all of the factors required for their expression, an ability that is generally unavailable to organisms that have unmethylated genomes. PMID:25368180

  5. 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

  6. 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

  7. 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. PMID:26806703

  8. Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro

    PubMed Central

    Natesan, Senthilkumar A.; Bladon, Alex J.; Coskun, Serdar; Qubbaj, Wafa; Prates, Renata; Munne, Santiago; Coonen, Edith; Dreesen, Joseph C.F.M.; Stevens, Servi J.C.; Paulussen, Aimee D.C.; Stock-Myer, Sharyn E.; Wilton, Leeanda J.; Jaroudi, Souraya; Wells, Dagan; Brown, Anthony P.C.; Handyside, Alan H.

    2014-01-01

    Purpose: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. Methods: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. Results: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. Conclusion: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development. PMID:24810687

  9. 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.

  10. [Preimplantation genetic diagnosis (PGD) of hereditary cancer syndromes].

    PubMed

    Raszyková, L; Hořínová, V; Texl, P

    2012-01-01

    Advances in molecular biology techniques made possible genotype analysis from one or several cells. This can be used in preimplantation genetic diagnosis (PGD) not only of chromosomal aneuploidy but also of single gene diseases as well as hereditary cancer syndromes. PGD can be a benefit for those cases when the risk of transfer of pathological alteration from parent to offspring is unwelcome. We submit three cases of PGD with the results.

  11. Behavior and Brain Gene Expression Changes in Mice Exposed to Preimplantation and Prenatal Stress

    PubMed Central

    Strata, Fabrizio; Giritharan, Gnanaratnam; Sebastiano, Francesca Di; Piane, Luisa Delle; Kao, Chia-Ning; Donjacour, Annemarie

    2015-01-01

    Preimplantation culture of mouse embryos has been suggested to result in reduced anxiety-like behavior in adulthood. Here, we investigated the effects of in vitro fertilization (IVF), embryo culture, and different diets on anxiety-like behavior using the elevated plus maze (EPM). We hypothesized that exposure to suboptimal conditions during the preimplantation stage would interact with the suboptimal diet to alter behavior. The expression of genes related to anxiety was then assessed by quantitative real-time polymerase chain reaction in various brain regions. When fed a normal diet during gestation and a moderately high-fat Western diet (WD) postnatally, naturally conceived (NC) and IVF mice showed similar anxiety-like behavior on the EPM. However, when fed a low-protein diet prenatally and a high-fat diet postnatally (LP/HF), NC mice showed a modest increase in anxiety-like behavior, whereas IVF mice showed the opposite: a strongly reduced anxiety-like behavior on the EPM. The robust reduction in anxiety-like behavior in IVF males fed the LP/HF diets was, intriguingly, associated with reduced expression of MAO-A, CRFR2, and GABA markers in the hypothalamus and cortex. These findings are discussed in relation to the developmental origin of health and disease hypothesis and the 2-hit model, which suggests that 2 events, occurring at different times in development, can act synergistically with long-term consequences observed during adulthood. PMID:25398605

  12. 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

  13. 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

  14. PRMT5 Protects Genomic Integrity during Global DNA Demethylation in Primordial Germ Cells and Preimplantation Embryos

    PubMed Central

    Kim, Shinseog; Günesdogan, Ufuk; Zylicz, Jan J.; Hackett, Jamie A.; Cougot, Delphine; Bao, Siqin; Lee, Caroline; Dietmann, Sabine; Allen, George E.; Sengupta, Roopsha; Surani, M. Azim

    2014-01-01

    Summary Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation. PMID:25457166

  15. 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.

  16. 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. PMID:26685865

  17. Relationship between Pre-Implant Interleukin-6 Levels, Inflammatory Response, and Early Outcome in Patients Supported by Left Ventricular Assist Device: A Prospective Study

    PubMed Central

    Caruso, Raffaele; Botta, Luca; Verde, Alessandro; Milazzo, Filippo; Vecchi, Irene; Trivella, Maria Giovanna; Martinelli, Luigi; Paino, Roberto; Frigerio, Maria; Parodi, Oberdan

    2014-01-01

    Purpose 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. Methods 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. Results 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. Conclusions LVAD-candidates with elevated pre-implant levels of IL-6 are associated, after

  18. 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

    2016-01-01

    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.

  19. 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

  20. Preimplantation genetic diagnosis of skin fragility-ectodermal dysplasia syndrome.

    PubMed

    Fassihi, H; Grace, J; Lashwood, A; Whittock, N V; Braude, P R; Pickering, S J; McGrath, J A

    2006-03-01

    Skin fragility-ectodermal dysplasia syndrome is an autosomal recessive disorder caused by loss-of-function mutations in the desmosomal protein, plakophilin 1. Clinically, there may be considerable morbidity from extensive skin erosions and painful fissures on the palms and soles. In the absence of any specific treatment, prenatal diagnosis is an option for couples at reproductive risk of recurrence. In 2000, we developed and applied a single cell nested polymerase chain reaction protocol to test one couple for compound heterozygous plakophilin 1 gene mutations by preimplantation genetic diagnosis (PGD). Although pregnancy was established, an unrelated trisomy 22 led to a spontaneous abortion. However, eight embryos of known genetic status were cryopreserved at that stage, and we planned to undertake subsequent frozen embryo replacement cycles that might lead to the birth of an unaffected child in this family. Embryo cryopreservation was carried out in June 2000 using standard protocols in a three-step freezing procedure. Four embryos were thawed in March 2003, one of which was viable and was used in a frozen embryo replacement cycle, but pregnancy did not occur. The remaining four embryos were thawed in February 2004, two of which were viable (both carriers of the paternal mutation) and these were used in a second frozen embryo replacement cycle, and a singleton pregnancy was established. The child's plakophilin 1 genotype was assessed by direct nucleotide sequencing across the site of both potential mutations. Following two frozen embryo replacement cycles, and almost 4 years after the initial embryo biopsy and mutation analysis, a pregnancy was achieved that progressed to term with the birth of a healthy baby girl. Nucleotide sequencing of cord blood DNA, taken immediately after delivery, showed that the child was a heterozygous carrier of the paternal mutation but not of the maternal mutation. This case demonstrates the value of embryo cryopreservation, which

  1. 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

  2. Live-cell quantification and comparison of mammalian oocyte cytosolic lipid content between species, during development, and in relation to body composition using nonlinear vibrational microscopy.

    PubMed

    Jasensky, Joshua; Boughton, Andrew P; Khmaladze, Alexander; Ding, Jun; Zhang, Chi; Swain, Jason E; Smith, George W; Chen, Zhan; Smith, Gary D

    2016-08-01

    Cytosolic lipids participate in the growth, development, and overall health of mammalian oocytes including many roles in cellular homeostasis. Significant emphasis has been placed on the study of lipids as a dynamic organelle, which in turn requires the development of tools and techniques to quantitate and compare how lipid content relates to cellular structure, function, and normalcy. Objectives of this study were to determine if nonlinear vibrational microscopy (e.g., coherent anti-Stokes Raman scattering or CARS microscopy) could be used for live-cell imaging to quantify and compare lipid content in mammalian oocytes during development and in relation to body composition; and compare its efficacy to methods involving cellular fixation and staining protocols. Results of this study demonstrate that CARS is able to identify lipids in live mammalian oocytes, and there exists quantifiable and consistent differences in percent lipid composition across ooctyes of different species, developmental stages, and in relation to body composition. Such a method of live-cell lipid quantification has (i) experimental power in basic cell biology, (ii) practical utility for identifying developmental predictive biomarkers while advancing biology-based oocyte/embryo selection, and (iii) ability to yield rationally supporting technology for decision-making in rodents, domestic species, and human assisted reproduction and/or fertility preservation.

  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. 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

  5. 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

    2014-10-01

    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

  6. 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

  7. Chromosome abnormalities in human arrested preimplantation embryos: A multiple-probe FISH study

    SciTech Connect

    Munne, S.; Grifo, J.; Cohen, J. ); Weier, H.U.G. )

    1994-07-01

    Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embroys. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities. 44 refs., 1 fig., 4 tabs.

  8. Expression of microRNAs in bovine and human pre-implantation embryo culture media.

    PubMed

    Kropp, Jenna; Salih, Sana M; Khatib, Hasan

    2014-01-01

    MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy.

  9. Preimplantation genetic diagnosis and rational choice under risk or uncertainty.

    PubMed

    Zuradzki, Tomasz

    2014-11-01

    In this paper I present an argument in favour of a parental duty to use preimplantation genetic diagnosis (PGD). I argue that if embryos created in vitro were able to decide for themselves in a rational manner, they would sometimes choose PGD as a method of selection. Couples, therefore, should respect their hypothetical choices on a principle similar to that of patient autonomy. My thesis shows that no matter which moral doctrine couples subscribe to, they ought to conduct the PGD procedure in the situations when it is impossible to implant all of the created embryos and if there is a significant risk for giving birth to a child with a serious condition.

  10. Anticipating issues related to increasing preimplantation genetic diagnosis use: a research agenda.

    PubMed

    Klitzman, Robert; Appelbaum, Paul S; Chung, Wendy; Sauer, Mark

    2008-01-01

    Increasing use of preimplantation genetic diagnosis (PGD) poses numerous clinical, social, psychological, ethical, legal and policy dilemmas, many of which have received little attention. Patients and providers are now considering and using PGD for a widening array of genetic disorders, and patients may increasingly seek 'designer babies.' In the USA, although governmental oversight policies have been discussed, few specific guidelines exist. Hence, increasingly, patients and providers will face challenging ethical and policy questions of when and for whom to use PGD, and how it should be financed. These issues should be better clarified and addressed through collection of data concerning the current use of PGD in the USA, including factors involved in decision making about PGD use, as well as the education of the various communities that are, and should be, involved in its implementation. Improved understanding of these issues will ultimately enhance the development and implementation of future clinical guidelines and policies.

  11. Anticipating issues related to increasing preimplantation genetic diagnosis use: a research agenda.

    PubMed

    Klitzman, Robert; Appelbaum, Paul S; Chung, Wendy; Sauer, Mark

    2008-01-01

    Increasing use of preimplantation genetic diagnosis (PGD) poses numerous clinical, social, psychological, ethical, legal and policy dilemmas, many of which have received little attention. Patients and providers are now considering and using PGD for a widening array of genetic disorders, and patients may increasingly seek 'designer babies.' In the USA, although governmental oversight policies have been discussed, few specific guidelines exist. Hence, increasingly, patients and providers will face challenging ethical and policy questions of when and for whom to use PGD, and how it should be financed. These issues should be better clarified and addressed through collection of data concerning the current use of PGD in the USA, including factors involved in decision making about PGD use, as well as the education of the various communities that are, and should be, involved in its implementation. Improved understanding of these issues will ultimately enhance the development and implementation of future clinical guidelines and policies. PMID:18644221

  12. Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos

    PubMed Central

    Wang, HaiYang; Luo, YiBo; Lin, ZiLi; Lee, In-Won; Kwon, Jeongwoo; Cui, Xiang-Shun; Kim, Nam-Hyung

    2015-01-01

    Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos. PMID:26556501

  13. Pre-implantation diagnosis of aneuploidy by polar body and blastomere FISH analysis

    SciTech Connect

    Munne, S.; Cohen, J.; Grifo, J.

    1994-09-01

    For preimplantation genetic diagnosis (PGD) of aneuploidy in human in-vitro fertilization (IVF), two blastomeres per embryo should be analyzed to minimize errors caused by FISH and mosaicism. But the biopsy of two cells from an 8-cell embryo can be detrimental. This can be substituted by initial FISH analysis of the first polar body (PB) and subsequent single blastomere analysis. Simultaneous FISH analysis of chromosomes X, Y, 18, 13/21 was used for first polar body aneuploidy analysis. Normal divalents appeared as single-dotted signals corresponding to their two chromatids. We found that pre-division of chromatids increased dramatically with time in culture. All but three pre-division events involved separation of chromatids within the PB or the egg, with a total of two chromatids in each. We concluded that PB aneuploidy analysis is safe when performed within 6 hours after egg retrieval. For our first clinical case we chose a 39 year-old female carrier of an X-linked disease already selected for FISH pre-implantation diagnosis. Eight polar bodies from 12 eggs were analyzed: six showed a normal X181321 complement of divalents; one had an extra chromatid for 13/21 (egg {number_sign}8); and one had a missing chromatid for 13/21 (egg {number_sign}10). After insemination, six fertilized eggs developed into embryos, including egg {number_sign}10 but not egg {number_sign}8. At day 3 of development, a single blastomere per embryo was analyzed by FISH. According to the blastomere analysis, one embryo was haploid, one tetraploid. The two normal female embryos were replaced and pregnancy and CFS results are pending. These results suggest that this technique can be successfully applied for PGD of major aneuploidies in IVF patients over 35. In addition, it indicates that studies on pre-division should be performed on eggs within six hours of retrieval.

  14. Expression of proto-oncogenes in bovine preimplantation blastocysts.

    PubMed

    Tetens, F; Kliem, A; Tscheudschilsuren, G; Navarrete Santos, A; Fischer, B

    2000-05-01

    Proto-oncogenes are involved in the regulation of gene expression, for example after ligand binding to growth factor receptors. Expression of the proto-oncogenes c-fos, c-jun, c-ha-ras and c-myc was studied in in vivo grown and in vitro cultured bovine preimplantation blastocysts employing RT-PCR, ribonuclease protection assay and immunohistochemistry. Thirteen- and 14- day-old preimplantation blastocysts, i.e. stages before and during trophoblast elongation, were used. In in vivo-grown blastocysts c-fos, c-jun and c-ha-ras transcripts as well as c-Fos, c-Jun and c-Myc proteins were detected in all stages studied. Cultured blastocysts were treated with 10 nM epidermal growth factor and 10 nM transforming growth factor-alpha simultaneously. Epidermal growth factor and transforming growth factor-alpha treatment induced c-fos mRNA and c-Myc protein expression. The induction of downstream targets of the epidermal growth factor receptor by epidermal growth factor and transforming growth factor-alpha indicates a functional epidermal growth factor signal transduction pathway in elongating bovine blastocysts.

  15. Preimplantation genetic diagnosis: design or too much design

    PubMed Central

    Verpoest, W.

    2009-01-01

    Preimplantation genetic diagnosis (PGD) is a technique that was first applied in humans in 1990 (Handyside et al., 1990; Verlinsky et al., 1990). Thirty years on an estimated 15000 children have been conceived and born using PGD, a number dwarfed by the huge number of children already conceived via conventional in vitro fertilisation. In contrast to numerous reports on reproductive outcome in conventional IVF, data on reproductive outcome of PGD are scarse. There is ongoing debate about the diagnostic accuracy and clinical relevance of Preimplantation genetic screening for aneuploidy (PGS) (Shahine et al., 2006; Twisk et al., 2006), however well conducted prospective randomized studies are few. In this PhD summary, the author describes the reproductive results of a large PGD program and applies life table analysis with multiple regression analysis and comparative analysis where appropriate. Potential risks of PGD including misdiagnosis, perinatal mortality and monozygotic twinning rate are assessed. The aim is to provide both patients and physicians with adequate information on all reproductive aspects of PGD as a diagnostic and therapeutic tool. PMID:25489466

  16. DNA modifications in the mammalian brain

    PubMed Central

    Shin, Jaehoon; Ming, Guo-li; Song, Hongjun

    2014-01-01

    DNA methylation is a crucial epigenetic mark in mammalian development, genomic imprinting, X-inactivation, chromosomal stability and suppressing parasitic DNA elements. DNA methylation in neurons has also been suggested to play important roles for mammalian neuronal functions, and learning and memory. In this review, we first summarize recent discoveries and fundamental principles of DNA modifications in the general epigenetics field. We then describe the profiles of different DNA modifications in the mammalian brain genome. Finally, we discuss roles of DNA modifications in mammalian brain development and function. PMID:25135973

  17. Expression patterns of long noncoding RNAs from Dlk1-Dio3 imprinted region and the potential mechanisms of Gtl2 activation during blastocyst development.

    PubMed

    Han, Zhengbin; Yu, Changwei; Tian, Yijun; Zeng, Tiebo; Cui, Wei; Mager, Jesse; Wu, Qiong

    2015-07-31

    The function of long noncoding RNAs (lncRNAs) in cell differentiation and development have begun to be revealed in recent years. However, the expression pattern and mechanisms regulating lncRNAs are largely unknown during mammalian preimplantation development. LncRNAs expressed from Dlk1-Dio3 imprinted region have been linked to pluripotency of induced pluripotent cells (iPSCs). In this study we show that these lncRNAs (Gtl2, Rian and Mirg) are first expressed at the morula stage and gradually restricted to the inner cell mass (ICM) as the embryo differentiates into the blastocyst. Analysis of DNA methylation at IG-DMR and Gtl2-DMR showed no change during preimplantation while the presence of the activating histone modification H3K4me3 increased significantly from 8-cell to blastocyst stage, which may explain the expression activation. Additionally, knockdown of transcription factors (Oct4, Sox2 and Nanog) in blastocyst reduced the expression of Gtl2, indicating pluripotency factors regulate transcription of these lncRNAs. This study provides the spatiotemporal expression and dynamic changes of lncRNAs from Dlk1-Dio3 imprinted region in mouse preimplantation stage embryos and offers insight into the potential mechanisms responsible for Gtl2 activation. PMID:26005002

  18. Expression patterns of long noncoding RNAs from Dlk1-Dio3 imprinted region and the potential mechanisms of Gtl2 activation during blastocyst development.

    PubMed

    Han, Zhengbin; Yu, Changwei; Tian, Yijun; Zeng, Tiebo; Cui, Wei; Mager, Jesse; Wu, Qiong

    2015-07-31

    The function of long noncoding RNAs (lncRNAs) in cell differentiation and development have begun to be revealed in recent years. However, the expression pattern and mechanisms regulating lncRNAs are largely unknown during mammalian preimplantation development. LncRNAs expressed from Dlk1-Dio3 imprinted region have been linked to pluripotency of induced pluripotent cells (iPSCs). In this study we show that these lncRNAs (Gtl2, Rian and Mirg) are first expressed at the morula stage and gradually restricted to the inner cell mass (ICM) as the embryo differentiates into the blastocyst. Analysis of DNA methylation at IG-DMR and Gtl2-DMR showed no change during preimplantation while the presence of the activating histone modification H3K4me3 increased significantly from 8-cell to blastocyst stage, which may explain the expression activation. Additionally, knockdown of transcription factors (Oct4, Sox2 and Nanog) in blastocyst reduced the expression of Gtl2, indicating pluripotency factors regulate transcription of these lncRNAs. This study provides the spatiotemporal expression and dynamic changes of lncRNAs from Dlk1-Dio3 imprinted region in mouse preimplantation stage embryos and offers insight into the potential mechanisms responsible for Gtl2 activation.

  19. Prenatal and pre-implantation genetic diagnosis.

    PubMed

    Vermeesch, Joris Robert; Voet, Thierry; Devriendt, Koenraad

    2016-09-15

    The past decade has seen the development of technologies that have revolutionized prenatal genetic testing; that is, genetic testing from conception until birth. Genome-wide single-cell arrays and high-throughput sequencing analyses are dramatically increasing our ability to detect embryonic and fetal genetic lesions, and have substantially improved embryo selection for in vitro fertilization (IVF). Moreover, both invasive and non-invasive mutation scanning of the genome are helping to identify the genetic causes of prenatal developmental disorders. These advances are changing clinical practice and pose novel challenges for genetic counselling and prenatal care. PMID:27629932

  20. Cross-Species Genome Wide Expression Analysis during Pluripotent Cell Determination in Mouse and Rat Preimplantation Embryos

    PubMed Central

    Cinelli, Paolo

    2012-01-01

    The transition between morula and blastocyst stage during preimplantation development represents the first differentiation event of embryogenesis. Morula cells undergo the first cellular specialization and produce two well-defined populations of cells, the trophoblast and the inner cell mass (ICM). Embryonic stem cells (ESCs) with unlimited self-renewal capacity are believed to represent the in vitro counterpart of the ICM. Both mouse and rat ESCs can be derived from the ICM cells, but their in vitro stability differs. In this study we performed a microarray analysis in which we compared the transcriptome of mouse and rat morula, blastocyst, and ICM. This cross-species comparison represents a good model for understanding the differences in derivation and cultivation of ESCs observed in the two species. In order to identify alternative regulation of important molecular mechanisms the investigation of differential gene expression between the two species was extended at the level of signaling pathways, gene families, and single selected genes of interest. Some of the genes differentially expressed between the two species are already known to be important factors in the maintenance of pluripotency in ESCs, like for example Sox2 or Stat3, or play a role in reprogramming somatic cells to pluripotency like c-Myc, Klf4 and p53 and therefore represent interesting candidates to further analyze in vitro in the rat ESCs. This is the first study investigating the gene expression changes during the transition from morula to blastocyst in the rat preimplantation development. Our data show that in the pluripotent pool of cells of the rat and mouse preimplantation embryo substantial differential regulation of genes is present, which might explain the difficulties observed for the derivation and culture of rat ESCs using mouse conditions. PMID:23077551

  1. Transcriptome Encyclopedia of Early Human Development.

    PubMed

    Sahakyan, Anna; Plath, Kathrin

    2016-05-01

    Our understanding of human pre-implantation development is limited by the availability of human embryos and cannot completely rely on mouse studies. Petropoulos et al. now provide an extensive transcriptome analysis of a large number of human pre-implantation embryos at single-cell resolution, revealing previously unrecognized features unique to early human development.

  2. Genetic divergence in cellular resistance to heat shock in cattle: differences between breeds developed in temperate versus hot climates in responses of preimplantation embryos, reproductive tract tissues and lymphocytes to increased culture temperatures.

    PubMed

    Paula-Lopes, F F; Chase, C C; Al-Katanani, Y M; Krininger, C E; Rivera, R M; Tekin, S; Majewski, A C; Ocon, O M; Olson, T A; Hansen, P J

    2003-02-01

    The detrimental effects of heat stress on fertility in cattle are less pronounced in heat-tolerant breeds. Although these genetic differences reflect differences in thermoregulation, cells from heat-tolerant breeds are less adversely compromised by increased temperature (that is, heat shock) than cells from heat-sensitive breeds. Experiments were performed to test the hypothesis that cells and tissues from two thermotolerant breeds (Brahman and Senepol) are better able to survive and function after exposure to increased temperature than cells and tissues from two thermosensitive breeds (Holstein and Angus). Exposure of embryos at>eight-cell stage at day 5 after insemination to heat shock of 41.0 degrees C for 6 h decreased development to the blastocyst stage and the number of cells per embryo. However, the deleterious effect of heat shock on blastocyst formation and the number of cells per embryo was less pronounced for Brahman than for Holstein and Angus breeds. Embryos from Senepol cows had very low development and it was not possible to determine heat shock effects in this breed. In contrast to the sensitivity of embryos to heat shock, there was no effect of a 41.0 degrees C heat shock on [(3)H]leucine incorporation into proteins secreted by oviductal or endometrial explants. Lymphocytes from Brahman and Senepol cows were more resistant to heat-induced apoptosis than lymphocytes from other breeds. Heat shock reduced lymphocyte glutathione content but the magnitude of the decrease was not affected by breed. In conclusion, embryos from Brahman cows are more resistant to heat shock than embryos from Holstein or Angus cows. Genetic differences are also present in thermotolerance for apoptosis response in lymphocytes, with Brahman and Senepol cattle being more resistant to heat shock than Angus and Holstein breeds. It is likely that the evolutionary forces that led to the Brahman and Senepol breeds being adapted to hot climates resulted in the selection of genes

  3. Preimplantation genetic diagnosis: public policy and public attitudes.

    PubMed

    Hudson, Kathy L

    2006-06-01

    This paper summarizes the regulatory framework surrounding preimplantation genetic diagnosis (PGD) in the United States. In addition, the author reports results of surveys that reveal conflicting popular opinions about the moral acceptability of manipulating embryos during PGD. For example, some people who feel that an embryo has as much moral status as a born baby nonetheless feel that using PGD to screen embryos for certain diseases is morally acceptable. The national debate about technologies like PGD is stunted because it is currently cast in the same terms as the debate over abortion rights. If national leaders begin discussions about regulation of PGD and similar technologies, it could help depolarize the debate to more accurately consider the nuanced views of the public.

  4. [The Cagliari (Italy) Court authorizes the preimplantation genetic diagnosis].

    PubMed

    Jorqui Azofra, María

    2007-01-01

    Today, preimplantation genetic diagnosis (PGD) has been greatly accepted within the framework of positive law of many European countries. Nevertheless, in other countries, such as Italy, it is forbidden by law. The ruling of the Civil Court of Cagliari which has authorized its use to a Sardinian couple, has opened, in this way, a small crack to be able to asses possible modifications to the Italian regulation on this matter. This article analyses the ruling of the Civil Court of Cagliari (Italy) from an ethical and legal perspective. The criteria which is used to analyse the legitimacy or illegitimacy of the practice of PGD is analysed. That is, on reasons which could justify or not the transfer of embryos in vitro to the woman. With this objective in mind, the Italian and Spanish normative models which regulates this controversial subject are looked at. As a conclusion, a critical evaluation of the arguments presented is made.

  5. Environmental influences on the production of pre-implantation embryos.

    PubMed

    Diercks, Ann-Kathrin; Schwab, Anna; Rittgen, Werner; Kruspel, Andreas; Heuss, Edgar; Schenkel, Johannes

    2010-06-01

    Generation and cryopreservation of transgenic mice depend on reliable and continuous production of pre-implantation embryos. To suppress circannual and circadian rhythms driving the physiological and sexual behaviour of free living animals, laboratory animals are housed under standardized conditions. It remains to be elucidated if the artificial climate can cover all environmental effects. Here, we report that the humidity in an animal facility affects the embryo yield. The weather at the location of the facility, especially the temperature, influences the climate within an animal facility; weather peaks are obviously covered in part only, even if the facility is equipped with a powerful air-conditioning supply. Subsequently, external weather changes interact with the environment within the facility, influencing the production of embryos. Furthermore, noise and/or vibrations as generated by construction works, negatively affect the embryo yield. PMID:20171725

  6. Preimplantation sex selection for family balancing in India.

    PubMed

    Malpani, A; Malpani, A; Modi, D

    2002-01-01

    We describe our experience with the use of embryo biopsy and fluorescent in-situ hybridization (FISH) in order to sex embryos for the purpose of family balancing in a private IVF clinic in India from April 1999-April 2001. Embryos were biopsied and analysed on day 3, cultured in sequential media, and then transferred on day 4 or day 5 after morphological selection of the best embryos. From a total of 42 cycles started, we achieved 14 clinical pregnancies and have had nine live births so far with five ongoing pregnancies. This is the first report of the use of preimplantation sex selection for family balancing in India, where couples place a premium on having baby boys, and the social and ethical aspects of the use of this technology in this setting are discussed.

  7. Otx2 is a target of N-myc and acts as a suppressor of sensory development in the mammalian cochlea.

    PubMed

    Vendrell, Victor; López-Hernández, Iris; Durán Alonso, María Beatriz; Feijoo-Redondo, Ana; Abello, Gina; Gálvez, Héctor; Giráldez, Fernando; Lamonerie, Thomas; Schimmang, Thomas

    2015-08-15

    Transcriptional regulatory networks are essential during the formation and differentiation of organs. The transcription factor N-myc is required for proper morphogenesis of the cochlea and to control correct patterning of the organ of Corti. We show here that the Otx2 gene, a mammalian ortholog of the Drosophila orthodenticle homeobox gene, is a crucial target of N-myc during inner ear development. Otx2 expression is lost in N-myc mouse mutants, and N-myc misexpression in the chick inner ear leads to ectopic expression of Otx2. Furthermore, Otx2 enhancer activity is increased by N-myc misexpression, indicating that N-myc may directly regulate Otx2. Inactivation of Otx2 in the mouse inner ear leads to ectopic expression of prosensory markers in non-sensory regions of the cochlear duct. Upon further differentiation, these domains give rise to an ectopic organ of Corti, together with the re-specification of non-sensory areas into sensory epithelia, and the loss of Reissner's membrane. Therefore, the Otx2-positive domain of the cochlear duct shows a striking competence to develop into a mirror-image copy of the organ of Corti. Taken together, these data show that Otx2 acts downstream of N-myc and is essential for patterning and spatial restriction of the sensory domain of the mammalian cochlea. PMID:26160903

  8. Development and Aging of the Kisspeptin–GPR54 System in the Mammalian Brain: What are the Impacts on Female Reproductive Function?

    PubMed Central

    Franceschini, Isabelle; Desroziers, Elodie

    2012-01-01

    The prominent role of the G protein coupled receptor GPR54 and its peptide ligand kisspeptin in the progression of puberty has been extensively documented in many mammalian species including humans. Kisspeptins are very potent gonadotropin-releasing hormone secretagogues produced by two main populations of neurons located in two ventral forebrain regions, the preoptic area and the arcuate nucleus. Within the last 2 years a substantial amount of data has accumulated concerning the development of these neuronal populations and their timely regulation by central and peripheral factors during fetal, neonatal, and peripubertal stages of development. This review focuses on the development of the kisspeptin–GPR54 system in the brain of female mice, rats, sheep, monkeys, and humans. We will also discuss the notion that this system represents a major target through which signals from the environment early in life can reprogram reproductive function. PMID:23543285

  9. Casz1 is required for cardiomyocyte G1-to-S phase progression during mammalian cardiac development.

    PubMed

    Dorr, Kerry M; Amin, Nirav M; Kuchenbrod, Lauren M; Labiner, Hanna; Charpentier, Marta S; Pevny, Larysa H; Wessels, Andy; Conlon, Frank L

    2015-06-01

    Organ growth occurs through the integration of external growth signals during the G1 phase of the cell cycle to initiate DNA replication. Although numerous growth factor signals have been shown to be required for the proliferation of cardiomyocytes, genetic studies have only identified a very limited number of transcription factors that act to regulate the entry of cardiomyocytes into S phase. Here, we report that the cardiac para-zinc-finger protein CASZ1 is expressed in murine cardiomyocytes. Genetic fate mapping with an inducible Casz1 allele demonstrates that CASZ1-expressing cells give rise to cardiomyocytes in the first and second heart fields. We show through the generation of a cardiac conditional null mutation that Casz1 is essential for the proliferation of cardiomyocytes in both heart fields and that loss of Casz1 leads to a decrease in cardiomyocyte cell number. We further report that the loss of Casz1 leads to a prolonged or arrested S phase, a decrease in DNA synthesis, an increase in phospho-RB and a concomitant decrease in the cardiac mitotic index. Taken together, these studies establish a role for CASZ1 in mammalian cardiomyocyte cell cycle progression in both the first and second heart fields.

  10. The mammalian blastocyst.

    PubMed

    Frankenberg, Stephen R; de Barros, Flavia R O; Rossant, Janet; Renfree, Marilyn B

    2016-01-01

    The blastocyst is a mammalian invention that carries the embryo from cleavage to gastrulation. For such a simple structure, it exhibits remarkable diversity in its mode of formation, morphology, longevity, and intimacy with the uterine endometrium. This review explores this diversity in the light of the evolution of viviparity, comparing the three main groups of mammals: monotremes, marsupials, and eutherians. The principal drivers in blastocyst evolution were loss of yolk coupled with evolution of the placenta. An important outcome of blastocyst development is differentiation of two extraembryonic lineages (trophoblast and hypoblast) that contribute to the placenta. While in many species trophoblast segregation is often coupled with blastocyst formation, in marsupials and at least some Afrotherians, these events do not coincide. Thus, many questions regarding the conservation of molecular mechanisms controlling these events are of great interest but currently unresolved. For further resources related to this article, please visit the WIREs website. PMID:26799266

  11. DL-alpha-tocopherol acetate mitigates maternal hyperthermia-induced pre-implantation embryonic death accompanied by a reduction of physiological oxidative stress in mice.

    PubMed

    Sakamoto, Natsumi; Ozawa, Manabu; Yokotani-Tomita, Kaori; Morimoto, Aki; Matsuzuka, Takaya; Ijiri, Daichi; Hirabayashi, Miho; Ushitani, Atsuko; Kanai, Yukio

    2008-04-01

    Maternal hyperthermia induces pre-implantation embryo death, which is accompanied by enhanced physiological oxidative stress. We evaluated whether the administration of DL-alpha-tocopherol acetate (TA) to hyperthermic mothers mitigated pre-implantation embryo death. Mice were exposed to heat stress (35 degrees C, 60% relative humidity) for 12 h or not heated (25 degrees C) on the day of mating. Twelve hours before the beginning of temperature treatment, TA was injected intraperitoneally at a dose of 1 g/kg body weight. After the treatment, zygotes were recovered and the developmental abilities and intracellular glutathione (GSH) levels were evaluated. Another set of mice, with or without TA treatment, was exposed to heat stress for 12, 24 and 36 h, and the urinary levels of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured. Heat stress significantly decreased the blastocyst development rate and the GSH content in zygotes, as compared with the non-heat-stressed embryos, while TA administration significantly mitigated the deleterious effects of heat stress with regard to both parameters. Moreover, although the urinary levels of 8-OHdG gradually increased according to the duration of heat exposure, with or without TA administration, the levels were lower in the TA-administered group than in the placebo-injected mice. These results suggest that heat stress enhances physiological oxidative stress, and that TA administration alleviates the hyperthermia-induced death of pre-implantation embryos by reducing physiological oxidative stress.

  12. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    PubMed Central

    Costa, Liliana; Faustino, Maria Amparo F.; Neves, Maria Graça P. M. S.; Cunha, Ângela; Almeida, Adelaide

    2012-01-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. PMID:22852040

  13. Photodynamic inactivation of mammalian viruses and bacteriophages.

    PubMed

    Costa, Liliana; Faustino, Maria Amparo F; Neves, Maria Graça P M S; Cunha, Angela; Almeida, Adelaide

    2012-07-01

    Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  14. Recent advances in mammalian protein production

    PubMed Central

    Bandaranayake, Ashok D.; Almo, Steven C.

    2014-01-01

    Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones. PMID:24316512

  15. [Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

    PubMed

    Wu, Yan-qing; Chen, Li-yun; Zhang, Zheng-hong; wang, Zheng-chao

    2013-04-01

    In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

  16. The kinesin related motor protein, Eg5, is essential for maintenance of pre-implantation embryogenesis

    SciTech Connect

    Castillo, Andrew; Justice, Monica J. . E-mail: mjustice@bcm.tmc.edu

    2007-06-08

    Eg5 is a plus end directed kinesin related motor protein (KRP) previously shown to be involved in the assembly and maintenance of the mitotic spindle. KRPs are molecular motors capable of generating forces upon microtubules (MTs) in dividing cells and driving structural rearrangements necessary in the developing spindle. In vitro experiments demonstrate that loss of Eg5 results in cell cycle arrest and defective centrosome separation resulting in the development of monopolar spindles. Here we describe mice with a genetrap insertion in Eg5. Heterozygous mutant mice appear phenotypically normal. In contrast, embryos homozygous for the Eg5 null allele recovered at embryonic days 2.5-3.5 display signs of a proliferation defect as reduced cell numbers and failure of compaction and progression to the blastocyst stage was observed. These data, in conjunction with previous in vitro data, suggest that loss of Eg5 results in abnormal spindle structure, cell cycle arrest and thereby reduced cell proliferation of early cleavage pre-implantation embryos. These observations further support the conclusion that Eg5 is essential for cell division early in mouse development, and that maternal contribution may sustain the embryo through the maternal to zygotic transition at which point supplies of functional Eg5 are exhausted, preventing further cell cleavage.

  17. Responses to Cell Loss Become Restricted as the Supporting Cells in Mammalian Vestibular Organs Grow Thick Junctional Actin Bands That Develop High Stability

    PubMed Central

    Burns, Joseph C.

    2014-01-01

    Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin–GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin–GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia. PMID:24478379

  18. Preimplantation genetic diagnosis for gender selection in the United States

    SciTech Connect

    Colls, P.; Silver, L.; Olivera, G.; Weier, J.; Escudero, T.; Goodall, N.; Tomkin, G.; Munne, S.

    2009-08-20

    Preimplantation genetic diagnosis (PGD) of gender selection for non medical reasons has been considered an unethical procedure by several authors and agencies in the Western society on the basis of disrupting the sex ratio, being discriminatory againsts women and disposal of normal embryos of the non desired gender. In this study, the analysis of a large series of PGD procedures for gender selection from a wide geographical area in the United States, shows that in general there is no deviation in preference towards any specific gender except for a preference of males in some ethnic populations of Chinese, Indian and Middle Eastern origin that represent a small percentage of the US population. In cases where only normal embryos of the non-desired gender are available, 45.5% of the couples elect to cancel the transfer, while 54.5% of them are open to have transferred embryos of the non-desired gender, this fact being strongly linked to cultural and ethnical background of the parents. In addition this study adds some evidence to the proposition that in couples with previous children of a given gender there is no biological predisposition towards producing embryos of that same gender. Based on these facts, it seems that objections to gender selection formulated by ethics committees and scientific societies are not well-founded.

  19. Current status of preimplantation genetic diagnosis in Japan

    PubMed Central

    Sato, Kenji; Sueoka, Kou; Iino, Kotaro; Senba, Hiroshi; Suzuki, Mariko; Mizuguchi, Yuki; Izumi, Yoko; Sato, Suguru; Nakabayashi, Akira; Tanaka, Mamoru

    2015-01-01

    This is a retrospective study aimingto clarify the current status of preimplantation genetic diagnosis (PGD) in Japan. Our data were collected from 12 facilities between September 2004 and September 2012, and entered into a database. A majority of PGD in Japan was performed for balanced structural chromosomal abnormalities in couples with recurrent miscarriage. PGD for monogenic diseases was performed only in two facilities. The average maternal age was 38 years for monogenic diseases and 40 years for chromosomal abnormalities. Overall there have been671 cycles to oocyte retrieval reported. Of these cycles, 85% (572 cycles)were for chromosomal abnormalities, and 15% (99 cycles) for monogenic diseases. Diagnosis rates in the current study were 70.8% for monogenic diseases and 94.0% for chromosomal abnormalities. Rates of embryo transfer of PGD were 62.7% for monogenic diseases and 25.5% for chromosomal abnormalities. Clinical pregnancy rates per embryo transfer were 12.0% for monogenic diseases and 35.6% for chromosomal abnormalities. Our study is the first PGD report from all facilities which had the approval of the ethics committee of the Japanese Society of Obstetrics and Gynecology. We have built a basis for gathering continuous PGD data in Japan. PMID:26124570

  20. Attitude towards pre-implantation genetic diagnosis for hereditary cancer.

    PubMed

    Lammens, Chantal; Bleiker, Eveline; Aaronson, Neil; Vriends, Annette; Ausems, Margreet; Jansweijer, Maaike; Wagner, Anja; Sijmons, Rolf; van den Ouweland, Ans; van der Luijt, Rob; Spruijt, Liesbeth; Gómez García, Encarna; Ruijs, Mariëlle; Verhoef, Senno

    2009-01-01

    The use of pre-implantation genetic diagnosis (PGD) for hereditary cancer is subject to on-going debate, particularly among professionals. This study evaluates the attitude towards PGD and attitude-associated characteristics of those concerned: family members with a hereditary cancer predisposition. Forty-eight Von Hippel-Lindau and 18 Li-Fraumeni Syndrome families were identified via the 9 family cancer clinics in the Netherlands. In total, 216 high risk family members and partners were approached, of whom 179 (83%) completed a self-report questionnaire. Of the high risk family members, 35% expressed a positive attitude towards PGD. Those with a current desire to have children were significantly more likely to have a positive attitude: 48% would consider the use of PGD. No other sociodemographic, medical or psychosocial variables were associated significantly with a positive attitude. The most frequently reported advantage of PGD is the avoidance of a possible pregnancy termination. Uncertainty about late effects was the most frequently reported disadvantage. These results indicate that approximately half of those contemplating a future pregnancy would consider the use of PGD. The actual uptake, however, is expected to be lower. There is no indication that psychosocial factors affect interest in PGD.

  1. Preimplantation genetic diagnosis for early-onset torsion dystonia.

    PubMed

    Rechitsky, S; Verlinsky, O; Kuliev, A; Ozen, S; Laziuk, K; Beck, R; Gleicher, N; Verlinsky, Y

    2004-02-01

    Early-onset primary torsion dystonia (DYT1) is the most severe and common form of hereditary movement disorders, characterized by sustained twisting contractures that begin in childhood, which is caused in majority of cases by a 3-bp deletion of the DYT1 gene on chromosome 9q34 at the heterozygote state. As there is no effective treatment of this disease, preimplantation genetic diagnosis (PGD) may be a useful option for at-risk couples to establish an DYT1 mutation-free pregnancy. PGD was performed for two obligate carriers of the DYT1 3-bp deletion, using blastomere testing to preselect the mutation-free embryos, based on mutation analysis with simultaneous testing of the three closely linked markers, D9S62, D9S63 and ASS. Of 19 tested blastomeres in three cycles, 17 had conclusive information about the mutation and linked markers, of which eight were predicted to be free of 3-bp deletion. Six of these embryos were transferred back to patients, two in each cycle, yielding singleton DYT1 3-bp deletion-free clinical pregnancies in two. One of these pregnancies was terminated due to severe anencephaly and the other resulted in birth of a mutation-free child. This is the first PGD for primary torsion dystonia, providing an alternative for those at-risk couples who cannot accept prenatal diagnosis and termination of pregnancy as an option for avoiding early onset torsion dystonia.

  2. Views of Preimplantation Genetic Diagnosis (PGD) among Psychiatrists and Neurologists

    PubMed Central

    Abbate, Kristopher J.; Klitzman, Robert; Chung, Wendy K.; Ottman, Ruth; Leu, Cheng-Shiun; Appelbaum, Paul S.

    2014-01-01

    Objective As prenatal genetic testing (GT) and Preimplantation Genetic Diagnosis (PGD) use increase, providers in many specialties may play roles in patient discussions and referrals. Hence, we examined key aspects of neurologists’ and psychiatrists’ views and approaches. Study Design We surveyed attitudes and practices among 163 neurologists and 372 psychiatrists. Results 24.9% of neurologists and 31.9% of psychiatrists had discussed prenatal GT with patients, but 95.3% didn’t feel comfortable discussing PGD; only 2.9% discussed it; and only 1.8% had patients ask about PGD. Most would refer for PGD for Huntington’s disease (HD) and Tay-Sachs, fewer for Cystic Fibrosis (CF), and fewer still for autism, Alzheimer’s (AD), or gender selection for family balancing; in each of these cases, psychiatrists > neurologists. Providers who’d refer for PGD for HD, CF, or gender selection differed from others in proportions of patients with insurance, were more likely to have undergone a GT themselves, and be concerned about discrimination. Conclusions These data, the first to examine how neurologists and psychiatrists view PGD, suggest they don’t feel comfortable discussing PGD, but have strong views about its use. Potential PGD use is associated with concerns about discrimination, and less experience with GT. These data highlight needs for enhancing education about these technologies among various providers. PMID:25098029

  3. [Extending preimplantation genetic diagnosis to HLA typing: the French exception].

    PubMed

    Steffann, Julie; Frydman, Nelly; Burlet, Philippe; Gigarel, Nadine; Hesters, Laetitia; Kerbrat, Violaine; Lamazou, Frédéric; Munnich, Arnold; Frydman, René

    2011-01-01

    Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclère Hospital after embryo selection at the Paris preimplantation genetic diagnosis (PGD) center. His birth revived the controversy over "double PGD". This procedure, authorized in France since 2006, allows couples who already have a child with a serious, incurable genetic disease, to opt for PGD in order to select a healthy embryo that is HLA-matched to the affected sibling and who may thus serve as an ombilical cord blood donor. The procedure is particularly complex and the baby take-home rate is still very low. Double PGD is strictly regulated in France, and candidate couples must first receive individual authorization from the Biomedicine Agency. In our experience, these couples have a strong desire to have children, as reflected by the large number of prior spontaneous pregnancies (25% of couples). Likewise, most of these couples request embryo transfer even when there is no HLA-matched embryo, which accounts for more than half of embryo transfers. The controversy surrounding this practice has flared up again in recent weeks, over the concepts of "designer babies" and "double savior siblings" (the baby is selected to be free of the hereditary disease, and may also serve as a stem cell donor for the affected sibling).

  4. [Extending preimplantation genetic diagnosis to HLA typing: the French exception].

    PubMed

    Steffann, Julie; Frydman, Nelly; Burlet, Philippe; Gigarel, Nadine; Hesters, Laetitia; Kerbrat, Violaine; Lamazou, Frédéric; Munnich, Arnold; Frydman, René

    2011-01-01

    Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclère Hospital after embryo selection at the Paris preimplantation genetic diagnosis (PGD) center. His birth revived the controversy over "double PGD". This procedure, authorized in France since 2006, allows couples who already have a child with a serious, incurable genetic disease, to opt for PGD in order to select a healthy embryo that is HLA-matched to the affected sibling and who may thus serve as an ombilical cord blood donor. The procedure is particularly complex and the baby take-home rate is still very low. Double PGD is strictly regulated in France, and candidate couples must first receive individual authorization from the Biomedicine Agency. In our experience, these couples have a strong desire to have children, as reflected by the large number of prior spontaneous pregnancies (25% of couples). Likewise, most of these couples request embryo transfer even when there is no HLA-matched embryo, which accounts for more than half of embryo transfers. The controversy surrounding this practice has flared up again in recent weeks, over the concepts of "designer babies" and "double savior siblings" (the baby is selected to be free of the hereditary disease, and may also serve as a stem cell donor for the affected sibling). PMID:22375367

  5. Assessment of developmental retardation and abnormality of in vivo produced preimplantation embryos in rat.

    PubMed

    Rupasri, A; Shivakumar, K R; Sreenath, B R; Seshagiri, P B

    1995-12-01

    In most mammals studied, a substantial numbers of preimplantation embryos are believed to be lost in vivo. In vitro, embryos develop slowly and lose viability. Hence, there is a need to assess the extent and cause of embryonic loss both in vivo and in vitro. In this study, we assessed the quality of in vivo produced ovulation products/embryos, recovered on days 1-5 pregnancy, from naturally bred wistar rats. From day 1 pregnant rats (n = 24), 226 ovulation products were recovered which included 52% (117) unfertilized oocytes and empty zonae with/without cell debris (UFO-EZ:CD) and 48% (109) 1-cells. Flushings of day 2 rats (n = 27) contained 229 ovulation products, consisting of 70% (160) 2-cells and 30% (69) UFO-EZ:CD. Flushings of day 3 rats (n = 27) had 23% (56) 2-cells, 6% (15) 3-cells, 23% (57) 4-cells, 1% (2) 5-7 cells, 2% (5) 8-cells and 45% (112) UFO-EZ:CD, total being 247. Flushings of day 4 rats (n = 28) had 193 ovulation products comprising of one morula, 45% (86) 8-cells, 5% (9) 5-7-cells and the rest were 4-cells (2), 3-cells (2), 2-cells (1) and 48% (92) UFO-EZ:CD. Day 5 flushings (n = 27) had 202 ovulation products which included 13% (27) morulae, 17% (34) early, 36% (73) mid and 2% (5) late blastocysts; additionally, 4-cells (1), 8-cells (2) and 30% (60) UFO-EZ:CD were also recovered. On day 4, embryos (8-cells) migrated from the oviduct to the uterus. When pregnant rats (n = 25) were allowed to term, only 15 females (60%) delivered pups (128) with variable litter size (2-12). These results indicate that 56% (619/1097) of recovered rat preimplantation embryos are of expected developmental age with a mixture of asynchronously cleaving embryos. The remaining 44% (478) is comprised of 38% (417) UFO-EZ:CD and 6% (61) abnormal and developmentally retarded embryos, which are unlikely to produce viable pups at term.

  6. Detection of chromosomal aneuploidy in human preimplantation embryos by next-generation sequencing.

    PubMed

    Wang, Li; Wang, Xiaohong; Zhang, Jianguang; Song, Zhuo; Wang, Shufang; Gao, Yang; Wang, Jun; Luo, Yaning; Niu, Ziru; Yue, Xiaojing; Xu, Genming; Cram, David S; Yao, Yuanqing

    2014-05-01

    Embryos produced by assisted reproductive technologies are commonly associated with a high level of aneuploidy. Currently, 24-chromosome profiling of embryo biopsy samples by array-based methods is available to identify euploid embryos for transfer that have a higher potential for implantation and development to term. From a laboratory and patient perspective, there is a need to explore the feasibility of developing an alternative method for routine aneuploidy assessment of embryos that would be more comprehensive, cost-effective, and efficient. We speculated that aneuploidy could be readily assessed in test single-cell biopsy samples by first performing whole genome amplification followed by library generation, massively parallel shot-gun sequencing, and finally bioinformatics analysis to quantitatively compare the ratio of uniquely mapped reads to reference cells. Using Down syndrome as an example, the copy number change for chromosome 21 was consistently 1.5-fold higher in multiple cell and single-cell samples with a 47,XX,+21 karyotype. Applying the validated sequencing strategy to 10 sister blastomeres from a single human embryo, we showed that the aneuploidy status called by sequencing was consistent with short tandem repeat allelic profiling. These validation studies indicate that aneuploidy detection using sequencing-based methodology is feasible for further improving the practice of preimplantation genetic diagnosis. PMID:24648399

  7. Whole genome amplification from single cells in preimplantation genetic diagnosis and prenatal diagnosis.

    PubMed

    Peng, Wen; Takabayashi, Haruo; Ikawa, Kazumi

    2007-03-01

    The literature on whole genome amplification (WGA) techniques and their application to preimplantation genetic diagnosis (PGD) and prenatal diagnosis is reviewed. General polymerase chain reaction (PCR) fails to provide adequate information from limited cells in PGD and non-invasive prenatal diagnosis. Therefore several WGA techniques, such as primer extension preamplification (PEP) and degenerate oligonucleotide primed PCR (DOP-PCR), have been developed and successfully applied to clinical work during the past decade, especially in PGD and prenatal diagnosis. These techniques can provide ample amplification of genetic sequences from single cells for a series of subsequent PCR analyses such as restriction fragment length polymorphisms (RFLP) and comparative genomic hybridization (CGH), thus opening up a new area for prenatal diagnosis. However, several problems have been reported in the application of these techniques. The ideal WGA technique should have high yield, faithful representation of the original template, complete coverage of the genome, and simply performed procedure. In order to make good use of these techniques in future research and clinical work, it is undoubtedly necessary for an extensive understanding of the merits and pitfalls of these recently developed techniques.

  8. Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

    PubMed

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

  9. Uniformity of nucleosome preservation pattern in Mammalian sperm and its connection to repetitive DNA elements.

    PubMed

    Samans, Birgit; Yang, Yang; Krebs, Stefan; Sarode, Gaurav Vilas; Blum, Helmut; Reichenbach, Myriam; Wolf, Eckhard; Steger, Klaus; Dansranjavin, Temuujin; Schagdarsurengin, Undraga

    2014-07-14

    Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5'-UTR, 3'-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes.

  10. Reproductive abnormalities in adult male mice following preimplantation exposures to estradiol or pesticide methoxychlor.

    PubMed

    Amstislavsky, Sergei Ya; Amstislavskaya, Tamara G; Amstislavsky, Vjacheslav S; Tibeikina, Marina A; Osipov, Kiril V; Eroschenko, Victor P

    2006-02-01

    Adult females of ICR strain of mice were bred, separated into different experimental groups, and treated as follows. On Days 2-4 of pregnancy, the mice received daily subcutaneous injections of either 0.05 ml sesame oil (vehicle) or same volume of 5.0mg of purified methoxychlor (MXC) suspended in the vehicle. Another group received a single subcutaneous injection of 1.0 microg of estradiol-17beta (E) on Day 2 of pregnancy only. Male offspring were tested at 3 and 6 months of age. At 3 months, E or MXC did not alter the weights of seminal vesicles, preputial glands, or testes, although after exposure for 30 min to a female in estrus behind a partition, testosterone levels were significantly reduced in treated males in comparison to control males exposed to the same partition test. At 6 months, the preputial glands and testes weight remained unchanged, while the seminal vesicles were significantly heavier in E- and MXC-treated males. Same partition tests again revealed that in E and MXC groups, testosterone levels remained significantly lower in comparison to control males. MXC or E exposures during preimplantation appear to induce long-term effects on the sexual development in 3 and 6 month-old-males by compromising their sexual arousal and altering seminal vesicles weights in the older group.

  11. Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination.

    PubMed

    Ray, Pierre F; Frydman, Nelly; Attié, Tania; Hamamah, Samir; Kerbrat, Violaine; Tachdjian, Gérard; Romana, Serge; Vekemans, Michel; Frydman, René; Munnich, Arnold

    2002-07-01

    Two healthy sisters with a familial history of mental retardation were referred to our centre for preimplantation genetic diagnosis (PGD). Their two brothers showed severe mental retardation. The molecular basis for their disorder could not be identified, but one of the sisters and the mother presented a highly skewed pattern of X-inactivation reinforcing the likelihood of an X-linked mode of inheritance. Both sisters requested PGD to avoid the abortion of potentially affected male fetuses. PGD for sex by fluorescent in-situ hybridization was carried out for the first sister and resulted in the birth of a female child. The second sister and her partner, whose niece had cystic fibrosis (CF), were tested for CF mutations, and were both found to be deltaF508 heterozygous. We developed an efficient single cell PCR protocol for the simultaneous amplification of the CF (deltadeltaF508) locus as well as the X-linked amelogenin gene and its highly homologous pseudogene on the Y chromosome. Two PGD cycles were carried out to screen against male and deltaF508 homozygous deleted embryos. In each case several embryos could be selected for transfer and the second cycle resulted in a twin pregnancy followed by the birth of two healthy female infants.

  12. Hereditary cancer predisposition syndromes and preimplantation genetic diagnosis: where are we now?

    PubMed

    Konstantopoulou, I; Pertesi, M; Fostira, F; Grivas, A; Yannoukakos, D

    2009-09-01

    Hereditary cancer predisposition syndromes have been model diseases in order to understand carcinogenesis in many different organs such as colon, breast, ovaries, stomach and others. Better understanding and follow up of these diseases have led to the increasing acceptance of cancer genetic testing and the improving survival of young patients with cancer. Once the mutation is identified in the gene, patients and their relatives have the option of preimplantation genetic diagnosis (PGD) in order to select embryos without familial cancer-predisposing mutations. This procedure has already been performed in several syndromes, including the common syndromes of genetic predisposition to colon and breast cancer. Despite the numerous ethical objections and legal arguments, PGD for adult-onset cancers is today a reality and couples with an inherited predisposing mutation deserve the same respect, support and right to choose if their child will be born having an extremely high risk for cancer development as in the case of other life-threatening diseases for which prenatal screening has become a standard.

  13. Birth of a healthy female after preimplantation genetic diagnosis for Charcot-Marie-Tooth type X.

    PubMed

    Iacobelli, M; Greco, E; Rienzi, L; Ubaldi, F; Podini, D; Nuccitelli, A; Tesarik, J; Baldi, M; Fiorentino, F

    2003-11-01

    The X-linked dominant form of Charcot-Marie-Tooth syndrome (CMTX) is a clinically and genetically heterogeneous hereditary disorder of the peripheral nerves caused by mutations in the GJB1 gene that encodes a gap junction protein named connexin 32 (Cx32). Clinically, CMTX is characterized by peripheral motor and sensory deficit with muscle atrophy. A couple with a previous history of pregnancy termination after being diagnosed positive for CMTX by chorionic villus sampling, was referred for preimplantation genetic diagnosis (PGD). The female partner carried the causative H94Q, characterized by a C-->G substitution in codon 94 of exon 2 of the GJB1 gene. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the mother's mutation using polymerase chain reaction (PCR), followed by mutation analysis performed using the minisequencing method. Amelogenin sequences on the X and Y chromosomes were also co-amplified to provide a correlation between embryo gender and mutation presence. A single PGD cycle was performed, involving nine fertilized oocytes, five of which developed into good quality embryos useful for biopsy. Two unaffected embryos were transferred, resulting in a singleton pregnancy followed by the birth of a healthy female.

  14. Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

    SciTech Connect

    Thomson, M.S.

    1988-01-01

    Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture and used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two {sup 32}P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos.

  15. Patterning of the mammalian cochlea

    PubMed Central

    Cantos, Raquel; Cole, Laura K.; Acampora, Dario; Simeone, Antonio; Wu, Doris K.

    2000-01-01

    The mammalian cochlea is sophisticated in its function and highly organized in its structure. Although the anatomy of this sense organ has been well documented, the molecular mechanisms underlying its development have remained elusive. Information generated from mutant and knockout mice in recent years has increased our understanding of cochlear development and physiology. This article discusses factors important for the development of the inner ear and summarizes cochlear phenotypes of mutant and knockout mice, particularly Otx and Otx2. We also present data on gross development of the mouse cochlea. PMID:11050199

  16. Dynamic changes in leptin distribution in the progression from ovum to blastocyst of the pre-implantation mouse embryo.

    PubMed

    Schulz, Laura C; Roberts, R Michael

    2011-06-01

    The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally derived leptin from the oocyte becomes differentially distributed among the blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased toward a particular fate as early as the two-cell stage. In this study, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 and 76.9% respectively) collected following superovulation but was reduced, particularly in CF1 embryos (29.8%; P<0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division and was not affected by removal of the zona pellucida. The presence or absence of leptin polarization was not linked to differences in the ability of embryos to normally develop to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm, but on blastocoel expansion, it was lost from the cells. Throughout development, leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development.

  17. Preimplantation Exposure to Bisphenol A and Triclosan May Lead to Implantation Failure in Humans

    PubMed Central

    Yuan, Mu; Bai, Ming-Zhu; Huang, Xu-Feng; Zhang, Yue; Liu, Jing; Hu, Min-Hao; Zheng, Wei-Qian; Jin, Fan

    2015-01-01

    Endocrine disrupting chemicals (EDCs) are chemicals that have the capacity to interfere with normal endocrine systems. Two EDCs, bisphenol A (BPA) and triclosan (TCS), are mass-produced and widespread. They both have estrogenic properties and similar chemical structures and pharmacokinetic features and have been detected in human fluids and tissues. Clinical evidence has suggested a positive association between BPA exposure and implantation failure in IVF patients. Studies in mouse models have suggested that preimplantation exposure to BPA and TCS can lead to implantation failure. This paper reviews the relationship between preimplantation exposure to BPA and TCS and implantation failure and discusses the remaining problems and possible solutions. PMID:26357649

  18. Preimplantation genetic screening 2.0: the theory.

    PubMed

    Geraedts, Joep; Sermon, Karen

    2016-08-01

    During the last few years a new generation of preimplantation genetic screening (PGS) has been introduced. In this paper, an overview of the different aspects of this so-called PGS 2.0 with respect to the why (what are the indications), the when (which developmental stage, i.e. which material should be studied) and the how (which molecular technique should be used) is given. With respect to the aims it is clear that PGS 2.0 can be used for a variety of indications. However, the beneficial effect of PGS 2.0 has not been proved yet in RCTs. It is clear that cleavage stage is not the optimal stage for biopsy. Almost all advocates of PGS 2.0 prefer trophectoderm biopsy. There are many new methods that allow the study of complete aneuploidy with respect to one or more of the 24 chromosomes. Because of the improved vitrification methods, selection of fresh embryos for transfer is more and more often replaced by frozen embryo transfer. The main goal of PGS has always been the improvement of IVF success. However, success is defined by different authors in many different ways. This makes it very difficult to compare the outcomes of different studies. In conclusion, the introduction of PGS 2.0 will depend on the success of the new biopsy strategies in combination with the analysis of all 24 chromosomes. It remains to be seen which approach will be the most successful and for which specific groups of patients. PMID:27256482

  19. Simplified Bioreactor For Growing Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F.

    1995-01-01

    Improved bioreactor for growing mammalian cell cultures developed. Designed to support growth of dense volumes of mammalian cells by providing ample, well-distributed flows of nutrient solution with minimal turbulence. Cells relatively delicate and, unlike bacteria, cannot withstand shear forces present in turbulent flows. Bioreactor vessel readily made in larger sizes to accommodate greater cell production quantities. Molding equipment presently used makes cylinders up to 30 centimeters long. Alternative sintered plastic techniques used to vary pore size and quantity, as necessary.

  20. Mammalian development in a changing environment: exposure to endocrine disruptors reveals the developmental plasticity of steroid-hormone target organs.

    PubMed

    Markey, Caroline M; Coombs, Macall A; Sonnenschein, Carlos; Soto, Ana M

    2003-01-01

    Recent findings in the field of environmental endocrine disruption have revealed that developmental exposure to estrogenic chemicals induces morphological, functional, and behavioral anomalies associated with reproduction. The aim of the present study was to determine the effects of in utero exposure to low doses of the estrogenic chemical bisphenol A (BPA) on the development of the female reproductive tissues and mammary glands in CD-1 mice. Humans are exposed to BPA, which leaches from dental materials and plastic food and beverage containers. Here we report that prenatal exposure to BPA induces alterations in tissue organization within the ovaries and mammary glands and disrupts estrous cyclicity in adulthood. Because estrogen receptors are expressed developmentally in these estrogen-target organs, we propose that BPA may directly affect the expression of genes involved in their morphogenesis. In addition, alterations in the sexual differentiation of the brain, and thus the hypothalamic-pituitary-gonadal axis, may further contribute to the observed phenotype. The emerging field of endocrine disruptors promises to provide new insights into the mechanisms underlying the development of hormone-target organs and demonstrates that the environment plays important roles in the making of phenotypes.

  1. The Wellcome Prize Lecture. A map of auditory space in the mammalian brain: neural computation and development.

    PubMed

    King, A J

    1993-09-01

    The experiments described in this review have demonstrated that the SC contains a two-dimensional map of auditory space, which is synthesized within the brain using a combination of monaural and binaural localization cues. There is also an adaptive fusion of auditory and visual space in this midbrain nucleus, providing for a common access to the motor pathways that control orientation behaviour. This necessitates a highly plastic relationship between the visual and auditory systems, both during postnatal development and in adult life. Because of the independent mobility of difference sense organs, gating mechanisms are incorporated into the auditory representation to provide up-to-date information about the spatial orientation of the eyes and ears. The SC therefore provides a valuable model system for studying a number of important issues in brain function, including the neural coding of sound location, the co-ordination of spatial information between different sensory systems, and the integration of sensory signals with motor outputs. PMID:8240794

  2. Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

    PubMed

    Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

    2008-07-01

    The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

  3. Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development.

    PubMed

    Kobayashi, Akio; Valerius, M Todd; Mugford, Joshua W; Carroll, Thomas J; Self, Michelle; Oliver, Guillermo; McMahon, Andrew P

    2008-08-01

    Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.

  4. The ART of studying early embryo development: progress and challenges in ruminant embryo culture.

    PubMed

    Lonergan, Pat; Fair, Trudee

    2014-01-01

    The study of preimplantation mammalian embryo development is challenging due to difficulties in accessing in vivo-derived embryos in large numbers at the early stages and the inability to culture embryos in vitro much beyond the blastocyst stage. Nonetheless, embryos exhibit an amazing plasticity and tolerance when it comes to adapting to the environment in which they are cultured. They are capable of developing in media ranging in composition from simple balanced salt solutions to complex systems involving serum and somatic cells. At least a proportion of the blastocysts that develop in culture are developmentally competent as evidenced by the fact that live offspring have resulted following transfer. However, several studies using animal models have shown that such embryos are sensitive to environmental conditions that can affect future pre- and post-natal growth and developmental potential. This review summarises some key aspects of early embryo development and the approaches taken to study this important window in early life.

  5. Protein degradation in preimplantation mouse embryos and the lethality of tritiated amino acids

    SciTech Connect

    Wielbold, J.L.

    1982-01-01

    The role of protein degradation in preimplantation development in the mouse was studied. Proteins of morulae and blastocysts (M and B) cultured in vitro after labeling for 1 hour (h) in /sup 3/H-leucine exhibit a mean half-life (t/sub 1///sub 2/) of 8.1 h. The t/sub 1///sub 2/ tends to increase (9.5 h) when 10% fetal calf serum is added to the chase medium. This decrease in protein degradation in the presence of serum is associated with an increase in the percentage of B that are hatching (P<0.02). This rate of protein degradation in vivo was affected by the stage of pseudopregnancy (PSP) of the recipient. Day 4 embryos in a Day 4 uterus (Day 1=vaginal plug) retained more of the /sup 3/H-leucine in their proteins than did Day 4 embryos remaining in culture (P<0.02), while Day 4 embryos in a Day 3 uterus retained the same amount of radioactivity as did Day 4 embryos in culture. This differential effect of uterine environment was also seen when Day 4 embryos were transferred to recipients. More fetuses developed to term when the recipient was in Day 3 of PSP (50.8%) than when the recipient was in Day 4 PSP (25.9%, P<0.001), regardless of the age of the recipient. Age of the recipient does affect the percentage of transferred embryos developing to term. Thus, protein degradation may vary with the stage of embryo development and the conditions to which the embryos are exposed. However, even low levels of incorporated tritiated leucine can have lethal effects on the embryos and compromise the validity of the protein half-lives determined.

  6. Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams.

    PubMed

    Fabian, Dušan; Kačmarová, Martina; Kubandová, Janka; Čikoš, Štefan; Koppel, Juraj

    2016-06-01

    The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors. PMID:27288336

  7. Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development.

    PubMed

    Liu, Xinyu; Fernandes, Roxanne; Gertsenstein, Marina; Perumalsamy, Alagammal; Lai, Ingrid; Chi, Maggie; Moley, Kelle H; Greenblatt, Ellen; Jurisica, Igor; Casper, Robert F; Sun, Yu; Jurisicova, Andrea

    2011-01-01

    Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.

  8. Ethical Attitudes of German Specialists in Reproductive Medicine and Legal Regulation of Preimplantation Sex Selection in Germany

    PubMed Central

    Wilhelm, Miriam; Dahl, Edgar; Alexander, Henry; Brähler, Elmar; Stöbel-Richter, Yve

    2013-01-01

    Background Because of its ethical and social implications, preimplantation sex selection is frequently the subject of debates. Methods In 2006, we surveyed specialists in reproductive medicine in Germany using an anonymous questionnaire, including sociodemographic data and questions regarding ethical problems occurring in the practice of reproductive medicine. Most questions focused on preimplantation sex selection, including 10 case vignettes, since these enabled us to describe the most difficult and ethically controversial situations. This is the first survey among specialists in reproductive medicine regarding this topic in Germany. Results 114 specialists in reproductive medicine participated, 72 males (63%) and 42 females (37%), average age was 48 years (age range 29–67 years). The majority of respondents (79%) favoured a regulation that limits the use of preimplantation sex selection only for medical reasons, such as X-linked diseases (including 18%: summoning an ethics commission for every case). A minority of 18% approved of the use of sex selection for non-medical reasons (4% generally and further 14% for family balancing). 90% had received obvious requests from patients. The highest approval (46%) got the counselling guideline against a preimplantation sex selection and advising a normal pregnancy, if preimplantation sex selection would be allowed in Germany. The majority (67%) was opposed the personal use of preimplantation sex selection for non-medical reasons, but would think about it in medical cases. In opposite to woman, 14% of the men were in favour of personal use for non-medical reasons (p = 0,043). 25% of specialists in reproductive medicine feared that an allowance of preimplantation sex selection would cause a shift in the sex ratio. Conclusions The majority of German specialists in reproductive medicine opposes preimplantation sex selection for non-medical reasons while recommending preimplantation sex selection for medical reasons, e

  9. Ethical challenges in assisted reproduction: the place of preimplantation genetic diagnosis in a just society.

    PubMed

    Whetstine, Leslie M

    2015-04-01

    The purpose of this article is to provide an overview of preimplantation genetic diagnosis and identify the relevant moral questions it raises. In the course of this discussion, the scope of parental rights and the inherent difficulty in defining disease/disability will be considered.

  10. Ethical challenges in assisted reproduction: the place of preimplantation genetic diagnosis in a just society.

    PubMed

    Whetstine, Leslie M

    2015-04-01

    The purpose of this article is to provide an overview of preimplantation genetic diagnosis and identify the relevant moral questions it raises. In the course of this discussion, the scope of parental rights and the inherent difficulty in defining disease/disability will be considered. PMID:24334349

  11. Toward an ethical eugenics: the case for mandatory preimplantation genetic selection.

    PubMed

    Appel, Jacob M

    2012-01-01

    Preimplantation genetic diagnosis offers the possibility of screening and terminating embryos with severe and life-threatening disabilities. This article argues that under certain conditions, the use of this technology is not merely desirable as a means to reduce human suffering but also an ethically required duty of a parent to a potential child.

  12. Ultrastructural changes in granulosa lutein cells and progesterone levels during preimplantation, implatation, and early placentation in the western spotted skunk.

    PubMed

    Sinha, A A; Mead, R A

    1975-12-01

    The ultrastructure of corpora lutea obtained during the preimplantation implantation and early post-implantation periods has been studied in 20 western spotted skunks. Fine structure of granulosa lutein cells was correlated with progesterone levels. The corpus luteum of the prolonged (7 month) preimplantaion period contained undifferentiated small granulosa cells and differentiated large granulosa lutein cells. The former ranged in size between 12 and 20 mu and the latter between 20 and 45 mu. The ratio of small and large cells was about equal in an animal 2 days prior to nidation whereas only few small cells and numerous large cells were observed in an animal estimated to be 8 to 12 hours from nidation. Occasionally small cells were observed amidst large ones during the 24 hour nidation period, i.e. adhesion of trophoblast with the luminal uterine epithelium, but small cells were absent in animals after this period. Small cells had some smooth and rough endoplasmic reticulum rod-shaped mitochondria with plate-like cristae, small Golgi complex, and relatively smooth plasma membranes. Large lutein cells had abundant smooth endoplasmic reticulum, membranous whorls of smooth endoplasmic reticulum, usually round mitochondria with tubular and lamellar cristae, a well developed Golgi complex, variable amounts of lipid droplets, and highly plicated and ruffled plasma membranes. Peripheral plasma progesterone levels during the prolonged preimplantation period ranged between 1.1 and 7.9 ng/ml, but during implantation it was between 8 and 16.6 ng/ml. It is suggested that plasma progesterone levels fluctuate during the time of implantation and should not be regarded as a basis to predict actual nidation in the western spotted skunk.

  13. Genetic variation in resistance of the preimplantation bovine embryo to heat shock.

    PubMed

    Hansen, Peter J

    2014-12-01

    Reproduction is among the physiological functions in mammals most susceptible to disruption by hyperthermia. Many of the effects of heat stress on function of the oocyte and embryo involve direct effects of elevated temperature (i.e. heat shock) on cellular function. Mammals limit the effects of heat shock by tightly regulating body temperature. This ability is genetically controlled: lines of domestic animals have been developed with superior ability to regulate body temperature during heat stress. Through experimentation in cattle, it is also evident that there is genetic variation in the resistance of cells to the deleterious effects of elevated temperature. Several breeds that were developed in hot climates, including Bos indicus (Brahman, Gir, Nelore and Sahiwal) and Bos taurus (Romosinuano and Senepol) are more resistant to the effects of elevated temperature on cellular function than breeds that evolved in cooler climates (Angus, Holstein and Jersey). Genetic differences are expressed in the preimplantation embryo by Day 4-5 of development (after embryonic genome activation). It is not clear whether genetic differences are expressed in cells in which transcription is repressed (oocytes >100 µm in diameter or embryos at stages before embryonic genome activation). The molecular basis for cellular thermotolerance has also not been established, although there is some suggestion for involvement of heat shock protein 90 and the insulin-like growth factor 1 system. Given the availability of genomic tools for genetic selection, identification of genes controlling cellular resistance to elevated temperature could be followed by progress in selection for those genes within the populations in which they exist. It could also be possible to introduce genes from thermotolerant breeds into thermally sensitive breeds. The ability to edit the genome makes it possible to design new genes that confer protection of cells from stresses like heat shock. PMID:25472041

  14. Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos

    PubMed Central

    Park, Chi-Hun; Uh, Kyung-Jun; Mulligan, Brendan P.; Jeung, Eui-Bae; Hyun, Sang-Hwan; Shin, Taeyoung; Ka, Hakhyun; Lee, Chang-Kyu

    2011-01-01

    In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos. PMID:21804912

  15. Mammalian airborne allergens.

    PubMed

    Aalberse, Rob C

    2014-01-01

    Historically, horse dandruff was a favorite allergen source material. Today, however, allergic symptoms due to airborne mammalian allergens are mostly a result of indoor exposure, be it at home, at work or even at school. The relevance of mammalian allergens in relation to the allergenic activity of house dust extract is briefly discussed in the historical context of two other proposed sources of house dust allergenic activity: mites and Maillard-type lysine-sugar conjugates. Mammalian proteins involved in allergic reactions to airborne dust are largely found in only 2 protein families: lipocalins and secretoglobins (Fel d 1-like proteins), with a relatively minor contribution of serum albumins, cystatins and latherins. Both the lipocalin and the secretoglobin family are very complex. In some instances this results in a blurred separation between important and less important allergenic family members. The past 50 years have provided us with much detailed information on the genomic organization and protein structure of many of these allergens. However, the complex family relations, combined with the wide range of post-translational enzymatic and non-enzymatic modifications, make a proper qualitative and quantitative description of the important mammalian indoor airborne allergens still a significant proteomic challenge. PMID:24925404

  16. Preimplantation genetic diagnosis (embryo screening) for enlarged vestibular aqueduct due to SLC26A4 mutation.

    PubMed

    Wu, Chen-Chi; Lin, Shin-Yu; Su, Yi-Nin; Fang, Mei-Ya; Chen, Shee-Uan; Hsu, Chuan-Jen

    2010-01-01

    Preimplantation genetic diagnosis (PGD) is used to analyze embryos genetically before their transfer into the uterus. For families with genetic diseases, PGD offers a chance to have an unaffected child, without facing termination of pregnancy. Although PGD has been performed for many monogenic disorders, such as cystic fibrosis and beta-thalassemia, the application of PGD to hereditary hearing impairment has not been explored. In the present study, we reported the development and application of PGD protocols to address enlarged vestibular aqueduct (EVA), which is a common type of hereditary hearing impairment associated with mutations in the SLC26A4 gene. The family requesting PGD had a history of EVA, segregating the SLC26A4 c.919-2A-->G mutation. In short, the PGD process was composed of two steps: the development of a single-cell testing protocol and clinical PGD cycles (i.e., selection and implantation of unaffected embryos using the single-cell testing protocol). First, protocols for genetic testing in a single cell were established for the c.919-2A-->G mutation using GenomiPhi technology and primer extension mini-sequencing. These protocols were validated on single lymphocytes collected from both parents and their affected child. Two clinical PGD cycles were then performed for the parents, with the second cycle successfully leading to a singleton pregnancy. The baby was homozygous for the wild-type SLC26A4 allele and revealed a normal audiological phenotype after birth. To our knowledge, this is the first report in the literature describing successful PGD in families with genetic hearing impairment. In our opinion, the application of PGD in the field of hereditary hearing impairment involves fewer ethical controversies than other novel applications of PGD and traditional indications for PGD for other monogenic diseases. Therefore, the approach demonstrated in the present study can also be used in a large number of families with other types of hereditary

  17. Survival Assessment of Mouse Preimplantation Embryos After Exposure to Cell Phone Radiation

    PubMed Central

    Safian, Fereshteh; Khalili, Mohammad Ali; Khoradmehr, Arezoo; Anbari, Fatemeh; Soltani, Saeedeh; Halvaei, Iman

    2016-01-01

    Background: Using cellular phone has rapidly increased all over the world. Also, the concern on the possible health hazards of electromagnetic fields (EMF) induced from cell phones to reproduction has been growing in many countries. The aim of this study was to assess the consequences and effects of exposure to the cell phone radiation on the quality and survival rates of preimplantation embryos in mice. Methods: A total of 40 mice (20 females and 20 males), 6 weeks old and sexually mature BALB/c, were used for control and experimental groups. The ovary burses were removed and the zygotes were dissected in the morning after mating. Next, 2-cell embryos were divided into two groups of control (n=150) and experimental (n=150). EMF (900–1800 MHz) was used for four days in experimental group for 30 min/day in culture at 37°C in a CO 2 incubator. The quality of embryos was recorded daily and the fluorescent staining was used for identification of viable blastocysts. All data were compared by Student’s t-test and Mann-Whitney test (p<0.05). Results: The rate of embryo survival to the blastocysts stage was similar in both groups. However, the percentage of dead embryos at the 2-cell stage was significantly higher in EMF-exposed group compared with controls (p=0.03). Also, the loss of cell viability significantly increased in experimental blastocysts (p=0.002). Conclusion: The normal embryonic development up to the blastocyst stage indicates that EMF-exposure commonly did not have adverse effect on embryo development in mice. But, it caused loss of blastocysts cell viability. PMID:27478766

  18. Evolutionary paths to mammalian cochleae.

    PubMed

    Manley, Geoffrey A

    2012-12-01

    Evolution of the cochlea and high-frequency hearing (>20 kHz; ultrasonic to humans) in mammals has been a subject of research for many years. Recent advances in paleontological techniques, especially the use of micro-CT scans, now provide important new insights that are here reviewed. True mammals arose more than 200 million years (Ma) ago. Of these, three lineages survived into recent geological times. These animals uniquely developed three middle ear ossicles, but these ossicles were not initially freely suspended as in modern mammals. The earliest mammalian cochleae were only about 2 mm long and contained a lagena macula. In the multituberculate and monotreme mammalian lineages, the cochlea remained relatively short and did not coil, even in modern representatives. In the lineage leading to modern therians (placental and marsupial mammals), cochlear coiling did develop, but only after a period of at least 60 Ma. Even Late Jurassic mammals show only a 270 ° cochlear coil and a cochlear canal length of merely 3 mm. Comparisons of modern organisms, mammalian ancestors, and the state of the middle ear strongly suggest that high-frequency hearing (>20 kHz) was not realized until the early Cretaceous (~125 Ma). At that time, therian mammals arose and possessed a fully coiled cochlea. The evolution of modern features of the middle ear and cochlea in the many later lineages of therians was, however, a mosaic and different features arose at different times. In parallel with cochlear structural evolution, prestins in therian mammals evolved into effective components of a new motor system. Ultrasonic hearing developed quite late-the earliest bat cochleae (~60 Ma) did not show features characteristic of those of modern bats that are sensitive to high ultrasonic frequencies.

  19. Evolutionary paths to mammalian cochleae.

    PubMed

    Manley, Geoffrey A

    2012-12-01

    Evolution of the cochlea and high-frequency hearing (>20 kHz; ultrasonic to humans) in mammals has been a subject of research for many years. Recent advances in paleontological techniques, especially the use of micro-CT scans, now provide important new insights that are here reviewed. True mammals arose more than 200 million years (Ma) ago. Of these, three lineages survived into recent geological times. These animals uniquely developed three middle ear ossicles, but these ossicles were not initially freely suspended as in modern mammals. The earliest mammalian cochleae were only about 2 mm long and contained a lagena macula. In the multituberculate and monotreme mammalian lineages, the cochlea remained relatively short and did not coil, even in modern representatives. In the lineage leading to modern therians (placental and marsupial mammals), cochlear coiling did develop, but only after a period of at least 60 Ma. Even Late Jurassic mammals show only a 270 ° cochlear coil and a cochlear canal length of merely 3 mm. Comparisons of modern organisms, mammalian ancestors, and the state of the middle ear strongly suggest that high-frequency hearing (>20 kHz) was not realized until the early Cretaceous (~125 Ma). At that time, therian mammals arose and possessed a fully coiled cochlea. The evolution of modern features of the middle ear and cochlea in the many later lineages of therians was, however, a mosaic and different features arose at different times. In parallel with cochlear structural evolution, prestins in therian mammals evolved into effective components of a new motor system. Ultrasonic hearing developed quite late-the earliest bat cochleae (~60 Ma) did not show features characteristic of those of modern bats that are sensitive to high ultrasonic frequencies. PMID:22983571

  20. Cryopreservation of Mammalian Oocytes by Using Sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates

    PubMed Central

    Eroglu, Ali

    2009-01-01

    Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me2SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1 M raffinose, and then cooled to -196°C in the presence of either 0.3 M raffinose and 0.5 M Me2SO (cryopreservation group 1) or 0.3 M raffinose and 1.0 M Me2SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity. PMID:19596315

  1. Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay.

    PubMed

    Dorion-Thibaudeau, July; St-Laurent, Gilles; Raymond, Céline; De Crescenzo, Gregory; Durocher, Yves

    2016-02-01

    We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin-biosensor surfaces. For receptor biotinylation, we first describe an in-cell protocol based on the co-transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in-cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers. Finally, biotin-tagged FcγRs produced with the in-cell approach are successfully applied to the development of SPR-based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM-gal), non-glycosylated trastuzumab (TZM-NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM-NG binding to CD16a was not detected, TZM-gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non-glycosylated form with CD64 was characterized by a higher dissociation rate. PMID:26762306

  2. Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates.

    PubMed

    Eroglu, Ali

    2010-07-01

    Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me(2)SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1M raffinose, and then cooled to -196 degrees C in the presence of either 0.3M raffinose and 0.5M Me(2)SO (cryopreservation group 1) or 0.3M raffinose and 1.0M Me(2)SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.

  3. Reprogramming of microRNAs by adenosine-to-inosine editing and the selective elimination of edited microRNA precursors in mouse oocytes and preimplantation embryos.

    PubMed

    García-López, Jesús; Hourcade, Juan de Dios; Del Mazo, Jesús

    2013-05-01

    Adenosine deaminases-acting-on-RNA (ADAR) proteins induce adenosine-to-inosine editing in double-stranded RNA molecules. This editing generates RNA diversity at the post-transcriptional level, and it has been implicated in the control of cell differentiation and development. The editing of microRNA (miRNA) precursors, along with Tudor-SN (Snd1) activity, could lead to the elimination of selected miRNAs and reprogram miRNA activity. Here, we report the dynamics of adenosine-to-inosine editing in miRNA precursors and their selected elimination during mouse preimplantation development. Adar1p110 and Snd1 were found to be strongly but differentially expressed in oocytes and zygotes with respect to later pre-implantation stages. When the biogenesis of miR-151 was assessed, the majority of miR-151 precursors was edited and subsequently eliminated during early development. Deep sequencing of this and other miRNAs confirmed that, in general, edited precursors were selectively eliminated at early post-zygotic stages. Moreover, in oocytes and throughout the zygote-to-blastocyst stages, Tudor-SN accumulated in newly discovered aggregates termed 'T bodies'. These results provide new insight into how editing and Tudor-SN-mediated elimination of miRNA precursors is regulated during early development.

  4. Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

    PubMed

    Barnea, E R; Almogi-Hazan, O; Or, R; Mueller, M; Ria, F; Weiss, L; Paidas, M J

    2015-12-01

    Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented.

  5. PreImplantation factor prevents atherosclerosis via its immunomodulatory effects without affecting serum lipids.

    PubMed

    Chen, Yung Chih; Rivera, Jennifer; Fitzgerald, Melissa; Hausding, Christian; Ying, Ya-Lan; Wang, Xiaowei; Todorova, Krassimira; Hayrabedyan, Soren; Barnea, Eytan R; Peter, Karlheinz

    2016-05-01

    PreImplantation factor (PIF) is a 15-amino acid peptide endogenously secreted by viable embryos, regulating/enabling maternal (host) acceptance/tolerance to the "invading" embryo (allograft) all-while preserving maternal immunity to fight infections. Such attributes make PIF a potential therapeutic agent for chronic inflammatory diseases. We investigated whether PIF's immunomodulatory properties prevent progression of atherosclerosis in the hyper-cholesterolaemic ApoE-deficient murine model. Male, high-fat diet fed, ApoE-deficient (ApoE-/-) mice were administered either PBS, scrambled PIF (0.3-3 mg/kg) or PIF (0.3-3 mg/kg) for seven weeks. After treatment, PIF (3 mg/kg)-treated ApoE-/- mice displayed significantly reduced atherosclerosis lesion burden in the aortic sinus and aortic arch, without any effect on lipid profile. PIF also caused a significant reduction in infiltration of macrophages, decreased expression of pro-inflammatory adhesion molecules, cytokines and chemokines in the plaque, and reduced circulating IFN-γ levels. PIF preferentially binds to monocytes/neutrophils. In vitro, PIF attenuated monocyte migration (MCP-1-induced chemotaxis assay) and in vivo in LPS peritonitis model. Also PIF prevented leukocyte extravasation (peritonitis thioglycollate-induced model), demonstrating that PIF exerts its effect in part by modulation of monocyte function. Inhibition of the potassium channel KCNAB3 (Kv1.3) and of the insulin degrading enzyme (IDE) was demonstrated as potential mechanism of PIF's immunomodulatory effects. In conclusion, PIF regulates/lowers inflammation and prevents atherosclerosis development without affecting circulating lipids. Overall our findings establish PIF as a strong immunomodulatory drug candidate for atherosclerosis therapy.

  6. Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A.

    PubMed

    De Vos, A; Sermon, K; De Rijcke, M; Goossens, V; Henderix, P; Van Ranst, N; Platteau, P; Lissens, W; Devroey, P; Van Steirteghem, A; Liebaers, I

    2003-07-01

    Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.

  7. Immune regulatory and neuroprotective properties of preimplantation factor: From newborn to adult.

    PubMed

    Barnea, E R; Almogi-Hazan, O; Or, R; Mueller, M; Ria, F; Weiss, L; Paidas, M J

    2015-12-01

    Embryonic-maternal interaction from the earliest stages of gestation has a key, sustained role in neurologic development, persisting into adulthood. Early adverse events may be detrimental in adulthood. Protective factors present during gestation could significantly impact post-natal therapy. The role of PreImplantation Factor (PIF) within this context is herein examined. Secreted by viable early embryos, PIF establishes effective embryonic-maternal communication and exerts essential trophic and protective roles by reducing oxidative stress and protein misfolding and by blunting the nocive let-7 microRNA related pathway. PIF's effects on systemic immunity lead to comprehensive immune modulation, not immune suppression. We examine PIF's role in protecting embryos from adverse maternal environment, which can lead to neurological disorders that may only manifest post-nataly: Synthetic PIF successfully translates endogenous PIF features in both pregnant and non-pregnant clinically relevant models. Specifically PIF has neuroprotective effects in neonatal prematurity. In adult relapsing-remitting neuroinflammation, PIF reverses advanced paralysis while promoting neurogenesis. PIF reversed Mycobacterium smegmatis induced brain infection. In graft-vs.-host disease, PIF reduced skin ulceration, liver inflammation and colon ulceration while maintaining beneficial anti-cancer, graft-vs.-leukemia effect. Clinical-grade PIF has high-safety profile even at supraphysiological doses. The FDA awarded Fast-Track designation, and university-sponsored clinical trials for autoimmune disorder are ongoing. Altogether, PIF properties point to its determining regulatory role in immunity, inflammation and transplant acceptance. Specific plans for using PIF for the treatment of complex neurological disorders (ie. traumatic brain injury, progressive paralysis), including neuroprotection from newborn to adult, are presented. PMID:26546485

  8. PreImplantation factor prevents atherosclerosis via its immunomodulatory effects without affecting serum lipids.

    PubMed

    Chen, Yung Chih; Rivera, Jennifer; Fitzgerald, Melissa; Hausding, Christian; Ying, Ya-Lan; Wang, Xiaowei; Todorova, Krassimira; Hayrabedyan, Soren; Barnea, Eytan R; Peter, Karlheinz

    2016-05-01

    PreImplantation factor (PIF) is a 15-amino acid peptide endogenously secreted by viable embryos, regulating/enabling maternal (host) acceptance/tolerance to the "invading" embryo (allograft) all-while preserving maternal immunity to fight infections. Such attributes make PIF a potential therapeutic agent for chronic inflammatory diseases. We investigated whether PIF's immunomodulatory properties prevent progression of atherosclerosis in the hyper-cholesterolaemic ApoE-deficient murine model. Male, high-fat diet fed, ApoE-deficient (ApoE-/-) mice were administered either PBS, scrambled PIF (0.3-3 mg/kg) or PIF (0.3-3 mg/kg) for seven weeks. After treatment, PIF (3 mg/kg)-treated ApoE-/- mice displayed significantly reduced atherosclerosis lesion burden in the aortic sinus and aortic arch, without any effect on lipid profile. PIF also caused a significant reduction in infiltration of macrophages, decreased expression of pro-inflammatory adhesion molecules, cytokines and chemokines in the plaque, and reduced circulating IFN-γ levels. PIF preferentially binds to monocytes/neutrophils. In vitro, PIF attenuated monocyte migration (MCP-1-induced chemotaxis assay) and in vivo in LPS peritonitis model. Also PIF prevented leukocyte extravasation (peritonitis thioglycollate-induced model), demonstrating that PIF exerts its effect in part by modulation of monocyte function. Inhibition of the potassium channel KCNAB3 (Kv1.3) and of the insulin degrading enzyme (IDE) was demonstrated as potential mechanism of PIF's immunomodulatory effects. In conclusion, PIF regulates/lowers inflammation and prevents atherosclerosis development without affecting circulating lipids. Overall our findings establish PIF as a strong immunomodulatory drug candidate for atherosclerosis therapy. PMID:26842698

  9. Mammalian touch catches up

    PubMed Central

    Walsh, Carolyn M.; Bautista, Diana M.; Lumpkin, Ellen A.

    2015-01-01

    An assortment of touch receptors innervate the skin and encode different tactile features of the environment. Compared with invertebrate touch and other sensory systems, our understanding of the molecular and cellular underpinnings of mammalian touch lags behind. Two recent breakthroughs have accelerated progress. First, an arsenal of cell-type-specific molecular markers allowed the functional and anatomical properties of sensory neurons to be matched, thereby unraveling a cellular code for touch. Such markers have also revealed key roles of non-neuronal cell types, such as Merkel cells and keratinocytes, in touch reception. Second, the discovery of Piezo genes as a new family of mechanically activated channels has fueled the discovery of molecular mechanisms that mediate and mechanotransduction in mammalian touch receptors. PMID:26100741

  10. Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media.

    PubMed

    Sadeesh, E M; Selokar, N L; Balhara, A K; Yadav, P S

    2016-10-01

    The objective of this study was to compare effects of in vitro culture systems on embryonic development and expression patterns of developmentally important genes in preimplantation buffalo embryos. After IVM/IVF presumptive zygotes were cultured in one of three systems: undefined TCM-199, mCR2aa medium supplemented with 10 % FBS and defined PVA-myo-inositol-phosphate-EGF medium. No (P > 0.05) differences at 2-cell, 4-cell and 8-cell to 16- cell stages were observed among the three cultured media used, however, increased (P < 0.05) blastocyst yield, cell number and hatching rate were found in defined medium compared to undefined media. The expression patterns of genes implicated in embryo metabolism (GLUT-1), anti-apoptosis (BCL-2), imprinting (IGF-2R), DNA methylation (DNMT-3A) and maternal recognition of pregnancy (IFNT) were increased (P < 0.05) in hatched blastocysts derived from defined medium compared to undefined media. In conclusion, serum-free, defined medium improved developmental competence of in vitro cultured buffalo embryos. Whether these differences in morphological development and gene expression have long-term effects on buffalo calves born after embryo transfer remains unknown. However, it is possible that early adaptations of the preimplantation embryo to its environment persist during fetal and post-natal development. PMID:27481470

  11. Rheotaxis guides mammalian sperm

    PubMed Central

    Miki, Kiyoshi; Clapham, David E

    2013-01-01

    Background In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals. Results Here, we show that positive rheotaxis, the ability of organisms to orient and swim against the flow of surrounding fluid, is a major taxic factor for mouse and human sperm. This flow is generated within 4 hours of sexual stimulation and coitus in female mice; prolactin-triggered oviductal fluid secretion clears the oviduct of debris, lowers viscosity, and generates the stream that guides sperm migration in the oviduct. Rheotaxic movement is demonstrated in capacitated and uncapacitated spermatozoa in low and high viscosity medium. Finally, we show that a unique sperm motion we quantify using the sperm head's rolling rate reflects sperm rotation that generates essential force for positioning the sperm in the stream. Rotation requires CatSper channels, presumably by enabling Ca2+ influx. Conclusions We propose that rheotaxis is a major determinant of sperm guidance over long distances in the mammalian female reproductive tract. Coitus induces fluid flow to guide sperm in the oviduct. Sperm rheotaxis requires rotational motion during CatSper channel-dependent hyperactivated motility. PMID:23453951

  12. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

    PubMed Central

    Carvacho, Ingrid; Ardestani, Goli; Lee, Hoi Chang; McGarvey, Kaitlyn; Fissore, Rafael A.; Lykke-Hartmann, Karin

    2016-01-01

    The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. PMID:27681336

  13. Preimplantation genetic diagnosis: technological advances to improve accuracy and range of applications.

    PubMed

    Kuliev, Anver; Verlinsky, Yury

    2008-04-01

    Preimplantation genetic diagnosis (PGD) is an option for couples who are at risk that enables them to have unaffected progeny without facing the risk of pregnancy termination after prenatal diagnosis as currently practiced. It is also one of the practical tools used in assisted reproduction technology to improve the chance of conception for infertility cases with poor prognosis. Because PGD is performed using a single biopsied cell, technological advances are important to improving PGD accuracy. This has contributed to the avoidance of misdiagnosis in PGD for single gene disorders, and extensive experience in PGD for chromosomal disorders suggests strategies for more reliable evaluation of the chromosomal status of the preimplantation embryo. This paper describes the present status of PGD for genetic and chromosomal disorders, its accuracy and range, and how PGD is an integral part of IVF and genetic practices.

  14. A legal jurisprudential deliberation on lineage and inheritance of the pre-implantation embryo.

    PubMed

    Rafiei, Mohammad Taqi

    2012-01-01

    Today, in vitro fertilization (IVF) is not just a medical issue, but certainly also a complex legal issue, in which lawyers can play an important role by establishing the suitable legal conditions to regulate legal relations by introducing necessary theories. One of the important and controversial issues, which can be approached legally, is the study of the pre-implantation embryo with regard to property law, especially the inheritance of the pre-implantation embryo. Article 3 of the Conditions of Embryo Donation to Infertile Couples Act of the year 2003 only stipulates the responsibilities of the intended couple and born child in terms of support, upbringing, maintenance and respect; it does not specify any law regarding the other financial outcomes of lineage like "inheritance", which makes this law imperfect. Also, in view of the fact that lineage is one of the causes of inheritance; studying inheritance without analyzing lineage in the pre-implantation embryo is not possible. Therefore, it is recommended to study lineage and inheritance simultaneously. Some questions arise in this regard, including whether it is possible to prove lineage between the genetic father and mother with a laboratory child, and between the owner of the womb (that is intended wife) and the child. Supposing the lineage is proved, what is the state of inheritance between them? Lawyers and Islamic jurists have different opinions regarding the lineage of the pre-implantation embryo and inheritance. The author believes that the owner of the sperm is regarded as the genetic father of the child and in terms of lineage between laboratory child and mother two genetic and carrying factors must be considered. Thereby, considering inheritance between the genetic father and the child is possible according to inheritance law. Regarding the inheritance state of a laboratory child from two mothers the problem can be solved by using the equality rule within the framework of inheritance law.

  15. Mammalian glycosylation in immunity

    PubMed Central

    Marth, Jamey D.; Grewal, Prabhjit K.

    2009-01-01

    Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous of receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome. PMID:18846099

  16. Analyses of Long Non-Coding RNA and mRNA profiling using RNA sequencing during the pre-implantation phases in pig endometrium.

    PubMed

    Wang, Yueying; Xue, Songyi; Liu, Xiaoran; Liu, Huan; Hu, Tao; Qiu, Xiaotian; Zhang, Jinlong; Lei, Minggang

    2016-01-01

    Establishment of implantation in pig is accompanied by a coordinated interaction between the maternal uterine endometrium and conceptus development. We investigated the expression profiles of endometrial tissue on Days 9, 12 and 15 of pregnancy and on Day 12 of non-pregnancy in Yorkshire, and performed a comprehensive analysis of long non-coding RNAs (lncRNAs) in endometrial tissue samples by using RNA sequencing. As a result, 2805 novel lncRNAs, 2,376 (301 lncRNA and 2075 mRNA) differentially expressed genes (DEGs) and 2149 novel transcripts were obtained by pairwise comparison. In agreement with previous reports, lncRNAs shared similar characteristics, such as shorter in length, lower in exon number, lower at expression level and less conserved than protein coding transcripts. Bioinformatics analysis showed that DEGs were involved in protein binding, cellular process, immune system process and enriched in focal adhesion, Jak-STAT, FoxO and MAPK signaling pathway. We also found that lncRNAs TCONS_01729386 and TCONS_01325501 may play a vital role in embryo pre-implantation. Furthermore, the expression of FGF7, NMB, COL5A3, S100A8 and PPP1R3D genes were significantly up-regulated at the time of maternal recognition of pregnancy (Day 12 of pregnancy). Our results first identified the characterization and expression profile of lncRNAs in pig endometrium during pre-implantation phases. PMID:26822553

  17. Pregnancy after preimplantation diagnosis for a deletion in the dystrophin gene by polymerase chain reaction in embryos obtained after intracytoplasmic sperm injection

    SciTech Connect

    Lissens, W.; Liu, J.; Van Broeckhoven, C.

    1994-09-01

    Duchenne muscular dystrophy (DMD) is one of the most common X-linked recessive diseases. In order to be able to perform a DMD-specific preimplantation diagnosis (PID) in a female carrier of a deletion of exons 3 to 18 in the dystrophin gene, we have developed a PCR assay to detect the deletion based on sequences of exon 17. The efficiency of this PCR was evaluated on 50 single blastomeres from 12 normal control embryos and on 41 blastomeres for 9 male and 3 female embryos from the female DMD carrier, obtained after a first preimplantation diagnosis by sexing. The exon 17 region was amplified with 100% efficiency, except in all 21 blastomeres from 6 male embryos from the carrier where no PCR signals were observed. The negative results in these blastomeres were interpreted as being found only in male embryos carrying the deletion. Intracytoplasmic sperm injection was carried out on the carrier`s metaphase II oocytes retrieved after ovarian stimulation. Embryos were analyzed for the presence of exon 17 and 2 male embryos were found to be deleted, while 4 embryos showed normal amplification signals. Three of the latter embryos were replaced, resulting in a singleton pregnancy. Amniotic cell analysis showed a normal female karyotype and DNA analysis indicated a non-carrier.

  18. Mammalian diversity: gametes, embryos and reproduction.

    PubMed

    Behringer, Richard R; Eakin, Guy S; Renfree, Marilyn B

    2006-01-01

    The class Mammalia is composed of approximately 4800 extant species. These mammalian species are divided into three subclasses that include the monotremes, marsupials and eutherians. Monotremes are remarkable because these mammals are born from eggs laid outside of the mother's body. Marsupial mammals have relatively short gestation periods and give birth to highly altricial young that continue a significant amount of 'fetal' development after birth, supported by a highly sophisticated lactation. Less than 10% of mammalian species are monotremes or marsupials, so the great majority of mammals are grouped into the subclass Eutheria, including mouse and human. Mammals exhibit great variety in morphology, physiology and reproduction. In the present article, we highlight some of this remarkable diversity relative to the mouse, one of the most widely used mammalian model organisms, and human. This diversity creates challenges and opportunities for gamete and embryo collection, culture and transfer technologies.

  19. Odor Coding by a Mammalian Receptor Repertoire

    PubMed Central

    Saito, Harumi; Chi, Qiuyi; Zhuang, Hanyi; Matsunami, Hiro; Mainland, Joel D.

    2009-01-01

    Deciphering olfactory encoding requires a thorough description of the ligands that activate each odorant receptor (OR). In mammalian systems, however, ligands are known for fewer than 50 of over 1400 human and mouse ORs, greatly limiting our understanding of olfactory coding. We performed high-throughput screening of 93 odorants against 464 ORs expressed in heterologous cells and identified agonists for 52 mouse and 10 human ORs. We used the resulting interaction profiles to develop a predictive model relating physicochemical odorant properties, OR sequences, and their interactions. Our results provide a basis for translating odorants into receptor neuron responses and unraveling mammalian odor coding. PMID:19261596

  20. An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening.

    PubMed

    Chang, Li-Jung; Chen, Shee-Uan; Tsai, Yi-Yi; Hung, Chia-Cheng; Fang, Mei-Ya; Su, Yi-Ning; Yang, Yu-Shih

    2011-09-01

    Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium. PMID:22384431

  1. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

    1993-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  2. Mitochondria and mammalian reproduction.

    PubMed

    Ramalho-Santos, João; Amaral, Sandra

    2013-10-15

    Mitochondria are cellular organelles with crucial roles in ATP synthesis, metabolic integration, reactive oxygen species (ROS) synthesis and management, the regulation of apoptosis (namely via the intrinsic pathway), among many others. Additionally, mitochondria in different organs or cell types may have distinct properties that can decisively influence functional analysis. In terms of the importance of mitochondria in mammalian reproduction, and although there are species-specific differences, these aspects involve both energetic considerations for gametogenesis and fertilization, control of apoptosis to ensure the proper production of viable gametes, and ROS signaling, as well as other emerging aspects. Crucially, mitochondria are the starting point for steroid hormone biosynthesis, given that the conversion of cholesterol to pregnenolone (a common precursor for all steroid hormones) takes place via the activity of the cytochrome P450 side-chain cleavage enzyme (P450scc) on the inner mitochondrial membrane. Furthermore, mitochondrial activity in reproduction has to be considered in accordance with the very distinct strategies for gamete production in the male and female. These include distinct gonad morpho-physiologies, different types of steroids that are more prevalent (testosterone, estrogens, progesterone), and, importantly, the very particular timings of gametogenesis. While spermatogenesis is complete and continuous since puberty, producing a seemingly inexhaustible pool of gametes in a fixed environment; oogenesis involves the episodic production of very few gametes in an environment that changes cyclically. These aspects have always to be taken into account when considering the roles of any common element in mammalian reproduction.

  3. Nuclear Distribution of RNA Polymerase II and mRNA Processing Machinery in Early Mammalian Embryos

    PubMed Central

    Bogolyubova, Irina O.; Bogolyubov, Dmitry S.

    2014-01-01

    Spatial distribution of components of nuclear metabolism provides a significant impact on regulation of the processes of gene expression. While distribution of the key nuclear antigens and their association with the defined nuclear domains were thoroughly traced in mammalian somatic cells, similar data for the preimplantation embryos are scanty and fragmental. However, the period of cleavage is characterized by the most drastic and dynamic nuclear reorganizations accompanying zygotic gene activation. In this minireview, we try to summarize the results of studies concerning distribution of major factors involved in RNA polymerase II-dependent transcription, pre-mRNA splicing mRNA export that have been carried out on early embryos of mammals. PMID:24868542

  4. Current and future assisted reproductive technologies for mammalian farm animals.

    PubMed

    Hansen, Peter J

    2014-01-01

    technologies that have the potential to improve efficiency of livestock production. The focus will be on technologies that manipulate male and female gametes as well as the stem cells from which they are derived and the preimplantation embryo. While technology is crucial to other interventions in the reproductive process like control of seasonal breeding, hormonal regulation of ovulation, estrous cyclicity and pregnancy establishment, feeding to optimize reproduction, minimizing environmental stress, and selection of genes controlling reproduction, these will not be considered here. Rather the reader is directed to other chapters in this volume as well as some reviews on other aspects of artificial manipulation of reproduction (Reprod Fertil Dev 24:258-266, 2011; Reprod Domest Anim 43:40-47, 2008; Reprod Domest Anim 43:122-128, 2008; Soc Reprod Fertil Suppl 66:87-102, 2009; Comprehensive biotechnology, Amsterdam, pp 477-485; Dairy production medicine, Chichester, pp 153-163; Theriogenology 76:1619-1631, 2011; Theriogenology 76:1568-1582, 2011; Theriogenology 77:1-11, 2012). Given the large number of mammalian species used for production of products useful for man and the diversity in their biology and management, the review will not be comprehensive but instead will use results from species that are most illustrative of the opportunities generated by assisted reproductive technologies.

  5. Current and future assisted reproductive technologies for mammalian farm animals.

    PubMed

    Hansen, Peter J

    2014-01-01

    technologies that have the potential to improve efficiency of livestock production. The focus will be on technologies that manipulate male and female gametes as well as the stem cells from which they are derived and the preimplantation embryo. While technology is crucial to other interventions in the reproductive process like control of seasonal breeding, hormonal regulation of ovulation, estrous cyclicity and pregnancy establishment, feeding to optimize reproduction, minimizing environmental stress, and selection of genes controlling reproduction, these will not be considered here. Rather the reader is directed to other chapters in this volume as well as some reviews on other aspects of artificial manipulation of reproduction (Reprod Fertil Dev 24:258-266, 2011; Reprod Domest Anim 43:40-47, 2008; Reprod Domest Anim 43:122-128, 2008; Soc Reprod Fertil Suppl 66:87-102, 2009; Comprehensive biotechnology, Amsterdam, pp 477-485; Dairy production medicine, Chichester, pp 153-163; Theriogenology 76:1619-1631, 2011; Theriogenology 76:1568-1582, 2011; Theriogenology 77:1-11, 2012). Given the large number of mammalian species used for production of products useful for man and the diversity in their biology and management, the review will not be comprehensive but instead will use results from species that are most illustrative of the opportunities generated by assisted reproductive technologies. PMID:24170352

  6. [Placental developmental defects in cloned mammalian animals].

    PubMed

    Ao, Zheng; Liu, Dewu; Cai, Gengyuan; Wu, Zhenfang; Li, Zicong

    2016-05-01

    The cloning technique, also called somatic cell nuclear transfer (SCNT), has been successfully established and gradually applied to various mammalian species. However, the developmental rate of SCNT mammalian embryos is very low, usually at 1% to 5%, which limits the application of SCNT. Placental developmental defects are considered as the main cause of SCNT embryo development inhibition. Almost all of SCNT-derived mammalian placentas exhibit various abnormalities, such as placental hyperplasia, vascular defects and umbilical cord malformation. Mechanistically, these abnormalities result from failure of establishment of correct epigenetic modification in the trophectoderm genome, which leads to erroneous expression of important genes for placenta development-related, particularly imprinted genes. Consequently, aberrant imprinted gene expression gives rise to placental morphologic abnormalities and functional defects, therefore decreases developmental competence of cloned embryos. Currently, although numerous methods that can improve the developmental ability of SCNT-derived embryos have been reported, most of them are unable to substantially enhance the success rate of SCNT due to failure to eliminate the placental development defects. In this review, we summarize placental abnormalities and imprinted gene expression in mammalian cloning, and propose directions for the future research aiming to improve the cloning efficiency. PMID:27232488

  7. Producing Newborn Synchronous Mammalian Cells

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R.; Helmstetter, Charles E.; Thornton, Maureen

    2008-01-01

    A method and bioreactor for the continuous production of synchronous (same age) population of mammalian cells have been invented. The invention involves the attachment and growth of cells on an adhesive-coated porous membrane immersed in a perfused liquid culture medium in a microgravity analog bioreactor. When cells attach to the surface divide, newborn cells are released into the flowing culture medium. The released cells, consisting of a uniform population of synchronous cells are then collected from the effluent culture medium. This invention could be of interest to researchers investigating the effects of the geneotoxic effects of the space environment (microgravity, radiation, chemicals, gases) and to pharmaceutical and biotechnology companies involved in research on aging and cancer, and in new drug development and testing.

  8. Suspension culture of mammalian cells.

    PubMed

    Birch, J R; Arathoon, R

    1990-01-01

    Mammalian cell suspension culture systems are being used increasingly in the biotechnology industry. This is due to their many advantages including simplicity and homogeneity of culture. Suspension systems are very adaptable (e.g., for microcarrier, microencapsulation, or other methods of culture). Their engineering is thoroughly understood and standardized at large scale, and automation and cleaning procedures are well established. Suspension systems offer the possibility of quick implementation of production protocols due to their ability to be scaled easily once the basic culture parameters are understood. The only main disadvantage of the suspension culture systems to date is their inapplicability for the production of human vaccines from either primary cell lines or from normal human diploid cell lines (Hayflick et al., 1987 and references therein). One of the great advantages of suspension culture is the opportunity it provides to study interactions of metabolic and production phenomena in chemostat or turbidostat steady-state systems. Furthermore, in suspension culture systems from which cell number and cell mass measurements are easy to obtain, rigorous and quantitative estimations of the effects of growth conditions or perturbations of metabolic homeostasis can be made. Such studies can speed up the development of optimal processes. With our increasing understanding of factors influencing expression in mammalian cells (Cohen and Levinson, 1988; Santoro et al., 1988) and the direct application of new methods in suspension culture (Rhodes and Birch, 1988), its usefulness and importance is likely to increase in the future. In this chapter, we have described some of the potential uses of the various suspension culture systems and have covered most of the established technology and literature. Due to the rapid developments and needs in the biotechnology industry and the versatility of suspension culture systems, it is probable that many more variations on this

  9. Involvement of Cl(-)/HCO3(-) exchanger SLC26A3 and SLC26A6 in preimplantation embryo cleavage.

    PubMed

    Lu, Yong Chao; Yang, Jing; Fok, Kin Lam; Ye, Ying Hui; Jin, Liang; Chen, Zheng Yun; Zhang, Xin Mei; Huang, He Feng; Chan, Hsiao Chang

    2016-01-01

    Bicarbonate (HCO3(-)) is essential for preimplantation embryo development. However, the mechanism underlying the HCO3(-) transport into the embryo remains elusive. In the present study, we examined the possible involvement of Cl(-)/HCO3(-) exchanger in mediating HCO3(-) transport into the embryo. Our results showed that depletion of extracellular Cl(-), even in the presence of HCO3(-), suppressed embryo cleavage in a concentration-dependent manner. Cleavage-associated HCO3(-)-dependent events, including increase of intracellular pH, upregulation of miR-125b and downregulation of p53, also required Cl(-). We further showed that Cl(-)/HCO3(-) exchanger solute carrier family 26 (SLC26) A3 and A6 were expressed at 2-cell through blastocyst stage. Blocking individual exchanger's activity by inhibitors or gene knockdown differentially decreased embryo cleavage and inhibited HCO3(-)-dependent events, while inhibiting/knocking down both produced an additive effect to an extent similar to that observed when CFTR was inhibited. These results indicate the involvement of SLC26A3 and A6 in transporting HCO3(-) essential for embryo cleavage, possibly working in concert with CFTR through a Cl(-) recycling pathway. The present study sheds light into our understanding of molecular mechanisms regulating embryo cleavage by the female reproductive tract. PMID:27346053

  10. Single‐Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells

    PubMed Central

    Briggs, Sharon F.; Dominguez, Antonia A.; Chavez, Shawn L.

    2015-01-01

    Abstract The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well‐understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte‐to‐embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4‐cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells. Stem Cells 2015;33:1771–1781 PMID:25753947

  11. Involvement of Cl(-)/HCO3(-) exchanger SLC26A3 and SLC26A6 in preimplantation embryo cleavage.

    PubMed

    Lu, Yong Chao; Yang, Jing; Fok, Kin Lam; Ye, Ying Hui; Jin, Liang; Chen, Zheng Yun; Zhang, Xin Mei; Huang, He Feng; Chan, Hsiao Chang

    2016-06-27

    Bicarbonate (HCO3(-)) is essential for preimplantation embryo development. However, the mechanism underlying the HCO3(-) transport into the embryo remains elusive. In the present study, we examined the possible involvement of Cl(-)/HCO3(-) exchanger in mediating HCO3(-) transport into the embryo. Our results showed that depletion of extracellular Cl(-), even in the presence of HCO3(-), suppressed embryo cleavage in a concentration-dependent manner. Cleavage-associated HCO3(-)-dependent events, including increase of intracellular pH, upregulation of miR-125b and downregulation of p53, also required Cl(-). We further showed that Cl(-)/HCO3(-) exchanger solute carrier family 26 (SLC26) A3 and A6 were expressed at 2-cell through blastocyst stage. Blocking individual exchanger's activity by inhibitors or gene knockdown differentially decreased embryo cleavage and inhibited HCO3(-)-dependent events, while inhibiting/knocking down both produced an additive effect to an extent similar to that observed when CFTR was inhibited. These results indicate the involvement of SLC26A3 and A6 in transporting HCO3(-) essential for embryo cleavage, possibly working in concert with CFTR through a Cl(-) recycling pathway. The present study sheds light into our understanding of molecular mechanisms regulating embryo cleavage by the female reproductive tract.

  12. Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

    PubMed

    Briggs, Sharon F; Dominguez, Antonia A; Chavez, Shawn L; Reijo Pera, Renee A

    2015-06-01

    The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well-understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte-to-embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4-cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells.

  13. AB033. Preimplantation genetic diagnosis of spinal muscular atrophy in Vietnam

    PubMed Central

    Khoa, Tran Van; Nga, Nguyen Thi Thanh; Tao, Nguyen Dinh; Sang, Trieu Tien; Giang, Ngo Truong; Dung, Vu Chi

    2015-01-01

    Objective Spinal muscular atrophy (SMA) is a severe neurodegenerative autosomal recessive disorder. Most of patients are caused by the homozygous absence of exon 7 of the telomeric copy of the SMN gene (SMNt) on chromosome 5. Setting up a molecular diagnostic protocol for detecting exon 7 gen SMNT homozygous deletion in single cell is basic to preimplantation genetic diagnosis of spinal muscular atrophy. Methods This study was carried out on 17 patients and their parents. Firstly, lymphocytes of patients and their parents were isolated from fresh blood by ficoll. Taking a lymphocyte on stereoscopic microscope, lysing the cell, amplifying whole genome, then amplifying exon 7 of SMNT gene by using a polymerase chain reaction, followed by HinfI restriction digest enzyme of the PCR enabling the important SMNT gene to be distinguished from the centromic SMN gene (SMNc) which has no clinical phenotype to detect mutation. Electrophoresis PCR products after digesting by restriction enzyme and analysis. Besides, the minisequencing technique has also been used to detect the absence of exon 7 of SMNT gene based on the difference of one nucleotide at 214-position in exon 7 (C-SMNT, T-SMNc). Secondly, the application of the protocol was set up on one lymphocyte to preimplantation genetic diagnosis of spinal muscular atrophy on biopsied blastomeres. Results Two different protocols which were PCR-RFLP and minisequencing, were set up on 200 lymphocytes from 17 patients and their parents to screen the homozygous deletion in exon 7 SMNT gene with the PCR efficiency in 96%. The results were similar with the gene diagnosed from fresh blood. The methods were also efficient, providing interpretable result in 96.55% (28/29) of the blastomeres tested. Three couples were treated using this method. Three normal embryos were transfer which resulted in one clinical pregnancy. Conclusions We have successfully applied the technique of PCR-RFLP and minisequencing for the preimplantation genetic

  14. Mammalian Wax Biosynthesis

    PubMed Central

    Cheng, Jeffrey B.; Russell, David W.

    2009-01-01

    Wax monoesters are synthesized by the esterification of fatty alcohols and fatty acids. A mammalian enzyme that catalyzes this reaction has not been isolated. We used expression cloning to identify cDNAs encoding a wax synthase in the mouse preputial gland. The wax synthase gene is located on the X chromosome and encodes a member of the acyltransferase family of enzymes that synthesize neutral lipids. Expression of wax synthase in cultured cells led to the formation of wax monoesters from straight chain saturated, unsaturated, and polyunsaturated fatty alcohols and acids. Polyisoprenols also were incorporated into wax monoesters by the enzyme. The wax synthase had little or no ability to synthesize cholesteryl esters, diacylglycerols, or triacylglycerols, whereas other acyltransferases, including the acyl-CoA:monoacylglycerol acyltransferase 1 and 2 enzymes and the acyl-CoA:diacylglycerol acyltransferase 1 and 2 enzymes, exhibited modest wax monoester synthesis activities. Confocal light microscopy indicated that the wax synthase was localized in membranes of the endoplasmic reticulum. Wax synthase mRNA was abundant in tissues rich in sebaceous glands such as the preputial gland and eyelid and was present at lower levels in other tissues. Coexpression of cDNAs specifying fatty acyl-CoA reductase 1 and wax synthase led to the synthesis of wax monoesters. The data suggest that wax monoester synthesis in mammals involves a two step biosynthetic pathway catalyzed by fatty acyl-CoA reductase and wax synthase enzymes. PMID:15220349

  15. Mammalian Wax Biosynthesis

    PubMed Central

    Cheng, Jeffrey B.; Russell, David W.

    2009-01-01

    The conversion of fatty acids to fatty alcohols is required for the synthesis of wax monoesters and ether lipids. The mammalian enzymes that synthesize fatty alcohols have not been identified. Here, an in silico approach was used to discern two putative reductase enzymes designated FAR1 and FAR2. Expression studies in intact cells showed that FAR1 and FAR2 cDNAs encoded isozymes that reduced fatty acids to fatty alcohols. Fatty acyl-CoA esters were the substrate of FAR1, and the enzyme required NADPH as a cofactor. FAR1 preferred saturated and unsaturated fatty acids of 16 or 18 carbons as substrates, whereas FAR2 preferred saturated fatty acids of 16 or 18 carbons. Confocal light microscopy indicated that FAR1 and FAR2 were localized in the peroxisome. The FAR1 mRNA was detected in many mouse tissues with the highest level found in the preputial gland, a modified sebaceous gland. The FAR2 mRNA was more restricted in distribution and most abundant in the eyelid, which contains wax-laden meibomian glands. Both FAR mRNAs were present in the brain, a tissue rich in ether lipids. The data suggest that fatty alcohol synthesis in mammals is accomplished by two fatty acyl-CoA reductase isozymes that are expressed at high levels in tissues known to synthesize wax monoesters and ether lipids. PMID:15220348

  16. Mammalian Gut Immunity

    PubMed Central

    Chassaing, Benoit; Kumar, Manish; Baker, Mark T.; Singh, Vishal; Vijay-Kumar, Matam

    2016-01-01

    The mammalian intestinal tract is the largest immune organ in the body and comprises cells from non-hemopoietic (epithelia, Paneth cells, goblet cells) and hemopoietic (macrophages, dendritic cells, T-cells) origin, and is also a dwelling for trillions of microbes collectively known as the microbiota. The homeostasis of this large microbial biomass is prerequisite to maintain host health by maximizing beneficial symbiotic relationships and minimizing the risks of living in such close proximity. Both microbiota and host immune system communicate with each other to mutually maintain homeostasis in what could be called a “love–hate relationship.” Further, the host innate and adaptive immune arms of the immune system cooperate and compensate each other to maintain the equilibrium of a highly complex gut ecosystem in a stable and stringent fashion. Any imbalance due to innate or adaptive immune deficiency or aberrant immune response may lead to dysbiosis and low-grade to robust gut inflammation, finally resulting in metabolic diseases. PMID:25163502

  17. Light exposure of the ovum and preimplantation embryo during ART procedures.

    PubMed

    Ottosen, Lars D M; Hindkjaer, Johnny; Ingerslev, Jakob

    2007-01-01

    Exposure to visible light (400-700 nm wavelengths) is an unnatural stress factor to preimplantation embryos cultured in vitro. This study investigated the spectral composition and intensity of light during IVF procedures, and calculated radiation doses reaching the embryo during handling and manipulation. The study shows that normal IVF procedure may result in stressing radiation doses, unless filters are applied. This is at present not sufficiently recognised. No Danish IVF clinics use filters to protect embryos against visible light. 95% of the radiation was from microscopes. Ambient light, in contrast, was not a significant contributor to light stress and the use of dark laboratories is not justified.

  18. Amorphous clusters in Co implanted ZnO induced by boron pre-implantation

    SciTech Connect

    Potzger, K.; Shalimov, A.; Zhou, S.; Schmidt, H.; Mucklich, A.; Helm, M.; Fassbender, J.; Liberati, M.; Arenholz, E.

    2009-02-09

    We demonstrate the formation of superparamagnetic/ferromagnetic regions within ZnO(0001) single crystals sequently implanted with B and Co. While the pre-implantation with B plays a minor role for the electrical transport properties, its presence leads to the formation of amorphous phases. Moreover, B acts strongly reducing on the implanted Co. Thus, the origin of the ferromagnetic ordering in local clusters with large Co concentration is itinerant d-electrons as in the case of metallic Co. The metallic amorphous phases are non-detectable by common X-ray diffraction.

  19. Trim43a, Trim43b, and Trim43c: Novel mouse genes expressed specifically in mouse preimplantation embryos.

    PubMed

    Stanghellini, Ilaria; Falco, Geppino; Lee, Sung-Lim; Monti, Manuela; Ko, Minoru S H

    2009-12-01

    We describe the identification and characterization of Trim43a, Trim43b, and Trim43c genes, whose expression are restricted to preimplantation stages and peak at the 8-cell to morula stage. We identified a 5kb DNA fragment that covers upstream region of Trim43a as a putative promoter, which can drive the expression of mStrawberry fluorescent protein in a manner similar to endogenous Trim43 genes. Trim43 genes will be useful stage-specific markers for the study of preimplantation embryos.

  20. Cheating death at the dawn of life: developmental control of apoptotic repression in the preimplantation embryo.

    PubMed

    Hansen, Peter J; Fear, Justin M

    2011-09-23

    During early development, the mammalian embryo is resistant to pro-apoptotic signals because of biochemical properties of the mitochondrion and nucleus. Mitochondria of the bovine two-cell embryo are resistant to depolarization because of low amounts of the proapoptotic protein BAX and high concentrations of the anti-apoptotic protein BCL2. As development proceeds, BAX content increases, BCL2 content declines, and mitochondria becomes capable of pore formation and depolarization in response to pro-apoptotic signals. The nucleus of the two-cell embryo is resistant to degradation by the DNase DFFB because epigenetic modifications, including DNA methylation and histone deacetylation, mask internucleosomal sites for DNA cleavage. Blastomere DNA becomes progressively less methylated during development so that DNA becomes accessible to cleavage by DFFB.

  1. Stem Cells in Mammalian Gonads.

    PubMed

    Wu, Ji; Ding, Xinbao; Wang, Jian

    2016-01-01

    Stem cells have great value in clinical application because of their ability to self-renew and their potential to differentiate into many different cell types. Mammalian gonads, including testes for males and ovaries for females, are composed of germline and somatic cells. In male mammals, spermatogonial stem cells maintain spermatogenesis which occurs continuously in adult testis. Likewise, a growing body of evidence demonstrated that female germline stem cells could be found in mammalian ovaries. Meanwhile, prior studies have shown that somatic stem cells exist in both testes and ovaries. In this chapter, we focus on mammalian gonad stem cells and discuss their characteristics as well as differentiation potentials.

  2. Positive Selection Linked with Generation of Novel Mammalian Dentition Patterns.

    PubMed

    Machado, João Paulo; Philip, Siby; Maldonado, Emanuel; O'Brien, Stephen J; Johnson, Warren E; Antunes, Agostinho

    2016-01-01

    A diverse group of genes are involved in the tooth development of mammals. Several studies, focused mainly on mice and rats, have provided a detailed depiction of the processes coordinating tooth formation and shape. Here we surveyed 236 tooth-associated genes in 39 mammalian genomes and tested for signatures of selection to assess patterns of molecular adaptation in genes regulating mammalian dentition. Of the 236 genes, 31 (∼13.1%) showed strong signatures of positive selection that may be responsible for the phenotypic diversity observed in mammalian dentition. Mammalian-specific tooth-associated genes had accelerated mutation rates compared with older genes found across all vertebrates. More recently evolved genes had fewer interactions (either genetic or physical), were associated with fewer Gene Ontology terms and had faster evolutionary rates compared with older genes. The introns of these positively selected genes also exhibited accelerated evolutionary rates, which may reflect additional adaptive pressure in the intronic regions that are associated with regulatory processes that influence tooth-gene networks. The positively selected genes were mainly involved in processes like mineralization and structural organization of tooth specific tissues such as enamel and dentin. Of the 236 analyzed genes, 12 mammalian-specific genes (younger genes) provided insights on diversification of mammalian teeth as they have higher evolutionary rates and exhibit different expression profiles compared with older genes. Our results suggest that the evolution and development of mammalian dentition occurred in part through positive selection acting on genes that previously had other functions. PMID:27613398

  3. Positive Selection Linked with Generation of Novel Mammalian Dentition Patterns.

    PubMed

    Machado, João Paulo; Philip, Siby; Maldonado, Emanuel; O'Brien, Stephen J; Johnson, Warren E; Antunes, Agostinho

    2016-09-11

    A diverse group of genes are involved in the tooth development of mammals. Several studies, focused mainly on mice and rats, have provided a detailed depiction of the processes coordinating tooth formation and shape. Here we surveyed 236 tooth-associated genes in 39 mammalian genomes and tested for signatures of selection to assess patterns of molecular adaptation in genes regulating mammalian dentition. Of the 236 genes, 31 (∼13.1%) showed strong signatures of positive selection that may be responsible for the phenotypic diversity observed in mammalian dentition. Mammalian-specific tooth-associated genes had accelerated mutation rates compared with older genes found across all vertebrates. More recently evolved genes had fewer interactions (either genetic or physical), were associated with fewer Gene Ontology terms and had faster evolutionary rates compared with older genes. The introns of these positively selected genes also exhibited accelerated evolutionary rates, which may reflect additional adaptive pressure in the intronic regions that are associated with regulatory processes that influence tooth-gene networks. The positively selected genes were mainly involved in processes like mineralization and structural organization of tooth specific tissues such as enamel and dentin. Of the 236 analyzed genes, 12 mammalian-specific genes (younger genes) provided insights on diversification of mammalian teeth as they have higher evolutionary rates and exhibit different expression profiles compared with older genes. Our results suggest that the evolution and development of mammalian dentition occurred in part through positive selection acting on genes that previously had other functions.

  4. Mammalian DNA Repair. Final Report

    SciTech Connect

    2003-01-24

    The Gordon Research Conference (GRC) on Mammalian DNA Repair was held at Harbortown Resort, Ventura Beach, CA. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  5. Polysome analysis of mammalian cells.

    PubMed

    He, Shan L; Green, Rachel

    2013-01-01

    To assess the global translational level of mammalian cells (see similar protocols for bacteria and yeast on Analysis of polysomes from bacteria, Polysome Profile Analysis - Yeast and Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

  6. Maturation of the mammalian secretome

    PubMed Central

    Simpson, Jeremy C; Mateos, Alvaro; Pepperkok, Rainer

    2007-01-01

    A recent use of quantitative proteomics to determine the constituents of the endoplasmic reticulum and Golgi complex is discussed in the light of other available methodologies for cataloging the proteins associated with the mammalian secretory pathway. PMID:17472737

  7. Mammalian cell cultivation in space

    NASA Astrophysics Data System (ADS)

    Gmünder, Felix K.; Suter, Robert N.; Kiess, M.; Urfer, R.; Nordau, C.-G.; Cogoli, A.

    Equipment used in space for the cultivation of mammalian cells does not meet the usual standard of earth bound bioreactors. Thus, the development of a space worthy bioreactor is mandatory for two reasons: First, to investigate the effect on single cells of the space environment in general and microgravity conditions in particular, and second, to provide researchers on long term missions and the Space Station with cell material. However, expertise for this venture is not at hand. A small and simple device for animal cell culture experiments aboard Spacelab (Dynamic Cell Culture System; DCCS) was developed. It provides 2 cell culture chambers, one is operated as a batch system, the other one as a perfusion system. The cell chambers have a volume of 200 μl. Medium exchange is achieved with an automatic osmotic pump. The system is neither mechanically stirred nor equipped with sensors. Oxygen for cell growth is provided by a gas chamber that is adjacent to the cell chambers. The oxygen gradient produced by the growing cells serves to maintain the oxygen influx by diffusion. Hamster kidney cells growing on microcarriers were used to test the biological performance of the DCCS. On ground tests suggest that this system is feasible.

  8. Cell-surface remodelling during mammalian erythropoiesis.

    PubMed

    Wraith, D C; Chesterton, C J

    1982-10-15

    Current evidence suggests that the major cell-surface modification occurring during mammalian erythropoiesis could be generated by two separate mechanisms: either selective loss of membrane proteins during enucleation or endocytosis at the subsequent reticulocyte and erythrocyte stages. The former idea was tested by collecting developing rabbit erythroid cells before and after the enucleation step and comparing their cell-surface protein composition via radiolabelling and electrophoresis. Few changes were observed. Our data thus lend support to the endocytosis mechanism.

  9. Can pre-implantation biopsies predict renal allograft function in pediatric renal transplant recipients?

    PubMed Central

    Kari, Jameela A.; Ma, Alison L.; Dufek, Stephanie; Mohamed, Ismail; Mamode, Nizam; Sebire, Neil J.; Marks, Stephen D.

    2015-01-01

    Objectives: To determine the utility of pre-implantation renal biopsy (PIB) to predict renal allograft outcomes. Methods: This is a retrospective review of all patients that underwent PIB from January 2003 to December 2011 at the Great Ormond Street Hospital for Children in London, United Kingdom. Thirty-two male patients (56%) aged 1.5-16 years (median: 10.2) at the time of transplantation were included in the study and followed-up for 33 (6-78) months. The results were compared with 33 controls. Results: The PIB showed normal histopathological findings in 13 patients (41%), mild chronic vascular changes in 8 (25%), focal tubular atrophy in one, moderate to severe chronic vascular change in 3, mild to moderate acute tubular damage in 6, and tissue was inadequate in one subject. Delayed graft function (DGF) was observed in 3 patients; 2 with vascular changes in PIB, and one with normal histopathological findings. Two subjects with PIB changes lost their grafts. The estimated glomerular filtration rate at 3-, and 6-months post-transplantation was lower in children with abnormal PIB changes compared with those with normal PIB. There was one case of DGF in the control group, and 4 children lost their grafts including the one with DGF. Conclusion: Pre-implantation renal biopsy can provide important baseline information of the graft with implications on subsequent medical treatment for pediatric renal transplant recipients. PMID:26593162

  10. Gray level Co-occurrence Matrices (GLCM) to assess microstructural and textural changes in pre-implantation embryos.

    PubMed

    Tan, Tiffany C Y; Ritter, Lesley J; Whitty, Annie; Fernandez, Renae C; Moran, Lisa J; Robertson, Sarah A; Thompson, Jeremy G; Brown, Hannah M

    2016-08-01

    The preimplantation embryo is extraordinarily sensitive to environmental signals and events such that perturbations can alter embryo metabolism and program an altered developmental trajectory, ultimately affecting the phenotype of the adult individual; indeed, the physical environment associated with in vitro embryo culture can attenuate development. Defining the underlying metabolic changes and mechanisms, however, has been limited by the imaging technology used to evaluate metabolites and structural features in the embryo. Here, we assessed the impact of in vitro fertilization and culture on mouse embryos using three metabolic markers: peroxyfluor 1 (a reporter of hydrogen peroxide), monochlorobimane (a reporter of glutathione), and Mitotracker Deep Red (a marker of mitochondria). We also evaluated the distribution pattern of histone 2AX gamma (γH2AX) in the nuclei of 2- and 8-cell embryos and blastocysts to investigate the degree of DNA damage caused by in vitro embryo culture. In vitro-fertilized embryos, in vivo-developed embryos, and in vivo-fertilized embryos recovered and cultured in vitro were compared at the 2-, 8-cell, and blastocyst stages. In addition to assessments based on fluorescence intensity, textural analysis using Gray Level Co-occurrence Matrix (GLCM), a statistical approach that assesses texture within an image, was used to evaluate peroxyfluor 1, monochlorobimane, and Mitotracker Deep Red staining in an effort to develop a robust metric of embryo quality. Our data provide strong evidence of modified metabolic parameters identifiable as altered fluorescence texture in embryos developed in vitro. Thus, texture-analysis approach may provide a means of gaining additional insight into embryo programming beyond conventional measurements of staining intensity for metabolic markers. Mol. Reprod. Dev. 83: 701-713, 2016 © 2016 Wiley Periodicals, Inc. PMID:27409576

  11. Structure of mammalian metallothionein.

    PubMed Central

    Kägi, J H; Vasák, M; Lerch, K; Gilg, D E; Hunziker, P; Bernhard, W R; Good, M

    1984-01-01

    All mammalian metallothioneins characterized contain a single polypeptide chain of 61 amino acid residues, among them 20 cysteines providing the ligands for seven metal-binding sites. Native metallothioneins are usually heterogeneous in metal composition, with Zn, Cd, and Cu occurring in varying proportions. However, forms containing only a single metal species, i.e., Zn, Cd, Ni, Co, Hg, Pb, Bi, have now been prepared by in vitro reconstitution from the metal-free apoprotein. By spectroscopic analysis of such derivatives it was established that all cysteine residues participate in metal binding, that each metal ion is bound to four thiolate ligands, and that the symmetry of each complex is close to that of a tetrahedron. To satisfy the requirements of the overall Me7(Cys-)20 stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. Experimental proof for the occurrence of such clusters comes from the demonstration of metal-metal interactions by spectroscopic and magnetic means. Thus, in Co(II)7-metallothionein, the Co(II)-specific ESR signals are effectively suppressed by antiferromagnetic coupling of juxtaposed paramagnetic metal ions. By monitoring changes in ESR signal size occurring on stepwise incorporation of Co(II) into the protein, it is possible to follow the building up of the clusters. This process is biphasic. Up to binding of four equivalents of Co(II), the ESR amplitude increases in proportion to the metal content, indicating generation of magnetically noninteracting high-spin complexes. However, upon addition of the remaining three equivalents of Co(II), these features are progressively suppressed, signaling the formation of clusters. The same mode of cluster formation has also been documented for Cd and Hg. The actual spatial organization of the clusters and the polypeptide chain remains to be established. An attractive possibility is the arrangement of the tetrahedral metal-thiolates in adamantane-like structures

  12. DNA replication and transcription in mammalian mitochondria.

    PubMed

    Falkenberg, Maria; Larsson, Nils-Göran; Gustafsson, Claes M

    2007-01-01

    The mitochondrion was originally a free-living prokaryotic organism, which explains the presence of a compact mammalian mitochondrial DNA (mtDNA) in contemporary mammalian cells. The genome encodes for key subunits of the electron transport chain and RNA components needed for mitochondrial translation. Nuclear genes encode the enzyme systems responsible for mtDNA replication and transcription. Several of the key components of these systems are related to proteins replicating and transcribing DNA in bacteriophages. This observation has led to the proposition that some genes required for DNA replication and transcription were acquired together from a phage early in the evolution of the eukaryotic cell, already at the time of the mitochondrial endosymbiosis. Recent years have seen a rapid development in our molecular understanding of these machineries, but many aspects still remain unknown.

  13. Freezing mammalian cells for production of biopharmaceuticals.

    PubMed

    Seth, Gargi

    2012-03-01

    Cryopreservation techniques utilize very low temperatures to preserve the structure and function of living cells. Various strategies have been developed for freezing mammalian cells of biological and medical significance. This paper highlights the importance and application of cryopreservation for recombinant mammalian cells used in the biopharmaceutical industry to produce high-value protein therapeutics. It is a primer that aims to give insight into the basic principles of cell freezing for the benefit of biopharmaceutical researchers with limited or no prior experience in cryobiology. For the more familiar researchers, key cell banking parameters such as the cell density and hold conditions have been reviewed to possibly help optimize their specific cell freezing protocols. It is important to understand the mechanisms underlying the freezing of complex and sensitive cellular entities as we implement best practices around the techniques and strategies used for cryopreservation. PMID:22226818

  14. High mobility group 1 (HMG1) protein in mouse preimplantation embryos.

    PubMed

    Spada, F; Brunet, A; Mercier, Y; Renard, J P; Bianchi, M E; Thompson, E M

    1998-08-01

    High mobility group 1 protein (HMG1) has traditionally been considered a structural component of chromatin, possibly similar in function to histone H1. In fact, at the onset of Xenopus and Drosophila development, HMG1 appears to substitute for histone H1: HMG1 is abundant when histone H1 is absent after the midblastula transition histone H1 largely replaces HMG1. We show that in early mouse embryos the expression patterns of HMG1 and histone H1 are not complementary. Instead, HMG1 content increases after zygotic genome activation at the same time as histone H1. HMG1 does not remain associated to mitotic chromosomes either in embryos or somatic cells. These results argue against a shared structural role for HMG1 and histone H1 in mammalian chromatin.

  15. Clinical Considerations of Preimplantation Genetic Diagnosis for Monogenic Diseases

    PubMed Central

    Hu, Xiaokun; Wang, Jing; Li, Yubin; Wang, Yizi; Ding, Chenhui; Zeng, Yanhong; Xu, Yanwen; Zhou, Canquan

    2015-01-01

    Purpose The aim of this study was to explore factors contribute to the success of PGD cycles for monogenic diseases. Methods During a 3-year period (January 2009 to December 2012), 184 consecutive ICSI-PGD cycles for monogenic diseases reaching the ovum pick-up and fresh embryo-transfer stage performed at the Reproductive Medicine Center of The First Affiliated Hospital Of Sun Yat-sen University were evaluated. Results ICSI was performed on 2206 metaphase II oocytes, and normal fertilization and cleavage rates were 83.4% (1840/2206) and 96.2% (1770/1840), respectively. In the present study, 60.5% (181/299) of day 3 good-quality embryos developed into good-quality embryos on day 4 after biopsy. Collectively, 42.9% clinical pregnancy rate (79/184) and 28.5% implantation rate (111/389) were presented. In the adjusted linear regression model, the only two significant factors affecting the number of genetically unaffected embryos were the number of biopsied embryos (coefficient: 0.390, 95%CI 0.317–0.463, P = 0.000) and basal FSH level (coefficient: 0.198, 95%CI 0.031–0.365, P = 0.021). In the adjusted binary logistic regression model, the only two significant factors affecting pregnancy outcome were the number of genetically available transferable embryos after PGD (adjusted OR 1.345, 95% CI 1.148–1.575, P = 0.000) and number of oocyte retrieved (adjusted OR 0.934, 95% CI 0.877–0.994, P = 0.031). Conclusion There should be at least four biopsied embryos to obtain at least one unaffected embryos in a PGD system for patients with single gene disorder and under the condition of basal FSH level smaller than 8.0mmol/L. Moreover, if only a low number (< 4) of biopsied embryos are available on day 3, the chance of unaffected embryos for transfer was small, with poor outcome. PMID:26421428

  16. Electroporation into Cultured Mammalian Embryos

    NASA Astrophysics Data System (ADS)

    Nomura, Tadashi; Takahashi, Masanori; Osumi, Noriko

    Over the last century, mammalian embryos have been used extensively as a common animal model to investigate fundamental questions in the field of developmental biology. More recently, the establishment of transgenic and gene-targeting systems in laboratory mice has enabled researchers to unveil the genetic mechanisms under lying complex developmental processes (Mak, 2007). However, our understanding of cell—cell interactions and their molecular basis in the early stages of mammalian embryogenesis is still very fragmentary. One of the major problems is the difficulty of precise manipulation and limited accessibility to mammalian embryos via uterus wall. Unfortunately, existing tissue and organotypic culture systems per se do not fully recapitulate three-dimensional, dynamic processes of organogenesis observed in vivo. Although transgenic animal technology and virus-mediated gene delivery are useful to manipulate gene expression, these techniques take much time and financial costs, which limit their use.

  17. Genes in mammalian reproduction

    SciTech Connect

    Gwatkin, R.B.L.

    1996-11-01

    This is an informative book which deals mainly with genomic imprinting, the role of steroid hormones in development, the expression of a variety of genes during development and the link to hereditary diseases. It is an up-to-date review in a field that is quickly changing and provides valuable basic information and current research trends.

  18. ESHRE PGD consortium best practice guidelines for organization of a PGD centre for PGD/preimplantation genetic screening.

    PubMed

    Harton, G; Braude, P; Lashwood, A; Schmutzler, A; Traeger-Synodinos, J; Wilton, L; Harper, J C

    2011-01-01

    In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. Subsequent years have seen the introduction of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written/formulated, the Consortium believed it was timely to update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four documents, including one relating to organization of the PGD centre and three relating to the methods used: DNA amplification, fluorescence in situ hybridization and biopsy/embryology. Here, we have updated the sections on organization of the PGD centre. One area that has continued to expand is Transport PGD, in which patients are treated at one IVF centre, whereas their gametes/embryos are tested elsewhere, at an independent PGD centre. Transport PGD/preimplantation genetic screening (PGS) has a unique set of challenges with respect to the nature of the sample and the rapid turn-around time required. PGS is currently controversial. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Current evidence suggests that PGS at cleavage stages is ineffective, but whether PGS at the blastocyst stage or on polar bodies might show improved delivery rates is still unclear. Thus, in this revision, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible.

  19. Optogenetics for gene expression in mammalian cells.

    PubMed

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  20. Susceptibility of porcine preimplantation embryos to viruses associated with reproductive failure.

    PubMed

    Zhao, Haijing; Zhao, Guangyuan; Wang, Wenjun

    2016-10-15

    In the modern biological area, the applications of pig as a laboratory model have extensive prospects, such as gene transfer, IVF, SCNT, and xenotransplantation. However, the risk of pathogen transmission by porcine embryos is always a topic to be investigated, especially the viruses related to reproductive failure, for instance, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, and porcine circovirus type 2. It should be mentioned that the zona pellucida (ZP) of porcine embryos can be a barrier against the viruses, but certain pathogens may stick to or even pass through the ZP. With intact, free, and damaged ZP, porcine preimplantation embryos are susceptible to these viruses in varying degrees, which may be associated with the virus-specific receptor on embryonic cell membrane. These topics are discussed in the present review. PMID:27423729

  1. Designer babies on tap? Medical students' attitudes to pre-implantation genetic screening.

    PubMed

    Meisenberg, Gerhard

    2009-03-01

    This paper describes two studies about the determinants of attitudes to pre-implantation genetic screening in a multicultural sample of medical students from the United States. Sample sizes were 292 in study 1 and 1464 in study 2. Attitudes were of an undifferentiated nature, but respondents did make a major distinction between use for disease prevention and use for enhancement. No strong distinctions were made between embryo selection and germ line gene manipulations, and between somatic gene therapy and germ line gene manipulations. Religiosity was negatively associated with acceptance of "designer baby" technology for Christians and Muslims but not Hindus. However, the strongest and most consistent influence was an apparently moralistic stance against active and aggressive interference with natural processes in general. Trust in individuals and institutions was unrelated to acceptance of the technology, indicating that fear of abuse by irresponsible individuals and corporations is not an important determinant of opposition.

  2. Preimplantation genetic diagnosis (PGD) according to medical ethics and medical law

    PubMed Central

    Lutz, Emine Elif Vatanoğlu

    2012-01-01

    Assisted reproductive techniques not only nourish great and sometimes illusive hopes of couples who yearn for babies, but also spark new debates by reversing opinions, beliefs and values. Applications made to infertility clinics are increasing due to the influences such as broadcasts made by the media concerning assisted reproductive techniques and other infertility treatments, increase in the knowledge that people have about these problems, late marriages and postponement of childbearing age owing to sociological changes. Pre-implantation genetic diagnosis (PGD) is a technique applied to couples who are known to carry genetic diseases or who have children with genetic diseases. This technique is conducted by doctors in Turkey for its important contribution to decreasing the risk of genetic diseases and in order to raise healthy generations. In this paper, the general ethical debates and the legal situation in Turkey will be discussed. PMID:24627675

  3. Case report: first successful application of preimplantation genetic diagnosis for hereditary angiooedema.

    PubMed

    Bautista-Llácer, Rosa; Alberola, Trinitat M; Vendrell, Xavier; Fernández, Esther; Pérez-Alonso, Manuel

    2010-11-01

    Hereditary angiooedema is an autosomal dominant disease caused by mutations in the SERPING1 gene. It is characterized by oedemas in different parts of the body, being particularly dangerous when swelling involves the upper airway. Preimplantation genetic diagnosis (PGD) was performed in a couple where the woman carries a deletion of 2.9Kb that includes exon 4 of the SERPING1 gene. Four polymorphic short tandem repeat markers were tested in order to establish the disease-bearing haplotype and three of them were fully informative. Amplification efficiency at the preclinical work up ranged from 71% to 100% for each locus and allele drop out rates were between 0% and 20% for the polymorphic markers. The couple underwent PGD using fluorescent multiplex heminested polymerase chain reaction. Six embryos were biopsied and five of them were diagnosed as healthy. Two embryos were transferred and a singleton pregnancy was achieved, resulting in the birth of a healthy boy.

  4. Preimplantation genetic diagnosis of a large pericentric inversion of chromosome 5.

    PubMed

    Iwarsson, E; Ahrlund-Richter, L; Inzunza, J; Rosenlund, B; Fridström, M; Hillensjö, T; Sjöblom, P; Nordenskjöld, M; Blennow, E

    1998-07-01

    We report the first established pregnancy using preimplantation genetic diagnosis in order to avoid chromosomal imbalance in the progeny of a woman carrying a large inversion of chromosome 5. This is also the first time where it has been possible to study the distribution of balanced and unbalanced gametes in a female inversion carrier. In total, 23 embryos were biopsied in two separate treatments and analysed by fluorescent in-situ hybridization. Of these, 10 were unbalanced, nine were balanced and for four the analysis was inconclusive. The diagnostic procedure was performed within 3.5 h. This allowed the biopsied embryos to be transferred the same day as the biopsy was taken (day 3). Two embryos were transferred each time, and in the second treatment a twin pregnancy with two chromosomally balanced fetuses was established. Healthy twins were delivered at 34 weeks of gestation.

  5. Designer babies on tap? Medical students' attitudes to pre-implantation genetic screening.

    PubMed

    Meisenberg, Gerhard

    2009-03-01

    This paper describes two studies about the determinants of attitudes to pre-implantation genetic screening in a multicultural sample of medical students from the United States. Sample sizes were 292 in study 1 and 1464 in study 2. Attitudes were of an undifferentiated nature, but respondents did make a major distinction between use for disease prevention and use for enhancement. No strong distinctions were made between embryo selection and germ line gene manipulations, and between somatic gene therapy and germ line gene manipulations. Religiosity was negatively associated with acceptance of "designer baby" technology for Christians and Muslims but not Hindus. However, the strongest and most consistent influence was an apparently moralistic stance against active and aggressive interference with natural processes in general. Trust in individuals and institutions was unrelated to acceptance of the technology, indicating that fear of abuse by irresponsible individuals and corporations is not an important determinant of opposition. PMID:19579681

  6. Chemosignals, Hormones and Mammalian Reproduction

    PubMed Central

    Petrulis, Aras

    2013-01-01

    Many mammalian species use chemosignals to coordinate reproduction by altering the physiology and behavior of both sexes. Chemosignals prime reproductive physiology so that individuals become sexually mature and active at times when mating is most probable and suppress it when it is not. Once in reproductive condition, odors produced and deposited by both males and females are used to find and select individuals for mating. The production, dissemination and appropriate responses to these cues are modulated heavily by organizational and activational effects of gonadal sex steroids and thereby intrinsically link chemical communication to the broader reproductive context. Many compounds have been identified as “pheromones” but very few have met the expectations of that term: a unitary, species-typical substance that is both necessary and sufficient for an experience-independent behavioral or physiological response. In contrast, most responses to chemosignals are dependent or heavily modulated by experience, either in adulthood or during development. Mechanistically, chemosignals are perceived by both main and accessory (vomeronasal) olfactory systems with the importance of each system tied strongly to the nature of the stimulus rather than to the response. In the central nervous system, the vast majority of responses to chemosignals are mediated by cortical and medial amygdala connections with hypothalamic and other forebrain structures. Despite the importance of chemosignals in mammals, many details of chemical communication differ even among closely related species and defy clear categorization. Although generating much research and public interest, strong evidence for the existence of a robust chemical communication among humans is lacking. PMID:23545474

  7. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    PubMed

    Liang, Lifeng; Wang, Cassie T; Sun, Xiaofang; Liu, Lian; Li, Man; Witz, Craig; Williams, Daniel; Griffith, Jason; Skorupski, Josh; Haddad, Ghassan; Gill, Jimmy; Wang, Wei-Hua

    2013-01-01

    A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.

  8. Mammalian-specific genomic functions: Newly acquired traits generated by genomic imprinting and LTR retrotransposon-derived genes in mammals

    PubMed Central

    KANEKO-ISHINO, Tomoko; ISHINO, Fumitoshi

    2015-01-01

    Mammals, including human beings, have evolved a unique viviparous reproductive system and a highly developed central nervous system. How did these unique characteristics emerge in mammalian evolution, and what kinds of changes did occur in the mammalian genomes as evolution proceeded? A key conceptual term in approaching these issues is “mammalian-specific genomic functions”, a concept covering both mammalian-specific epigenetics and genetics. Genomic imprinting and LTR retrotransposon-derived genes are reviewed as the representative, mammalian-specific genomic functions that are essential not only for the current mammalian developmental system, but also mammalian evolution itself. First, the essential roles of genomic imprinting in mammalian development, especially related to viviparous reproduction via placental function, as well as the emergence of genomic imprinting in mammalian evolution, are discussed. Second, we introduce the novel concept of “mammalian-specific traits generated by mammalian-specific genes from LTR retrotransposons”, based on the finding that LTR retrotransposons served as a critical driving force in the mammalian evolution via generating mammalian-specific genes. PMID:26666304

  9. Mammalian developmental genetics in the twentieth century.

    PubMed

    Artzt, Karen

    2012-12-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas.

  10. Mammalian Developmental Genetics in the Twentieth Century

    PubMed Central

    Artzt, Karen

    2012-01-01

    This Perspectives is a review of the breathtaking history of mammalian genetics in the past century and, in particular, of the ways in which genetic thinking has illuminated aspects of mouse development. To illustrate the power of that thinking, selected hypothesis-driven experiments and technical advances are discussed. Also included in this account are the beginnings of mouse genetics at the Bussey Institute, Columbia University, and The Jackson Laboratory and a retrospective discussion of one of the classic problems in developmental genetics, the T/t complex and its genetic enigmas. PMID:23212897

  11. Potential Mammalian Filovirus Reservoirs

    PubMed Central

    Carroll, Darin S.; Mills, James N.; Johnson, Karl M.

    2004-01-01

    Ebola and Marburg viruses are maintained in unknown reservoir species; spillover into human populations results in occasional human cases or epidemics. We attempted to narrow the list of possibilities regarding the identity of those reservoir species. We made a series of explicit assumptions about the reservoir: it is a mammal; it supports persistent, largely asymptomatic filovirus infections; its range subsumes that of its associated filovirus; it has coevolved with the virus; it is of small body size; and it is not a species that is commensal with humans. Under these assumptions, we developed priority lists of mammal clades that coincide distributionally with filovirus outbreak distributions and compared these lists with those mammal taxa that have been tested for filovirus infection in previous epidemiologic studies. Studying the remainder of these taxa may be a fruitful avenue for pursuing the identity of natural reservoirs of filoviruses. PMID:15663841

  12. The cell biology of neural stem and progenitor cells and its significance for their proliferation versus differentiation during mammalian brain development.

    PubMed

    Farkas, Lilla M; Huttner, Wieland B

    2008-12-01

    The switch of neural stem and progenitor cells from proliferation to differentiation during development is a crucial determinant of brain size. This switch is intimately linked to the architecture of the two principal classes of neural stem and progenitor cells, the apical (neuroepithelial, radial glial) and basal (intermediate) progenitors, which in turn is crucial for their symmetric versus asymmetric divisions. Focusing on the developing rodent neocortex, we discuss here recent advances in understanding the cell biology of apical and basal progenitors, place key regulatory molecules into subcellular context, and highlight their roles in the control of proliferation versus differentiation. PMID:18930817

  13. Modelling mammalian cellular quiescence

    PubMed Central

    Yao, Guang

    2014-01-01

    Cellular quiescence is a reversible non-proliferating state. The reactivation of ‘sleep-like’ quiescent cells (e.g. fibroblasts, lymphocytes and stem cells) into proliferation is crucial for tissue repair and regeneration and a key to the growth, development and health of higher multicellular organisms, such as mammals. Quiescence has been a primarily phenotypic description (i.e. non-permanent cell cycle arrest) and poorly studied. However, contrary to the earlier thinking that quiescence is simply a passive and dormant state lacking proliferating activities, recent studies have revealed that cellular quiescence is actively maintained in the cell and that it corresponds to a collection of heterogeneous states. Recent modelling and experimental work have suggested that an Rb-E2F bistable switch plays a pivotal role in controlling the quiescence–proliferation balance and the heterogeneous quiescent states. Other quiescence regulatory activities may crosstalk with and impinge upon the Rb-E2F bistable switch, forming a gene network that controls the cells’ quiescent states and their dynamic transitions to proliferation in response to noisy environmental signals. Elucidating the dynamic control mechanisms underlying quiescence may lead to novel therapeutic strategies that re-establish normal quiescent states, in a variety of hyper- and hypo-proliferative diseases, including cancer and ageing. PMID:24904737

  14. Amino acids in the cultivation of mammalian cells.

    PubMed

    Salazar, Andrew; Keusgen, Michael; von Hagen, Jörg

    2016-05-01

    Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure. PMID:26832172

  15. Amino acids in the cultivation of mammalian cells.

    PubMed

    Salazar, Andrew; Keusgen, Michael; von Hagen, Jörg

    2016-05-01

    Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.

  16. The mammalian ovary from genesis to revelation.

    PubMed

    Edson, Mark A; Nagaraja, Ankur K; Matzuk, Martin M

    2009-10-01

    Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago.

  17. The Mammalian Ovary from Genesis to Revelation

    PubMed Central

    Edson, Mark A.; Nagaraja, Ankur K.; Matzuk, Martin M.

    2009-01-01

    Two major functions of the mammalian ovary are the production of germ cells (oocytes), which allow continuation of the species, and the generation of bioactive molecules, primarily steroids (mainly estrogens and progestins) and peptide growth factors, which are critical for ovarian function, regulation of the hypothalamic-pituitary-ovarian axis, and development of secondary sex characteristics. The female germline is created during embryogenesis when the precursors of primordial germ cells differentiate from somatic lineages of the embryo and take a unique route to reach the urogenital ridge. This undifferentiated gonad will differentiate along a female pathway, and the newly formed oocytes will proliferate and subsequently enter meiosis. At this point, the oocyte has two alternative fates: die, a common destiny of millions of oocytes, or be fertilized, a fate of at most approximately 100 oocytes, depending on the species. At every step from germline development and ovary formation to oogenesis and ovarian development and differentiation, there are coordinated interactions of hundreds of proteins and small RNAs. These studies have helped reproductive biologists to understand not only the normal functioning of the ovary but also the pathophysiology and genetics of diseases such as infertility and ovarian cancer. Over the last two decades, parallel progress has been made in the assisted reproductive technology clinic including better hormonal preparations, prenatal genetic testing, and optimal oocyte and embryo analysis and cryopreservation. Clearly, we have learned much about the mammalian ovary and manipulating its most important cargo, the oocyte, since the birth of Louise Brown over 30 yr ago. PMID:19776209

  18. Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution.

    PubMed

    Block, J; Chase, C C; Hansen, P J

    2002-09-01

    Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than

  19. Mechanisms of mammalian iron homeostasis.

    PubMed

    Pantopoulos, Kostas; Porwal, Suheel Kumar; Tartakoff, Alan; Devireddy, L

    2012-07-24

    Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in the acquisition or distribution of the metal causes anemia, whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanisms to regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways as well as in mechanisms underlying intracellular iron trafficking, an important but less studied area of mammalian iron homeostasis.

  20. DAX1/NR0B1 was expressed during mammalian gonadal development and gametogenesis before it was recruited to the eutherian X chromosome.

    PubMed

    Stickels, Robert; Clark, Kevin; Heider, Thomas N; Mattiske, Deidre M; Renfree, Marilyn B; Pask, Andrew J

    2015-01-01

    The nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene is an orphan nuclear receptor that is X-linked in eutherian mammals and plays a critical role in the establishment and function of the hypothalamic-pituitary-adrenal-gonadal axis. Duplication or overexpression of NR0B1 in eutherian males causes male to female sex reversal, and mutation and deletions of NR0B1 cause testicular defects. Thus, gene dosage is critical for the function of NR0B1 in normal gonadogenesis. However, NR0B1 is autosomal in all noneutherian vertebrates, including marsupials and monotreme mammals, and two active copies of the gene are compatible with both male and female gonadal development. In the current study, we examined the evolution and expression of autosomal NR0B1 during gonadal development in a marsupial (the tammar wallaby) as compared to the role of its X-linked orthologues in a eutherian (the mouse). We show that NR0B1 underwent rapid evolutionary change when it relocated from its autosomal position in the nonmammalian vertebrates, monotremes, and marsupials to an X-linked location in eutherian mammals. Despite the acquisition of a novel genomic location and a unique N-terminal domain, NR0B1 protein distribution was remarkably similar between mice and marsupials both throughout gonadal development and during gamete formation. A conserved accumulation of NR0B1 protein was observed in developing oocytes, where its function appears to be critical in the early embryo, prior to zygotic genome activation. Together these findings suggest that NR0B1 had a conserved role in gonadogenesis that existed long before it moved to the X chromosome and despite undergoing significant evolutionary change.

  1. DAX1/NR0B1 was expressed during mammalian gonadal development and gametogenesis before it was recruited to the eutherian X chromosome.

    PubMed

    Stickels, Robert; Clark, Kevin; Heider, Thomas N; Mattiske, Deidre M; Renfree, Marilyn B; Pask, Andrew J

    2015-01-01

    The nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene is an orphan nuclear receptor that is X-linked in eutherian mammals and plays a critical role in the establishment and function of the hypothalamic-pituitary-adrenal-gonadal axis. Duplication or overexpression of NR0B1 in eutherian males causes male to female sex reversal, and mutation and deletions of NR0B1 cause testicular defects. Thus, gene dosage is critical for the function of NR0B1 in normal gonadogenesis. However, NR0B1 is autosomal in all noneutherian vertebrates, including marsupials and monotreme mammals, and two active copies of the gene are compatible with both male and female gonadal development. In the current study, we examined the evolution and expression of autosomal NR0B1 during gonadal development in a marsupial (the tammar wallaby) as compared to the role of its X-linked orthologues in a eutherian (the mouse). We show that NR0B1 underwent rapid evolutionary change when it relocated from its autosomal position in the nonmammalian vertebrates, monotremes, and marsupials to an X-linked location in eutherian mammals. Despite the acquisition of a novel genomic location and a unique N-terminal domain, NR0B1 protein distribution was remarkably similar between mice and marsupials both throughout gonadal development and during gamete formation. A conserved accumulation of NR0B1 protein was observed in developing oocytes, where its function appears to be critical in the early embryo, prior to zygotic genome activation. Together these findings suggest that NR0B1 had a conserved role in gonadogenesis that existed long before it moved to the X chromosome and despite undergoing significant evolutionary change. PMID:25395677

  2. Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.

    PubMed

    Pereyra-Bonnet, Federico; Bevacqua, Romina; La Rosa, Isabel; Sipowicz, Pablo; Radrizzani, Martin; Fernandez-Martin, Rafael; Salamone, Daniel

    2011-12-01

    The import of exogenous DNA (eDNA) from the cytoplasm to the nucleus represents a key intracellular obstacle for efficient gene delivery in mammalian cells. In this study, cumulus cells or oolemma vesicles previously incubated with eDNA, and naked eDNA were injected into the cytoplasm of MII oocytes to evaluate their efficiency for eDNA expressing bovine embryo production. Our study evaluated the potential of short time co-incubation (5 min) of eDNA with; (1) cumulus cells, to be used as donor cells for SCNT and (2) oolemma vesicles (vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group consisting of the injection of eDNA alone (plasmid) followed by parthenogenic activation. Two different pCX-EGFP plasmid concentrations (50 and 500 ng/μl) were employed. The results showed that embryos produced by SCNT and by vesicle injection assisted by chemical activation were able to express the eDNA in higher rates than embryos injected with plasmid alone. The lower plasmid concentration allowed the highest development rates in all groups. Using confocal microscopy, we analyzed the interaction of FITC- labeled eDNA with cumulus cells and vesicles as well as oocytes injected with labeled plasmid alone. Our images demonstrated that eDNA interacted with cumulus cells and vesicles, resulting an increase in its expression efficiency. In contrast, oocytes injected with DNA alone did not show signs of transgene accumulation, and their eDNA expression rates were lower. In a further experiment, we evaluated if transgene-expressing embryos could be produced by means of vesicle injection followed by IVF. The lower plasmid concentration (50 ng/μl) injected after IVF, produced the best results. Preliminary FISH analysis indicated detectable integration events in 1/5 of SCNT blastocysts treated. Our studies demonstrate for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT

  3. Olfactory sensitivity in mammalian species.

    PubMed

    Wackermannová, M; Pinc, L; Jebavý, L

    2016-07-18

    Olfaction enables most mammalian species to detect and discriminate vast numbers of chemical structures called odorants and pheromones. The perception of such chemical compounds is mediated via two major olfactory systems, the main olfactory system and the vomeronasal system, as well as minor systems, such as the septal organ and the Grueneberg ganglion. Distinct differences exist not only among species but also among individuals in terms of their olfactory sensitivity; however, little is known about the mechanisms that determine these differences. In research on the olfactory sensitivity of mammals, scientists thus depend in most cases on behavioral testing. In this article, we reviewed scientific studies performed on various mammalian species using different methodologies and target chemical substances. Human and non-human primates as well as rodents and dogs are the most frequently studied species. Olfactory threshold studies on other species do not exist with the exception of domestic pigs. Olfactory testing performed on seals, elephants, and bats focused more on discriminative abilities than on sensitivity. An overview of olfactory sensitivity studies as well as olfactory detection ability in most studied mammalian species is presented here, focusing on comparable olfactory detection thresholds. The basics of olfactory perception and olfactory sensitivity factors are also described. PMID:27070753

  4. Endocrinology of the mammalian fetal testis.

    PubMed

    O'Shaughnessy, Peter J; Fowler, Paul A

    2011-01-01

    The testes are essential endocrine regulators of fetal masculinization and male development and are, themselves, subject to hormonal regulation during gestation. This review focuses, primarily, on this latter control of testicular function. Data available suggest that, in most mammalian species, the testis goes through a period of independent function before the fetal hypothalamic-pituitary-gonadal axis develops at around 50% of gestation. This pituitary-independent phase coincides with the most critical period of fetal masculinization. Thereafter, the fetal testes appear to become pituitary hormone-dependent, concurrent with declining Leydig cell function, but increasing Sertoli cell numbers. The two orders of mammals most commonly used for these types of studies (rodents and primates) appear to represent special cases within this general hypothesis. In terms of testicular function, rodents are born 'early' before the pituitary-dependent phase of fetal development, while the primate testis is dependent upon placental gonadotropin released during the pituitary-independent phase of development.

  5. Preimplantation genetic diagnosis of chromosome balance in embryos from a patient with a balanced reciprocal translocation.

    PubMed

    Pierce, K E; Fitzgerald, L M; Seibel, M M; Zilberstein, M

    1998-02-01

    Duplications or deletions are present in a high percentage of the gametes produced by individuals carrying balanced translocations. Preimplantation genetic diagnosis was used to examine chromosome balance in embryos from a patient having a reciprocal translocation within the short arms of chromosomes 5 and 8 (46,XX,t(5;8)(p13;p23)). This woman has two sisters with the translocation unbalanced, resulting in a partial trisomy for chromosome 5 and partial monosomy for chromosome 8 (46,XX,-8, +der(8)t(5;8)(p13;p23)) with associated mental retardation and physical abnormalities. The patient and her husband desired to have children without the abnormal chromosome balance and wished to reduce the likelihood of spontaneous abortion or need for therapeutic abortion. Fluorescence in-situ hybridization (FISH) probes for the alpha-satellite region of chromosome 8 and for a region on the short arm of chromosome 5 (5p15.2) were tested initially on lymphocytes from the patient and her sisters. The hybridization signal for chromosome 5 was detected in the expected two copies for the patient and three copies for the sisters in 87% of the cells. Two hybridization signals for chromosome 8 were detected in 96% of the cells from all individuals. Additional probe testing was done using blastomeres from polyspermic embryos. The couple then proceeded with a stimulated in-vitro fertilization (IVF) cycle and biopsies were done on 13 embryos at the 7-10-cell stage using a method of zona drilling and fluid displacement. Diagnosis was possible on at least one blastomere for nine embryos. Three embryos had nuclei with three hybridization signals for chromosome 5, three had fewer than two signals for one or both chromosomes, one was mosaic, and two had two signals for each chromosome. The latter were transferred to the patient, but pregnancy was not achieved. The results demonstrate that preimplantation genetic diagnosis for patients with reciprocal translocations can be used to identify embryos

  6. A simplified approach for oocyte enucleation in mammalian cloning.

    PubMed

    Iuso, Domenico; Czernik, Marta; Zacchini, Federica; Ptak, Grazyna; Loi, Pasqualino

    2013-12-01

    Despite its success in almost all farm and laboratory animals, somatic cell nuclear transfer (SCNT) is still a low-efficiency technique. In this investigation, we determined the impact of each enucleation step on oocyte viability (assessed by parthenogenetic activation): Hoechst (HO) staining, cytochalasin B, ultraviolet (UV) exposure, and demecolcine. Our data showed that of all the factors analyzed, UV exposure impaired oocyte development (cleavage, 59% for untreated oocytes vs. 8% UV exposed; blastocyst stage, 32% untreated vs. 0% UV exposed). A minor toxicity was detected following demecolcine treatment (cleavage, 62%; blastocyst stage, 13%). Next, we compared HO/UV (canonical) and demecolcine-assisted enucleation (DAE), with a straight removal of metaphase chromosomes without any chemical or physical aid (straight enucleation). DAE improved the preimplantation development of sheep cloned embryos compared to HO/UV enucleation (cleavage, 38% vs. 19%; blastocysts, 17% vs. 4%), yet straight enucleation resulted in the highest cleavage and blastocysts rates (61% and 30%, respectively). We concluded that: (1) UV exposure harms sheep oocyte and embryo development; (2) DAE may represent an alternative approach, especially for unskilled operators; and (3) straight enucleation remains, in our estimation, the most reliable and least harmful protocol for SCNT.

  7. Dynamic regulation of DNA methyltransferases in human oocytes and preimplantation embryos after assisted reproductive technologies.

    PubMed

    Petrussa, Laetitia; Van de Velde, Hilde; De Rycke, Martine

    2014-09-01

    DNA methylation is a key epigenetic modification which is essential for normal embryonic development. Major epigenetic reprogramming takes place during gametogenesis and in the early embryo; the complex DNA methylation patterns are established and maintained by DNA methyltransferases (DNMTs). However, the influence of assisted reproductive technologies (ART) on DNA methylation reprogramming enzymes has predominantly been studied in mice and less so in human oocytes and embryos. The expression and localization patterns of the four known DNMTs were analysed in human oocytes and IVF/ICSI embryos by immunocytochemistry and compared between a reference group of good quality fresh embryos and groups of abnormally developing embryos or embryo groups after cryopreservation. In humans, DNMT1o rather than DNMT1s seems to be the key player for maintaining methylation in early embryos. DNMT3b, rather than DNMT3a and DNMT3L, appears to ensure global DNA remethylation in the blastocysts before implantation. DNMT3L, an important regulator of maternal imprint methylation in mouse, was not detected in human oocytes (GV, MI and MII stage). Our study confirms the existence of species differences for mammalian DNA methylation enzymes. In poor quality fresh embryos, the switch towards nuclear DNMT3b expression was delayed and nuclear DNMT1, DNMT1s and DNMT3b expression was less common. Compared with the reference embryos, a smaller number of cryopreserved embryos showed nuclear DNMT1, while a delayed switch to nuclear DNMT3b and an extended DNMT1s temporal expression pattern were also observed. The spatial and temporal expression patterns of DNMTs seem to be disturbed in abnormally developing embryos and in embryos that have been cryopreserved. Further research must be performed in order to understand whether the potentially disturbed embryonic DNMT expression after cryopreservation has any long-term developmental consequences. PMID:24994815

  8. Histone acetyl transferase 1 is essential for mammalian development, genome stability, and the processing of newly synthesized histones H3 and H4.

    PubMed

    Nagarajan, Prabakaran; Ge, Zhongqi; Sirbu, Bianca; Doughty, Cheryl; Agudelo Garcia, Paula A; Schlederer, Michaela; Annunziato, Anthony T; Cortez, David; Kenner, Lukas; Parthun, Mark R

    2013-06-01

    Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat1(-/-) neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1(-/-) mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1(-/-) MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly.

  9. Cis-Suppression to Arrest Protein Aggregation in Mammalian Cells

    PubMed Central

    Gregoire, Simpson; Zhang, Shaojie; Costanzo, Joseph; Wilson, Kelly; Fernandez, Erik J.; Kwon, Inchan

    2015-01-01

    Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1A4V) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1A4V as a key residue responsible for SOD1A4V conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1A4V variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1A4V variants were verified using cell fractionation and in vitro stability assays. PMID:24114411

  10. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  11. An Adaptive Threshold in Mammalian Neocortical Evolution

    PubMed Central

    Kalinka, Alex T.; Tomancak, Pavel; Huttner, Wieland B.

    2014-01-01

    Expansion of the neocortex is a hallmark of human evolution. However, determining which adaptive mechanisms facilitated its expansion remains an open question. Here we show, using the gyrencephaly index (GI) and other physiological and life-history data for 102 mammalian species, that gyrencephaly is an ancestral mammalian trait. We find that variation in GI does not evolve linearly across species, but that mammals constitute two principal groups above and below a GI threshold value of 1.5, approximately equal to 109 neurons, which may be characterized by distinct constellations of physiological and life-history traits. By integrating data on neurogenic period, neuroepithelial founder pool size, cell-cycle length, progenitor-type abundances, and cortical neuron number into discrete mathematical models, we identify symmetric proliferative divisions of basal progenitors in the subventricular zone of the developing neocortex as evolutionarily necessary for generating a 14-fold increase in daily prenatal neuron production, traversal of the GI threshold, and thus establishment of two principal groups. We conclude that, despite considerable neuroanatomical differences, changes in the length of the neurogenic period alone, rather than any novel neurogenic progenitor lineage, are sufficient to explain differences in neuron number and neocortical size between species within the same principal group. PMID:25405475

  12. Calcium Signaling in Mammalian Eggs at Fertilization.

    PubMed

    Shirakawa, Hideki; Kikuchi, Takashi; Ito, Masahiko

    2016-01-01

    The innovation and development of live-cell fluorescence imaging methods have revealed the dynamic aspects of intracellular Ca2+ in a wide variety of cells. The fertilized egg, the very first cell to be a new individual, has long been under extensive investigations utilizing Ca2+ imaging since its early days, and spatiotemporal Ca2+ dynamics and underlying mechanisms of Ca2+ mobilization, as well as physiological roles of Ca2+ at fertilization, have become more or less evident in various animal species. In this article, we illustrate characteristic patterns of Ca2+ dynamics in mammalian gametes and molecular basis for Ca2+ release from intracellular stores leading to the elevation in cytoplasmic Ca2+ concentration, and describe the identity and properties of sperm-borne egg-activating factor in relation to the induction of Ca2+ waves and Ca2+ oscillations, referring to its potential use in artificial egg activation as infertility treatment. In addition, a possible Ca2+ influx-driven mechanism for slow and long-lasting Ca2+ oscillations characteristic of mammalian eggs is proposed, based on the recent experimental findings and mathematical modeling. Cumulative knowledge about the roles of Ca2+ in the egg activation leading to early embryogenesis is summarized, to emphasize the diversity of functions that Ca2+ can perform in a single type of cell.

  13. The cellular code for mammalian thermosensation.

    PubMed

    Pogorzala, Leah A; Mishra, Santosh K; Hoon, Mark A

    2013-03-27

    Mammalian somatosenory neurons respond to thermal stimuli and allow animals to reliably discriminate hot from cold and to select their preferred environments. Previously, we generated mice that are completely insensitive to temperatures from noxious cold to painful heat (-5 to 55°C) by ablating several different classes of nociceptor early in development. In the present study, we have adopted a selective ablation strategy in adult mice to study this phenotype and have demonstrated that separate populations of molecularly defined neurons respond to hot and cold. TRPV1-expressing neurons are responsible for all behavioral responses to temperatures between 40 and 50°C, whereas TRPM8 neurons are required for cold aversion. We also show that more extreme cold and heat activate additional populations of nociceptors, including cells expressing Mrgprd. Therefore, although eliminating Mrgprd neurons alone does not affect behavioral responses to temperature, when combined with ablation of TRPV1 or TRPM8 cells, it significantly decreases responses to extreme heat and cold, respectively. Ablation of TRPM8 neurons distorts responses to preferred temperatures, suggesting that the pleasant thermal sensation of warmth may in fact just reflect reduced aversive input from TRPM8 and TRPV1 neurons. As predicted by this hypothesis, mice lacking both classes of thermosensor exhibited neither aversive nor attractive responses to temperatures between 10 and 50°C. Our results provide a simple cellular basis for mammalian thermosensation whereby two molecularly defined classes of sensory neurons detect and encode both attractive and aversive cues. PMID:23536068

  14. Embryo selection with preimplantation chromosomal screening in patients with recurrent pregnancy loss.

    PubMed

    Shahine, Lora K; Lathi, Ruth B

    2014-03-01

    Recurrent pregnancy loss (RPL) is a multifactorial disorder which is often challenging for both patients and providers. Guidelines for the evaluation and treatment of patients with RPL include screening for uterine abnormalities, parental chromosomes, and antiphospholipid antibodies, but approximately half of RPL patients remain unexplained. The current recommendation for patients with unexplained RPL is expectant management which offers most patients a 60 to 80% success rate over time. Genetic imbalances in the embryo, including inherited unbalanced translocations and de novo aneuploidy, are frequent causes of miscarriage. Preimplantation genetic screening (PGS) has been proposed as an effective method for selecting viable embryos for transfer that may result lower risk of miscarriage for patients with unexplained RPL and carriers of balanced translocations. The current evidence examining the use of in vitro fertilization with PGS in patients with RPL reveals variable results, due to differences in technologies used and variable patient populations. Newer approaches, which include blastocyst biopsy and the ability to screen for all 24 chromosomes, show the most promise in reducing miscarriage rates. Studies that identify which patients are most likely to benefit from PGS and include live birth rates per initiated cycles are needed before universally recommending this treatment to couples with RPL.

  15. Dehydroepiandrosterone (DHEA) reduces embryo aneuploidy: direct evidence from preimplantation genetic screening (PGS)

    PubMed Central

    2010-01-01

    Background Dehydroepiandrosterone (DHEA) has been reported to improve pregnancy chances in women with diminished ovarian reserve (DOR), and to reduce miscarriage rates by 50-80%. Such an effect is mathematically inconceivable without beneficial effects on embryo ploidy. This study, therefore, assesses effects of DHEA on embryo aneuploidy. Methods In a 1:2, matched case control study 22 consecutive women with DOR, supplemented with DHEA, underwent preimplantation genetic screening (PGS) of embryos during in vitro fertilization (IVF) cycles. Each was matched by patient age and time period of IVF with two control IVF cycles without DHEA supplementation (n = 44). PGS was performed for chromosomes X, Y, 13, 16, 18, 21 and 22, and involved determination of numbers and percentages of aneuploid embryos. Results DHEA supplementation to a significant degree reduced number (P = 0.029) and percentages (P < 0.001) of aneuploid embryos, adjusted for relevant covariates. Short term supplementation (4-12 weeks) resulted in greatest reduction in aneuploidy (21.6%, 95% CI -2.871-46.031). Discussion Beneficial DHEA effects on DOR patients, at least partially, are the likely consequence of lower embryo aneuploidy. DHEA supplementation also deserves investigation in older fertile women, attempting to conceive, where a similar effect, potentially, could positively affect public health. PMID:21067609

  16. Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome.

    PubMed

    Lee, H-S; Kim, M J; Lim, C K; Cho, J W; Song, I O; Kang, I S

    2011-01-01

    Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders. PMID:22095609

  17. Preimplantation genetic diagnosis for Pelizaeus-Merzbacher disease with testing for age-related aneuploidies.

    PubMed

    Verlinsky, Y; Rechitsky, S; Laziuk, K; Librach, C; Genovese, R; Kuliev, A

    2006-01-01

    Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive demyelinating disorder of the central nervous system, caused by mutations of the proteolipid protein 1 gene (PLP1 gene). As no specific therapy is available for PMD, preimplantation genetic diagnosis (PGD) may be a useful option for couples carrying this mutation. PGD was performed for a couple who had had one child with the L86P mutation in exon 3 of the PLP1 gene. Because of advanced maternal age, PGD for this single-gene disorder was performed together with testing for chromosomal abnormalities. Polar bodies and blastomeres were tested for the presence of maternal mutation and closely linked markers DXS8020 and PLP5' (CA)n. The same blastomeres were also tested for the copy number of chromosomes 13, 16, 18, 21, 22, X and Y, and five chromosomally abnormal embryos were identified. A total of three embryos predicted to be unaffected and free of chromosomal disorder were transferred back to the patient, resulting in a twin pregnancy and the birth of two healthy female infants confirmed to be free of PMD, representing the first PGD for PMD combined with aneuploidy testing.

  18. First successful application of preimplantation genetic diagnosis and haplotyping for congenital hyperinsulinism.

    PubMed

    Qubbaj, Wafa; Al-Swaid, Abdulrahman; Al-Hassan, Saad; Awartani, Khalid; Deek, Hesham; Coskun, Serdar

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