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Sample records for preimplantation mammalian development

  1. Apoptotic processes during mammalian preimplantation development.

    PubMed

    Fabian, Dusan; Koppel, Juraj; Maddox-Hyttel, Poul

    2005-07-15

    The paper provides a review of the current state of knowledge on apoptosis during normal preimplantation development based on the literature and on the authors' own findings. Information is focused on the occurrence and the characteristics of spontaneous apoptotic processes. Reports concerning the chronology and the incidence of programmed cell death in mouse, cow, pig and human embryos in early preimplantation stages up to the blastocyst stage are summarized. In addition, specific attributes of the apoptotic process in mammalian preimplantation development are provided, including the description of both morphological and biochemical features of cell death. PMID:15955348

  2. 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

  3. 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

  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. PMID:25498236

  5. GENE EXPRESSION IN PRE-IMPLANTATION MAMMALIAN EMBRYOS

    EPA Science Inventory

    The pre-implantation mammalian embryo is initially under the control of maternal informational macromolecules that are accumulated during oogenesis. ubsequently, the genetic program of development becomes dependent upon new transcription derived from activation of the embryonic g...

  6. Epigenetic Dynamics During Preimplantation Development

    PubMed Central

    Marcho, Chelsea; Cui, Wei; Mager, Jesse

    2015-01-01

    Successful mammalian development requires descendants of single-cell zygotes to differentiate into diverse cell types even though they contain the same genetic material. Preimplantation dynamics are first driven by the necessity of reprogramming haploid parental epigenomes to reach a totipotent state. This process requires extensive erasure of epigenetic marks shortly after fertilization. During the few short days after formation of the zygote, epigenetic programs are established and are essential for the first lineage decisions and differentiation. Here we review the current understanding of DNA methylation and histone modification dynamics responsible for these early changes during mammalian preimplantation development. In particular we highlight insights that have been gained through next generation sequencing technologies comparing human embryos to other models as well as the recent discoveries of active DNA demethylation mechanisms at play during preimplantation. PMID:26031750

  7. 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. PMID:26395495

  8. 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

  9. Preimplantation Stress and Development

    PubMed Central

    Feuer, Sky; Rinaudo, Paolo

    2013-01-01

    The developmental origins of health and disease hypothesis holds that inappropriate environmental cues in utero, a period marked by tremendous developmental sensitivity, facilitate cellular reprogramming to ultimately predispose disease in adulthood. In this review, we analyze if stress during early stages of development can affect future health. This has wide clinical importance, given that 5 million children have been conceived with assisted reproductive technologies (ART). Because the primary outcome of assisted reproduction procedures is delivery at term of a live, healthy baby, the postnatal effects occurring outside of the neonatal period are often overlooked. To this end, the long-term outcome of ART is appropriately the most relevant concern of the field today. Evidence of adverse consequences is controversial. The majority of studies have concluded no obvious problems in IVF-conceived children, although a number of isolated cases of imprinted diseases, cancers, or malformations have been reported. Given that animal studies suggest alteration of metabolic pathways following preimplantation stress, it will be of great importance to follow-up ART individuals as they enter later stages of adult life. PMID:24203919

  10. Apoptosis in mammalian preimplantation embryos: regulation by survival factors.

    PubMed

    Brison, Daniel R.

    2000-01-01

    The formation of a developmentally competent mammalian blastocyst requires the transition from a unicellular state, the fertilized zygote, to a differentiated multicellular structure. In common with other developing organisms, generation of the required cell population involves the processes of cell division, differentiation and cell death, all of which can be regulated by peptide growth factors. Cell death in the preimplantation embryo occurs by apoptosis and, by analogy with other systems, may serve to eliminate unwanted cells during the critical developmental transitions that take place during this period. Cells may be eliminated because they are abnormal or possess defects, including damaged DNA or chromosomal abnormalities. At the early cleavage stages, apoptosis may be associated with activation of the embryonic genome and may contribute to the blastomere fragmentation commonly observed in human IVF embryos. The major wave of apoptosis occurs in a number of species in the inner cell mass of the blastocyst, as identified using nuclear labelling including terminal transferase-mediated dUTP nick end labelling (TUNEL) and fluorescence and confocal microscopy. Apoptosis may protect the integrity and cellular composition of the inner cell mass, by eliminating damaged cells or possibly those with an inappropriate phenotype. Preimplantation embryos express genes involved in the regulation and execution of apoptosis and their cells can undergo this default pathway in the absence of exogenous survival signals. Evidence is now accumulating from several species that apoptosis in the embryo is regulated by soluble peptide growth factors acting as survival factors in an autocrine or paracrine manner. To date, these include transforming growth factor alpha and members of the insulin-like growth factor family. Apoptosis may also be affected by environmental factors, including culture conditions and the composition of media. The regulation of apoptosis in the preimplantation

  11. 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

  12. Epigenetics in preimplantation mammalian development.

    PubMed

    Canovas, Sebastian; Ross, Pablo Juan

    2016-07-01

    Fertilization is a very dynamic period of comprehensive chromatin remodeling, from which two specialized cells result in a totipotent zygote. The formation of a totipotent cell requires extensive epigenetic remodeling that, although independent of modifications in the DNA sequence, still entails a profound cell-fate change, supported by transcriptional profile modifications. As a result of finely tuned interactions between numerous mechanisms, the goal of fertilization is to form a full healthy new individual. To avoid the persistence of alterations in epigenetic marks, the epigenetic information contained in each gamete is reset during early embryogenesis. Covalent modification of DNA by methylation, as well as posttranslational modifications of histone proteins and noncoding RNAs, appears to be the main epigenetic mechanisms that control gene expression. These allow different cells in an organism to express different transcription profiles, despite each cell containing the same DNA sequence. In the context of replacement of spermatic protamine with histones from the oocyte, active cell division, and specification of different lineages, active and passive mechanisms of epigenetic remodeling have been revealed as critical for editing the epigenetic profile of the early embryo. Importantly, redundant factors and mechanisms are likely in place, and only a few have been reported as critical for fertilization or embryo survival by the use of knockout models. The aim of this review is to highlight the main mechanisms of epigenetic remodeling that ensue after fertilization in mammals. PMID:27165992

  13. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development.

    PubMed

    Lu, Falong; Liu, Yuting; Inoue, Azusa; Suzuki, Tsukasa; Zhao, Keji; Zhang, Yi

    2016-06-01

    How the chromatin regulatory landscape in the inner cell mass cells is established from differentially packaged sperm and egg genomes during preimplantation development is unknown. Here, we develop a low-input DNase I sequencing (liDNase-seq) method that allows us to generate maps of DNase I-hypersensitive site (DHS) of mouse preimplantation embryos from 1-cell to morula stage. The DHS landscape is progressively established with a drastic increase at the 8-cell stage. Paternal chromatin accessibility is quickly reprogrammed after fertilization to the level similar to maternal chromatin, while imprinted genes exhibit allelic accessibility bias. We demonstrate that transcription factor Nfya contributes to zygotic genome activation and DHS formation at the 2-cell stage and that Oct4 contributes to the DHSs gained at the 8-cell stage. Our study reveals the dynamic chromatin regulatory landscape during early development and identifies key transcription factors important for DHS establishment in mammalian embryos. PMID:27259149

  14. 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

  15. 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

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

    PubMed Central

    Torres-Padilla, Maria Elena

    2008-01-01

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

  17. Preimplantation genetic diagnosis: development and regulation.

    PubMed

    Thomas, C

    2006-06-01

    Pre-implantation genetic diagnosis (PGD) is used to biopsy and analyse embryos created through in vitro fertilisation (IVF) to avoid implanting an embryo affected by a mutation or chromosomal abnormality associated with serious illness. It reduces the chance that the parents will be faced with a difficult decision of whether to terminate the pregnancy, if the disorder is detected during the course of gestation. PGD is widely accepted for this purpose although there have been suggestions that such procedures have the effect of de-valuing persons in the community with disabilities. PGD potentially has other more controversial purposes, including the selection of the sex of the baby for personal preferences such as balancing the family, rather than to avoid a sex-linked disorder. Recently PGD has become available to create a donor child who is Human Leukocyte Antigen (HLA) matched with a sibling in need of stem cell transplant. In most cases the intention is to utilise the cord blood. However, an HLA-matched child could potentially be required to be a donor of tissues and organs throughout life. This may arise should the initial cord blood donation fail for any one of several reasons, such as inadequate cord blood cell dose, graft failure after cord blood transplant, or the recipient child experiencing a recurrence of the original illness after transplant. However, such on-going demands could also arise if a HLA-matched child was fortuitously conceived by natural means. As such, the issue is not PGD, but rather whether to harvest bone marrow or a solid organ from a child. This raises the question of whether there should be limits and procedures to protect such children from exploitation until they achieve sufficient competence to be able to make mature and autonomous decisions about whether to donate, even if the consequence may in some cases be that it is too late to save the sibling. Additionally, the parents may not be able to make a dispassionate decision, when

  18. 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

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

    PubMed

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

    2012-01-01

    One-carbon metabolism (OCM) can be seen as integrated metabolic pathways centered on the metabolism of two nutritional substances, folate and methionine. Mammalian oocytes and preimplantation embryos express almost all enzymes that participate in OCM, suggesting that they can independently metabolize OCM nutrients. A deficiency or excess of OCM nutrients and their metabolites during in vitro culture affects preimplantation development of mammalian embryos. Recent in vivo studies have demonstrated that specific OCM dietary interventions during the periconceptional (mainly oocyte growth and preimplantation) period can cause epigenetic alterations in DNA of offspring and program the long-term consequences in their health in adulthood. The epigenetic processes are likely to be implicated in the effects of OCM nutrients; however, understanding their effects at the level of specific genes and their implications in assisted reproductive technology will require further investigations. PMID:22450283

  20. Cell death is involved in sexual dimorphism during preimplantation development.

    PubMed

    Oliveira, C S; Saraiva, N Z; de Lima, M R; Oliveira, L Z; Serapião, R V; Garcia, J M; Borges, C A V; Camargo, L S A

    2016-02-01

    In bovine preimplantation development, female embryos progress at lower rates and originate smaller blastocysts than male counterparts. Although sex-specific gene expression patterns are reported, when and how sex dimorphism is established is not clear. Differences among female and male early development can be useful for human assisted reproductive medicine, when X-linked disorders risk is detected, and for genetic breeding programs, especially in dairy cattle, which requires female animals for milk production. The aim of this study was to characterize the development of female and male embryos, attempting to identify sex effects during preimplantation development and the role of cell death in this process. Using sex-sorted semen from three different bulls for fertilization, we compared kinetics of bovine sex-specific embryos in six time points, and cell death was assessed in viable embryos. For kinetics analysis, we detected an increased population of female embryos arrested at 48 and 120h.p.i., suggesting this time points as delicate stages of development for female embryos that should be considered for testing improvement strategies for assisted reproductive technologies. Assessing viable embryos quality, we found 144h.p.i. is the first time point when viable embryos are phenotypically distinct: cell number is decreased, and apoptosis and cell fragmentation are increased in female embryos at this stage. These new results lead us to propose that sex dimorphism in viable embryos is established during morula-blastocyst transition, and cell death is involved in this process. PMID:26752320

  1. Exogenous retroelement integration in sperm and embryos affects preimplantation development.

    PubMed

    Kitsou, C; Lazaros, L; Bellou, S; Vartholomatos, G; Sakaloglou, P; Hatzi, E; Markoula, S; Zikopoulos, K; Tzavaras, T; Georgiou, I

    2016-09-01

    Retroelement transcripts are present in male and female gametes, where they are typically regulated by methylation, noncoding RNAs and transcription factors. Such transcripts are required for occurrence of retrotransposition events, while failure of retrotransposition control may exert negative effects on cellular function and proliferation. In order to investigate the occurrence of retrotransposition events in mouse epididymal spermatozoa and to address the impact of uncontrolled retroelement RNA expression in early preimplantation embryos, we performed in vitro fertilization experiments using spermatozoa preincubated with plasmid vectors containing the human retroelements LINE-1, HERVK-10 or the mouse retroelement VL30, tagged with an enhanced green fluorescence (EGFP) gene-based cassette. Retrotransposition events in mouse spermatozoa and embryos were detected using PCR, FACS analysis and confocal microscopy. Our findings show that: (i) sperm cell incorporates exogenous retroelements and favors retrotransposition events, (ii) the inhibition of spermatozoa reverse transcriptase can decrease the retrotransposition frequency in sperm cells, (iii) spermatozoa can transfer exogenous human or mouse retroelements to the oocyte during fertilization and (iv) retroelement RNA overexpression affects embryo morphology and impairs preimplantation development. These findings suggest that the integration of exogenous retroelements in the sperm genome, as well as their transfer into the mouse oocyte, could give rise to new retrotransposition events and genetic alterations in mouse spermatozoa and embryos. PMID:27450800

  2. Characterization of SCF-Complex during Bovine Preimplantation Development

    PubMed Central

    Benesova, Veronika; Kinterova, Veronika; Kanka, Jiri; Toralova, Tereza

    2016-01-01

    The degradation of maternal proteins is one of the most important events during early development, and it is presumed to be essential for embryonic genome activation (EGA), but the precise mechanism is still not known. It is thought that a large proportion of the degradation of maternal proteins is mediated by the ubiquitin-proteolytic system. In this study we focused on the expression of the Skp1-Cullin1-F-box (SCF) complex, a modular RING-type E3 ubiquitin-ligase, during bovine preimplantation development. The complex consists of three invariable components—Cul1, Skp1, Rbx1 and F-box protein, which determines the substrate specificity. The protein level and mRNA expression of all three invariable members were determined. Cul1 and Skp1 mRNA synthesis was activated at early embryonic stages, at the 4c and early 8c stage, respectively, which suggests that these transcripts are necessary for preparing the embryo for EGA. CUL1 protein level increased from MII to the morula stage, with a significant difference between MII and L8c, and between MII and the morula. The CUL1 protein was localized primarily to nuclei and to a lesser extent to the cytoplasm, with a lower signal in the inner cell mass (ICM) compared to the trophectoderm (TE) at the blastocyst stage. The level of SKP1 protein significantly increased from MII oocytes to 4c embryos, but then significantly decreased again. The localization of the SKP1 protein was analysed throughout the cell and similarly to CUL1 at the blastocyst stage, the staining was less intensive in the ICM. There were no statistical differences in RBX1 protein level and localization. The active SCF-complex, which is determined by the interaction of Cul1 and Skp1, was found throughout the whole embryo during preimplantation development, but there was a difference at the blastocyst stage, which exhibits a much stronger signal in the TE than in the ICM. These results suggest that all these genes could play an important role during

  3. 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

  4. Requirement for nuclear autoantigenic sperm protein mRNA expression in bovine preimplantation development.

    PubMed

    Nagatomo, Hiroaki; Kohri, Nanami; Akizawa, Hiroki; Hoshino, Yumi; Yamauchi, Nobuhiko; Kono, Tomohiro; Takahashi, Masashi; Kawahara, Manabu

    2016-03-01

    Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle. PMID:26690724

  5. Cell fate regulation in early mammalian development

    NASA Astrophysics Data System (ADS)

    Oron, Efrat; Ivanova, Natalia

    2012-08-01

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

  6. 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

  7. 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...

  8. 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

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

    PubMed

    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

    2016-06-17

    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

  10. 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. PMID:25985793

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

    PubMed Central

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

    2015-01-01

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

  12. Similar kinetics for 5-methylcytosine and 5-hydroxymethylcytosine during human preimplantation development in vitro.

    PubMed

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

    2016-07-01

    After fertilization, the mammalian embryo undergoes epigenetic reprogramming with genome-wide DNA demethylation and subsequent remethylation. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) was suggested to be an intermediate step in the DNA demethylation pathway. Other evidence, such as the stability of 5hmC in specific tissues, suggests that 5hmC constitutes a new epigenetic modification with its own biological function. Since few studies have been conducted on human material compared to animal models and species-specific epigenetic differences have been reported, we studied global DNA methylation and hydroxymethylation patterns in human in vitro preimplantation embryos using immunocytochemistry, comparing these patterns in good-quality and abnormally developing embryos. Our data showed that DNA methylation and hydroxymethylation modifications co-exist. 5mC and 5hmC signals were found in oocytes and in paternal and maternal pronuclei of zygotes, present in non-reciprocal patterns-which contrasts published data for the mouse. These two epigenetic modifications are present between Days 1 and 7 of in vitro development, with 5mC levels declining over cell divisions without noticeable remethylation during this period. A main decline in 5mC and 5hmC occurred as the embryo progressed from compaction to the blastocyst stage. No difference in (hydroxy)methylation was found between the inner cell mass and trophectoderm. When comparing normally and abnormally developing embryos, DNA (hydroxy)methylation reprogramming was abnormal in poor-quality embryos, especially during the first cleavages. Mol. Reprod. Dev. 83: 594-605, 2016 © 2016 Wiley Periodicals, Inc. PMID:27163211

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

    PubMed

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

    2015-01-01

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

  14. 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

  15. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  18. 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

  19. Dynamics of histone H3 acetylation in the nucleosome core during mouse pre-implantation development.

    PubMed

    Ziegler-Birling, Céline; Daujat, Sylvain; Schneider, Robert; Torres-Padilla, Maria-Elena

    2016-08-01

    In mammals, the time period that follows fertilization is characterized by extensive chromatin remodeling, which enables epigenetic reprogramming of the gametes. Major changes in chromatin structure persist until the time of implantation, when the embryo develops into a blastocyst, which comprises the inner cell mass and the trophectoderm. Changes in DNA methylation, histone variant incorporation, and covalent modifications of the histones tails have been intensively studied during pre-implantation development. However, modifications within the core of the nucleosomes have not been systematically analyzed. Here, we report the first characterization and temporal analysis of 3 key acetylated residues in the core of the histone H3: H3K64ac, H3K122ac, and H3K56ac, all located at structurally important positions close to the DNA. We found that all 3 acetylations occur during pre-implantation development, but with different temporal kinetics. Globally, H3K64ac and H3K56ac were detected throughout cleavage stages, while H3K122ac was only weakly detectable during this time. Our work contributes to the understanding of the contribution of histone modifications in the core of the nucleosome to the "marking" of the newly established embryonic chromatin and unveils new modification pathways potentially involved in epigenetic reprogramming. PMID:26479850

  20. Leptin and ObRa/MEK signalling in mouse oocyte maturation and preimplantation embryo development.

    PubMed

    Ye, Yinghui; Kawamura, Kazuhiro; Sasaki, Mitsue; Kawamura, Nanami; Groenen, Peter; Sollewijn Gelpke, Maarten D; Kumagai, Jin; Fukuda, Jun; Tanaka, Toshinobu

    2009-08-01

    Recent studies indicate that LH stimulates production of ovarian paracrine factors that induce meiosis of the oocyte. DNA microarray analyses of ovarian transcripts were performed in mice and major increases of a short isoform of leptin receptor, ObRa, were identified by the preovulatory LH/human chorionic gonadotrophin (HCG) surge. In oocytes, the level of ObRa transcripts was increased shortly after HCG stimulation, whereas the level of ObRb transcripts was not changed. Leptin was produced by cumulus, granulosa, theca and interstitial cells of ovaries and its transcript level was not regulated during gonadotrophin treatment. Treatment with leptin promoted germinal vesicle breakdown (GVBD) in oocytes within preovulatory follicles, and enhance first polar body extrusion in both cumulus-oocyte complexes and denuded oocytes. The leptin-promoted GVBD and first polar body extrusion were blocked by a mitogen-activated protein kinase extracellular signal regulated kinase kinases (MEK)1/2 inhibitor, U0126, but not its inactive analogue U0124. Furthermore, leptin promoted fertilization of oocytes and the in-vitro development of zygotes to preimplantation embryos. These findings suggest paracrine roles of leptin in the enhancement of nuclear maturation of oocytes through MEK1/2 signalling, and in the promotion of cytoplasmic maturation essential for successful oocyte development to the preimplantation embryos. PMID:19712552

  1. Development of the Mammalian Kidney.

    PubMed

    McMahon, Andrew P

    2016-01-01

    The basic unit of kidney function is the nephron. In the mouse, around 14,000 nephrons form in a 10-day period extending into early neonatal life, while the human fetus forms the adult complement of nephrons in a 32-week period completed prior to birth. This review discusses our current understanding of mammalian nephrogenesis: the contributing cell types and the regulatory processes at play. A conceptual developmental framework has emerged for the mouse kidney. This framework is now guiding studies of human kidney development enabled in part by in vitro systems of pluripotent stem cell-seeded nephrogenesis. A near future goal will be to translate our developmental knowledge-base to the productive engineering of new kidney structures for regenerative medicine. PMID:26969971

  2. Cell death in mammalian development.

    PubMed

    Penaloza, C; Orlanski, S; Ye, Y; Entezari-Zaher, T; Javdan, M; Zakeri, Z

    2008-01-01

    During embryogenesis there is an exquisite orchestration of cellular division, movement, differentiation, and death. Cell death is one of the most important aspects of organization of the developing embryo, as alteration in timing, level, or pattern of cell death can lead to developmental anomalies. Cell death shapes the embryo and defines the eventual functions of the organs. Cells die using different paths; understanding which path a dying cell takes helps us define the signals that regulate the fate of the cell. Our understanding of cell death in development stems from a number of observations indicating genetic regulation of the death process. With today's increased knowledge of the pathways of cell death and the identification of the genes whose products regulate the pathways we know that, although elimination of some of these gene products has no developmental phenotype, alteration of several others has profound effects. In this review we discuss the types and distributions of cell death seen in developing mammalian embryos as well as the gene products that may regulate the process. PMID:18220829

  3. 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

  4. Polycomb gene expression and histone H3 lysine 27 trimethylation changes during bovine preimplantation development.

    PubMed

    Ross, Pablo J; Ragina, Neli P; Rodriguez, Ramon M; Iager, Amy E; Siripattarapravat, Kannika; Lopez-Corrales, Nestor; Cibelli, Jose B

    2008-12-01

    Trimethylation of histone H3 at lysine 27 (H3K27me3) is established by polycomb group genes and is associated with stable and heritable gene silencing. The aim of this study was to characterize the expression of polycomb genes and the dynamics of H3K27me3 during bovine oocyte maturation and preimplantation development. Oocytes and in vitro-produced embryos were collected at different stages of development. Polycomb gene expression was analyzed by real-time quantitative RT-PCR and immunofluorescence. Global H3K27me3 levels were determined by semiquantitative immunofluorescence. Transcripts for EZH2, EED, and SUZ12 were detected at all stages analyzed, with EZH2 levels being the highest of the three at early stages of development. By the time the embryo reached the blastocyst stage, the level of PcG gene mRNA levels significantly increased. Immunofluorescence staining indicated nuclear expression of EZH2 at all stages while nuclear localized EED and SUZ12 were only evident at the morula and blastocyst stages. Semiquantitative analysis of H3K27me3 levels showed that nuclear fluorescence intensity was the highest in immature oocytes, which steadily decreased after fertilization to reach a nadir at the eight-cell stage, and then increased at the blastocyst stage. These results suggest that the absence of polycomb repressive complex 2 proteins localized to the nucleus of early embryos could be responsible for the gradual decrease in H3K27me3 during early preimplantation development. PMID:18784248

  5. 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

  6. Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development.

    PubMed

    Jansen, Sarah; Esmaeilpour, Tahereh; Pantaleon, Marie; Kaye, Peter L

    2006-03-01

    Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. In the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (Km 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. In expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression. PMID:16514190

  7. 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. PMID:25932910

  8. Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection.

    PubMed

    Li, Xiao Xia; Lee, Kyung-Bon; Lee, Ji Hye; Kim, Keun Jung; Kim, Eun Young; Han, Kil-Woo; Park, Kang-Sun; Yu, Jung; Kim, Min Kyu

    2014-01-15

    Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis

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

    PubMed Central

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

    2015-01-01

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

  10. 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

  11. 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

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

    PubMed

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

    1996-05-01

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

  13. Inhibitory effects of preimplantation exposure to bisphenol-A on blastocyst development and implantation

    PubMed Central

    Pan, Xiaoyan; Wang, Xuenan; Sun, Yanmei; Dou, Zhaohua; Li, Zhixin

    2015-01-01

    The effect of preimplantation exposure to bisphenol-A (BPA) on blastocyst development and implantation is investigated. Mice were orally administered with BPA (200, 400, 600, and 800 mg/kg/day) from Day 0.5 to Day 3.5 of their pregnancy. Blastocyst development was examined on Day 4 of pregnancy. With 400 mg/kg/day BPA, implantation site number and implantation rate significantly reduced. With 600 and 800 mg/kg/day BPA, no implantation site was observed. BPA at 800 mg/kg/day significantly reduced blastocyst development rate and hatching rate. With 400 and 600 mg/kg/day BPA, Blastocyst development rate showed no significant difference whereas hatching rate was lower. With 400, 600, and 800 mg/kg/day BPA, some embryos were detected in the fallopian tube and hatched blastocysts showed greatly increased apoptosis level and endothelial nitric oxide synthase expression. In summary, high concentration BPA delayed the transfer of embryos to the uterus, damaged blastocyst development before implantation, and inhibited embryo implantation. PMID:26309523

  14. 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

  15. 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

  16. 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

  17. Functional characterization of CDX2 during bovine preimplantation development in vitro.

    PubMed

    Goissis, Marcelo D; Cibelli, Jose B

    2014-10-01

    Placental defects are common in bovine embryos produced using assisted reproductive techniques. A proper understanding of the events leading to inner cell mass (ICM) and trophectoderm (TE) specification could help identify the origins of such developmental failures. We focused on caudal-type homeobox transcription factor 2 (CDX2) since it has a specific role during TE differentiation in mouse embryos. Of all the preimplantation stages analyzed, CDX2 protein was present only at the blastocyst stage. To further understand the roles of CDX2 during bovine development, we depleted CDX2 mRNA; despite a significant loss of detectable protein, embryos were able to form blastocysts at the same rate as controls. Embryos lacking CDX2 did not show abnormalities in the number of TE, ICM, or total cells in the blastocyst. Expression of the developmentally important genes SOX2, POU5F1, and NANOG, or TE markers such as IFN-T and KRT18 were not affected by the reduction in CDX2 levels, nor was the localization of SOX2 and POU5F1 protein. Using a functional barrier assay, we observed that the TE epithelial layer of embryos lacking CDX2 had lost its integrity. Our results thus indicate that CDX2 is not required for TE formation during bovine development; nevertheless, it is necessary for maintaining TE integrity. PMID:25251051

  18. 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. PMID:26379100

  19. 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

  20. β-catenin-mediated adhesion is required for successful preimplantation mouse embryo development.

    PubMed

    Messerschmidt, Daniel; de Vries, Wilhelmine N; Lorthongpanich, Chanchao; Balu, Sathish; Solter, Davor; Knowles, Barbara B

    2016-06-01

    β-catenin (CTNNB1) is integral to cell adhesion and to the canonical Wnt signaling pathway. The effects of maternal and zygotic CTNNB1 on embryogenesis have each been separately assessed, whereas the effect of its total absence has not. As the 'traditional' conditional Ctnnb1 knockout alleles give rise to truncated CTNNB1 fragments, we designed a new knockout allele incapable of CTNNB1 production. Mouse embryos lacking intact maternal/zygotic CTNNB1 from two knockout strains were examined in detail. Preimplantation embryos are formed, yet abnormalities in their size and shape were found throughout pre- and early postimplantation development. In the absence of the zona pellucida, embryos lacking CTNNB1 undergo fission and these separated blastomeres can become small trophoblastic vesicles, which in turn induce decidual reactions. Comparing the severity of this defective adhesion phenotype in embryos bearing the null allele with those carrying the 'traditional' knockout allele suggests a hypomorphic effect of the truncated CTNNB1 protein fragment, an important observation with possible impact on previous and future studies. PMID:27246714

  1. Effects of T-2 mycotoxin on in vitro development and chromatin status of mouse embryos in preimplantation stages.

    PubMed

    Somoskői, Bence; Kovács, Melinda; Cseh, Sándor

    2016-07-01

    T-2 toxin is a mycotoxin produced by phytopathogenic fungi of the Fusarium genus and has many well-studied deleterious effects on mammalian cells and reproductive tract. Despite the wide scale studies, the effects on preimplantation stage embryos are lacking. The aim of our study was to investigate the impact of T-2 on the cleavage stage of mouse embryos with regard to development to blastocysts and nuclear chromatin status.Six-weeks-old BDF1 female mice were superovulated and placed together overnight with mature males. Zygotes were flushed 20 h after human chorionic gonadotropin injection and divided randomly into treated (supplemented with 0.5, 0.75, and 1 ng/ml T-2) and nontreated (control) groups. Embryos were cultured in vitro for 96 h. Developmental stage was evaluated in the 72(nd)- and 96(th)-h for assessment of development dynamics. At the end of culture period, blastocysts from treated and control groups with normal morphology were selected for nuclear chromatin analysis. Blastocysts were categorized (grade A, B, and C) depending on the proportion of blasomeres with micronuclei and/or lobulated nuclei.Our data show significant decrease in the proportions of blastocysts in the 0.75 and 1 ng/ml toxin-supplemented groups compared with the control group. Blastocyst rate did not differ in embryos treated with 0.5 ng/ml T-2 but 24 h delay was found in blastocoel formation in all the treated groups. Only grade A (21.1%) and B (78.9%) blastocysts were found in low-toxin-contaminated group similar to the control ones (50-50%). Grade C embryos appeared in the 0.75 ng/ml (10%) treated group and the rate increased significantly (33.3%) in the highest contaminated group.T-2 mycotoxin has a harmful effect on early embryo development which results in decreased blastocyst proportion, delayed blastulation, and increased rate of chromatin damage. PMID:25425537

  2. Detection of apoptosis in mammalian development.

    PubMed

    Lin, Lin; Penaloza, Carlos; Ye, Yixia; Lockshin, Richard A; Zakeri, Zahra

    2009-01-01

    Mammalian development is dependent on an intricate orchestration of cell proliferation and death. Deregulation in the levels, localization, and type of cell death can lead to disease and even death of the developing embryo. The mechanisms involved in such deregulation are many; alterations and or manipulations of these can aid in the detection, prevention and possible treatments of any effects this de-regulation may have. Here we describe how cell death can be detected during mammalian development, using diverse staining and microscopy methods, while taking advantage of the advancements in cell death mechanisms, derived from biochemical and teratological studies in the field. PMID:19609762

  3. 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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-02-14

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

  6. 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.

  7. l-Carnitine affects preimplantation embryo development toward infertility in mice.

    PubMed

    Kyvelidou, Christiana; Sotiriou, Dimitris; Antonopoulou, Tania; Tsagkaraki, Margarita; Tserevelakis, George J; Filippidis, George; Athanassakis, Irene

    2016-10-01

    l-Carnitine (l-Cn), despite the beneficial role as energy-generating substance delivering long-chain fatty acids to the β-oxidation pathway in mitochondria, has been accused to cause an endometriosis-like state to BALB/c mice manifested by increased inflammatory cytokines in serum and peritoneal fluid, accumulation of immune cells in the peritoneal cavity and uterine walls and most importantly, correlating to infertility. Exploring this type of infertility, the effect of l-Cn on preimplantation embryo development, ovarian integrity and systemic maternal immunity was studied. Using nonlinear microscopy analysis, which was shown to be a powerful tool for determining embryo quality by quantitatively estimating the lipid body (LB) content of the cells, it was shown that in vitro and in vivo administration of l-Cn significantly decreased LB mean area in zygotes. Daily intraperitoneal administration of 2.5mg l-Cn for 3, 4 and 7days to mice significantly decreased the percent of normal zygotes. However, only the 7-day treatment persisted by affecting 2- and 8-cell stage embryos, while almost abolishing blastocyst development. Such effects were accompanied by abnormal ovarian histology, showing increased numbers of corpora luteus and elevated progesterone concentration in the serum. In addition, it was shown that the 7-day l-Cn treatment pushed maternal systemic immunity toward inflammation and immunosuppression by increasing CD11b-, CD25- and CD11bGr1-positive cells in spleen, which opposed the necessity for immunostimulation at these early stages of pregnancy. In conclusion, the results presented here demonstrated that elevated doses of l-Cn affect early stages of embryo development, leading to infertility. PMID:27402869

  8. Lack of maternal glutamate cysteine ligase modifier subunit (Gclm) decreases oocyte glutathione concentrations and disrupts preimplantation development in mice.

    PubMed

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

    2011-07-01

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

  9. Glia in mammalian development and disease.

    PubMed

    Zuchero, J Bradley; Barres, Ben A

    2015-11-15

    Glia account for more than half of the cells in the mammalian nervous system, and the past few decades have witnessed a flood of studies that detail novel functions for glia in nervous system development, plasticity and disease. Here, and in the accompanying poster, we review the origins of glia and discuss their diverse roles during development, in the adult nervous system and in the context of disease. PMID:26577203

  10. Lanosterol influences cytoplasmic maturation of pig oocytes in vitro and improves preimplantation development of cloned embryos.

    PubMed

    Lee, Sanghoon; Jin, Jun-Xue; Khoirinaya, Candrani; Kim, Geon A; Lee, Byeong Chun

    2016-03-01

    Lanosterol is a precursor of meiosis-activating sterols in the cholesterol biosynthetic pathway and induces a physiological signal that instructs the oocyte to reinitiate meiosis. In this study, we examined the effect of lanosterol on IVM of porcine oocytes, specifically on nuclear maturation, cytoplasmic maturation by investigating intracellular glutathione (GSH) levels and lipid content, embryonic development after parthenogenetic activation and somatic cell nuclear transfer (SCNT), and on gene expression in cumulus cells, oocytes, and SCNT-derived blastocysts. There was no significant difference in nuclear maturation rates between the control and treatment groups (10, 50, and 100 μM of lanosterol added to IVM culture medium). Supplementation with 50-μM lanosterol significantly increased lipid content and GSH levels and decreased reactive oxygen species levels compared with the control. In addition, oocytes treated with 50 μM of lanosterol exhibited significantly increased blastocyst formation rates and total cell numbers after parthenogenetic activation (30.3% and 63.9 vs. 21.6% and 36.5, respectively) and SCNT (18.2% and 53.7 vs. 12.6% and 37.5, respectively), when compared with the control group. Cumulus cells treated with 50 μM of lanosterol showed significantly increased 14α-demethylase, Δ14-reductase, and Δ7-reductase mRNA transcript levels. Significantly increased PPARγ, SREBF1, GPX1, and Bcl-2 and decreased Bax transcript levels were observed in mature oocytes treated with 50 μM of lanosterol compared with the control. SCNT blastocysts derived from 50-μM lanosterol-treated oocytes had significantly higher POU5F1, FGFR2, and Bcl-2 transcript levels than control SCNT-derived blastocysts. In conclusion, supplementation with 50 μM of lanosterol during IVM improves preimplantation development of SCNT embryos by elevating lipid content of oocytes, increasing GSH levels, decreasing reactive oxygen species levels, and regulating genes related to the

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

    PubMed

    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

  12. 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

  13. 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.

  14. Neonatal Phytoestrogen Exposure Alters Oviduct Mucosal Immune Response to Pregnancy and Affects Preimplantation Embryo Development in the Mouse1

    PubMed Central

    Jefferson, Wendy N.; Padilla-Banks, Elizabeth; Phelps, Jazma Y.; Cantor, Amy M.; Williams, Carmen J.

    2012-01-01

    ABSTRACT Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are “posteriorized” as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype. PMID:22553218

  15. Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides.

    PubMed

    Siddall, Laura S; Barcroft, Lisa C; Watson, Andrew J

    2002-12-01

    Morpholino antisense oligonucleotides act by blocking translation of their target gene products and are effective tools for down-regulating gene expression. The current study was conducted to define treatment conditions for the use of morpholino oligonucleotides (MOs) in mammalian preimplantation embryos, and to employ MOs to target genes and study gene function in the early embryo. For the first time, ethoxylated polyethylenimine (EPEI), Lipofectin or Lysolecithin delivery agents were employed in combination with a fluorescent control MO and an alpha-catenin specific MO, to down-regulate gene expression during murine preimplantation development. Experiments applied to both two- and eight-cell stage murine preimplantation embryos contrasted the efficacy of MO concentrations of 1, 2, 5, 10, and 20 microM and treatment delivery times of 3, 6, 24, and 48 hr. Continuous treatment of two-cell embryos with Lipofectin and 20 microM alpha-catenin MO for 48 hr resulted in a significant (P < 0.05) reduction in development to the blastocyst stage and was accompanied by a marked reduction in alpha-catenin protein. These results indicate that morpholino antisense oligonucleotides are effective tools for down-regulating gene expression during mammalian preimplantation development. PMID:12412042

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

    PubMed Central

    Little, Melissa H.; McMahon, Andrew P.

    2012-01-01

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

  17. Light-sheet imaging of mammalian development.

    PubMed

    de Medeiros, Gustavo; Balázs, Bálint; Hufnagel, Lars

    2016-07-01

    Tackling modern cell and developmental biology questions requires fast 3D imaging with sub-cellular resolution over extended periods of time. Fluorescence microscopy has emerged as a powerful tool to image biological samples with high spatial and temporal resolution with molecular specificity. In particular, the highly efficient illumination and detection scheme of light-sheet fluorescence microscopy is starting to revolutionize the way we can monitor cellular and developmental processes in vivo. Here we summarize the state-of-the art of light-sheet imaging with a focus on mammalian development - from instrumentation, mounting and sample handling to data processing. PMID:27288888

  18. Actin Disorganization Plays a Vital Role in Impaired Embryonic Development of In Vitro-Produced Mouse Preimplantation Embryos

    PubMed Central

    Tan, Kun; An, Lei; Wang, Shu-Min; Wang, Xiao-Dong; Zhang, Zhen-Ni; Miao, Kai; Sui, Lin-Lin; He, Shu-Zhi; Nie, Jing-Zhou; Wu, Zhong-Hong; Tian, Jian-Hui

    2015-01-01

    Assisted reproductive technology (ART) is being increasingly applied to overcome infertility. However, the in vitro production process, the main procedure of ART, can lead to aberrant embryonic development and health-related problems in offspring. Understanding the mechanisms underlying the ART-induced side effects is important to improve the ART process. In this study, we carried out comparative transcriptome profiling between in vivo- (IVO) and in vitro- produced (IVP) mouse blastocysts. Our results suggested that aberrant actin organization might be a major factor contributing to the impaired development of IVP embryos. To test this, we examined the effect of actin disorganization on the development of IVP preimplantation embryos. Specific disruption of actin organization by cytochalasin B (CB) indicated that well-organized actin is essential for in vitro embryonic development. Supplementing the culture medium with 10–9 M melatonin, a cytoskeletal modulator in adult somatic cells, significantly reversed the disrupted expression patterns of genes related to actin organization, including Arhgef2, Bcl2, Coro2b, Flnc, and Palld. Immunofluorescence analysis showed that melatonin treatment of IVP embryos significantly improved the distribution and organization of actin filaments (F-actin) from the 8-cell stage onwards. More importantly, we found that melatonin alleviated the CB-mediated aberrant F-actin distribution and organization and rescued CB-induced impaired embryonic development. This is the first study to indicate that actin disorganization is implicated in impaired development of IVP embryos during the preimplantation stage. We also demonstrated that improving actin organization is a promising strategy to optimize existing IVP systems. PMID:26076347

  19. 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

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

    PubMed Central

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

    2015-01-01

    Traditional Chinese medicinal herbs containing berberine have been historically used to prevent miscarriage. Here, we investigated whether the anti-apoptotic effects of berberine on pre-implantation embryonic development are regulated by miRNA-21. Mouse pronuclear embryos were cultured in medium with or without berberine, and some were then microinjected with a miRNA-21 inhibitor. The in vitro developmental rates of 2- and 4-cell embryos and blastocysts, blastocyst cell numbers, apoptotic rates, and apoptotic cell numbers were measured in each group. Furthermore, we examined the transcription levels of miRNA-21 and its target genes (caspase-3, PTEN, and Bcl-2) and their translation levels. Comparisons were made with in vivo-developed and untreated embryos. We found that berberine significantly increased the developmental rates and cell numbers of mouse blastocysts and decreased apoptotic cell rates in vitro. Berberine also significantly increased miRNA-21 and Bcl-2 transcription levels and significantly decreased caspase-3 and PTEN transcription levels. In embryos treated with a miRNA-21 inhibitor, the results followed the opposite trend; PTEN and caspase-3 transcription levels increased significantly, while the transcription level of Bcl-2 decreased significantly. Additionally, berberine treatment significantly increased the Bcl-2 protein level and significantly decreased the caspase-3 and PTEN protein levels in blastocysts, but there were no significant differences observed in the levels of these proteins in 2- and 4-cell embryos. This study revealed that miRNA-21 is important for pre-implantation embryonic development, especially blastocyst development in vitro. Berberine elevates miRNA-21 expression, decreases PTEN and caspase-3 levels, increases Bcl-2 levels, and exerts anti-apoptotic and pro-growth effects. PMID:26042820

  1. Global Epigenomic Reconfiguration During Mammalian Brain Development

    PubMed Central

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

    2013-01-01

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

  2. Mammalian oocyte growth and development in vitro.

    PubMed

    Eppig, J J; O'Brien, M; Wigglesworth, K

    1996-06-01

    This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes form preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. PMID:9115726

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

    PubMed Central

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

    2015-01-01

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

  4. 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

  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. Three-Dimensional Distribution of UBF and Nopp140 in Relationship to Ribosomal DNA Transcription During Mouse Preimplantation Development.

    PubMed

    Koné, Maïmouna Coura; Fleurot, Renaud; Chebrout, Martine; Debey, Pascale; Beaujean, Nathalie; Bonnet-Garnier, Amélie

    2016-04-01

    The nucleolus is a dynamic nuclear compartment that is mostly involved in ribosome subunit biogenesis; however, it may also play a role in many other biological processes, such as stress response and the cell cycle. Mainly using electron microscopy, several studies have tried to decipher how active nucleoli are set up during early development in mice. In this study, we analyzed nucleologenesis during mouse early embryonic development using 3D-immunofluorescent detection of UBF and Nopp140, two proteins associated with different nucleolar compartments. UBF is a transcription factor that helps maintain the euchromatic state of ribosomal genes; Nopp140 is a phosphoprotein that has been implicated in pre-rRNA processing. First, using detailed image analyses and the in situ proximity ligation assay technique, we demonstrate that UBF and Nopp140 dynamic redistribution between the two-cell and blastocyst stages (time of implantation) is correlated with morphological and structural modifications that occur in embryonic nucleolar compartments. Our results also support the hypothesis that nucleoli develop at the periphery of nucleolar precursor bodies. Finally, we show that the RNA polymerase I inhibitor CX-5461: 1) disrupts transcriptional activity, 2) alters preimplantation development, and 3) leads to a complete reorganization of UBF and Nopp140 distribution. Altogether, our results underscore that highly dynamic changes are occurring in the nucleoli of embryos and confirm a close link between ribosomal gene transcription and nucleologenesis during the early stages of development. PMID:26984997

  7. 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

  8. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages. PMID:17141556

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

    PubMed Central

    Miao, Yi-Liang; Williams, Carmen J.

    2012-01-01

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

  10. Neonatal exposure to genistein disrupts ability of female mouse reproductive tract to support preimplantation embryo development and implantation.

    PubMed

    Jefferson, Wendy N; Padilla-Banks, Elizabeth; Goulding, Eugenia H; Lao, Shin-Ping C; Newbold, Retha R; Williams, Carmen J

    2009-03-01

    Female mice treated neonatally with the phytoestrogen genistein (50 mg/kg/day) have multioocyte follicles, lack regular estrous cyclicity, and are infertile even after superovulation. To determine the cause of their infertility, we examined oocyte developmental competence and timing of embryo loss. Eggs obtained by superovulation of genistein-treated or control females were equally capable of being fertilized in vitro and cultured to the blastocyst stage. However, if eggs were fertilized in vivo, retrieved at the pronucleus stage, and cultured, there was a significant reduction in the percentage of embryos from genistein-treated females reaching the blastocyst stage. When these blastocysts were transferred to pseudopregnant recipients, the number of live pups produced was similar to that in controls. Preimplantation embryo development in vivo was examined by flushing embryos from the oviduct and/or uterus. Similar numbers of one-cell and two-cell embryos were obtained from genistein-treated and control females. However, significantly fewer embryos (<50%) were obtained from genistein-treated females on postcoital Days 3 and 4. To determine if neonatal genistein treatment altered the ability of the uterus to support implantation, blastocysts from control donors were transferred to control and genistein-treated pseudopregnant recipients. These experiments demonstrated that genistein-treated females are not capable of supporting normal implantation of control embryos. Taken together, these results suggest that oocytes from mice treated neonatally with genistein are developmentally competent; however, the oviductal environment and the uterus have abnormalities that contribute to the observed reproductive failure. PMID:19005167

  11. Maternal diabetes promotes mTORC1 downstream signalling in rabbit preimplantation embryos.

    PubMed

    Gürke, Jacqueline; Schindler, Maria; Pendzialek, S Mareike; Thieme, René; Grybel, Katarzyna J; Heller, Regine; Spengler, Katrin; Fleming, Tom P; Fischer, Bernd; Navarrete Santos, Anne

    2016-05-01

    The mammalian target of rapamycin complex 1 (mTORC1) is known to be a central cellular nutrient sensor and master regulator of protein metabolism; therefore, it is indispensable for normal embryonic development. We showed previously in a diabetic pregnancy that embryonic mTORC1 phosphorylation is increased in case of maternal hyperglycaemia and hypoinsulinaemia. Further, the preimplantation embryo is exposed to increased L-leucine levels during a diabetic pregnancy. To understand how mTOR signalling is regulated in preimplantation embryos, we examined consequences of L-leucine and glucose stimulation on mTORC1 signalling and downstream targets in in vitro cultured preimplantation rabbit blastocysts and in vivo. High levels of L-leucine and glucose lead to higher phosphorylation of mTORC1 and its downstream target ribosomal S6 kinase 1 (S6K1) in these embryos. Further, L-leucine supplementation resulted in higher embryonic expression of genes involved in cell cycle (cyclin D1; CCND1), translation initiation (eukaryotic translation initiation factor 4E; EIF4E), amino acid transport (large neutral amino acid transporter 2; Lat2: gene SLC7A8) and proliferation (proliferating cell nuclear antigen; PCNA) in a mTORC1-dependent manner. Phosphorylation of S6K1 and expression patterns of CCND1 and EIF4E were increased in embryos from diabetic rabbits, while the expression of proliferation marker PCNA was decreased. In these embryos, protein synthesis was increased and autophagic activity was decreased. We conclude that mammalian preimplantation embryos sense changes in nutrient supply via mTORC1 signalling. Therefore, mTORC1 may be a decisive mediator of metabolic programming in a diabetic pregnancy. PMID:26836250

  12. 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

  13. 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

  14. 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

  15. Where hearing starts: the development of the mammalian cochlea.

    PubMed

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

    2016-02-01

    The mammalian cochlea is a remarkable sensory organ, capable of perceiving sound over a range of 10(12) 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

  16. 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

  17. 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. PMID:19051249

  18. 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

  19. Biocompatibility assessment of fibrous nanomaterials in mammalian embryos.

    PubMed

    Munk, Michele; Camargo, Luiz S A; Quintão, Carolina C R; Silva, Saulo R; Souza, Eliza D; Raposo, Nádia R B; Marconcini, Jose M; Jorio, Ado; Ladeira, Luiz O; Brandão, Humberto M

    2016-07-01

    Currently there is a growing interest in the use of nanotechnology in reproductive medicine and reproductive biology. However, their toxic effects on mammalian embryos remain poorly understood. In this work, we evaluate the biocompatibility of two fibrous nanomaterials (NMs): cotton cellulose nanofibers (CNF) and carboxylated multiwalled carbon nanotubes (MWCNT-COOH), by performing an investigation of the embryonic development, gene expression (biomarkers focused on cell stress, apoptosis and totipotency) and in situ apoptosis in bovine embryos. Exposure to NMs did not interfere in preimplantation development or in the incidence of apoptosis in the bovine embryo, but they did affect the gene expression. The results presented are important for an understanding of the toxicity of cotton CNF and MWCNT-COOH on mammalian embryos. To our knowledge, we report the first evaluation of biocompatibility between these NMs on preimplantation embryos, which may open a new window for reproductive biomedical applications. PMID:26949162

  20. Phylogenetic memory of developing mammalian dentition.

    PubMed

    Peterkova, Renata; Lesot, Hervé; Peterka, Miroslav

    2006-05-15

    Structures suppressed during evolution can be retraced due to atavisms and vestiges. Atavism is an exceptional emergence of an ancestral form in a living individual. In contrast, ancestral vestige regularly occurs in all members of an actual species. We surveyed data about the vestigial and atavistic teeth in mammals, updated them by recent findings in mouse and human embryos, and discussed their ontogenetic and evolutionary implications. In the mouse incisor and diastema regions, dental placodes are transiently distinct being morphologically similar to the early tooth primordia in reptiles. Two large vestigial buds emerge in front of the prospective first molar and presumably correspond to the premolars eliminated during mouse evolution. The incorporation of the posterior premolar vestige into the lower first molar illustrates the putative mechanism of evolutionary disappearance of the last premolar in the mice. In mutant mice, devious development of the ancestral tooth primordia might lead to their revivification and origin of atavistic supernumerary teeth. Similarity in the developmental schedule between three molars in mice and the respective third and fourth deciduous premolar and the first molar in humans raises a question about putative homology of these teeth. The complex patterning of the vestibular and dental epithelium in human embryos is reminiscent of the pattern of "Zahnreihen" in lower vertebrates. A hypothesis was presented about the developmental relationship between the structures at the external aspect of the dentition in mammals (oral vestibule, pre-lacteal teeth, paramolar cusps/teeth), the tooth glands in reptiles, and the earliest teeth in lower vertebrates. PMID:16463376

  1. The Effects of Calcitonin on the Development of and Ca2+ Levels in Heat-shocked Bovine Preimplantation Embryos In Vitro

    PubMed Central

    KAMANO, Shumpei; IKEDA, Shuntaro; SUGIMOTO, Miki; KUME, Shinichi

    2014-01-01

    Intracellular calcium homeostasis is essential for proper cell function. We investigated the effects of heat shock on the development of and the intracellular Ca2+ levels in bovine preimplantation embryos in vitro and the effects of calcitonin (CT), a receptor-mediated Ca2+ regulator, on heat shock-induced events. Heat shock (40.5 C for 10 h between 20 and 30 h postinsemination) of in vitro-produced bovine embryos did not affect the cleavage rate; however, it significantly decreased the rates of development to the 5- to 8-cell and blastocyst stages as compared with those of the control cultured for the entire period at 38.5 C (P < 0.05). The relative intracellular Ca2+ levels at the 1-cell stage (5 h after the start of heat shock), as assessed by Fluo-8 AM, a fluorescent probe for Ca2+, indicated that heat shock significantly lowered the Ca2+ level as compared with the control level. Semiquantitative reverse transcription PCR and western blot analyses revealed the expression of CT receptor in bovine preimplantation embryos. The addition of CT (10 nM) to the culture medium ameliorated the heat shock-induced impairment of embryonic development beyond the 5- to 8-cell stage. The Ca2+ level in the heat-shocked embryos cultured with CT was similar to that of the control embryos, suggesting that heat shock lowers the Ca2+ level in fertilized embryos in vitro and that a lower Ca2+ level is implicated in heat shock-induced impairment of embryonic development. Intracellular Ca2+-mobilizing agents, e.g., CT, may effectively circumvent the detrimental effects of heat shock on early embryonic development. PMID:24899099

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

    PubMed

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

    2016-02-26

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. PMID:26850853

  3. Studies toward birth and early mammalian development in space

    NASA Astrophysics Data System (ADS)

    Ronca, April E.

    2003-10-01

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

  4. The mammalian oocyte orchestrates the rate of ovarian follicular development

    PubMed Central

    Eppig, John J.; Wigglesworth, Karen; Pendola, Frank L.

    2002-01-01

    The development of both the mammalian oocyte and the somatic cell compartments of the ovarian follicle is highly coordinated; this coordination ensures that the ovulated oocyte is ready to undergo fertilization and subsequent embryogenesis. Disruption of this synchrony results in oocyte developmental failure. Communication between the oocyte and companion somatic cells is essential for successful development of both follicular compartments. However, it was not previously known whether one cell type, either the somatic or the germ cell compartment, determines the overall rate of follicular development. To test the hypothesis that the oocyte orchestrates the rate of follicle development, mid-sized oocytes isolated from secondary follicles were transferred back to primordial follicles, the earliest stage of follicular development. This transfer doubled the rate of follicular development and the differentiation of follicular somatic cells. Oocyte development in these accelerated follicles appeared normal; recovered oocytes were competent to undergo fertilization and embryonic development. These results demonstrate that oocytes orchestrate and coordinate the development of mammalian ovarian follicles and that the rate of follicular development is based on a developmental program intrinsic to the oocyte. PMID:11867735

  5. Linking development with generation of novelty in mammalian teeth

    PubMed Central

    Jernvall, Jukka

    2000-01-01

    The evolution of mammalian teeth is characterized by the frequent and convergent evolution of new cusps. The evolution of new cusps can be linked to tooth development via population-level variation. This allows testing whether development increases the capacity to evolve, or evolvability, by facilitating and even directing morphological change. In a population sample of living seals, variation in cusp number of individual teeth is from three to five cusps, the variably present cusps being the shortest ones that also develop last. By factoring in recent evidence on development, I show that the variation in cusp number can be explained by a patterning cascade mode of cusp development that cumulatively increases and directs height variation in short cusps. The biased variation in seal tooth cusps supports the recognition of teeth as highly evolvable because only small developmental changes are needed to produce large changes in size and number of small cusps. This evolvability of tooth cusps may have facilitated the fast and independent acquisition of new cusps in mammalian evolution. In phylogenetic studies, small cusps may be unreliable as phylogenetic signals. Population level variation can be a powerful tool in testing and generating hypotheses in developmental evolution studies. PMID:10706636

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

    PubMed

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

    2015-11-01

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

  7. The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts morphogenesis of the rat pre-implantation embryo

    PubMed Central

    Hutt, Karla J; Shi, Zhanquan; Albertini, David F; Petroff, Brian K

    2008-01-01

    Background Environmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR) pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation. Results We performed a systematic 3 dimensional (3D) confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months) and acute (50 ng/kg and 1 μg/kg at proestrus) maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell), with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts. Conclusion We have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses. PMID:18171477

  8. Evolution and development of the mammalian cerebral cortex

    PubMed Central

    Molnár, Zoltán; Kaas, Jon H.; de Carlos, Juan A.; Hevner, Robert F.; Lein, Ed; Němec, Pavel

    2014-01-01

    Comparative developmental studies of the mammalian brain can identify key changes that can generate the diverse structures and functions of brains. We have studied how the neocortex of early mammals became organized into functionally distinct areas, and how the current level of cortical cellular and laminar specialization arose from the simpler premammalian cortex. We demonstrate the neocortical organization in early mammals that is most informative for an understanding of how the large, complex human brain evolved from a long line of ancestors. The radial and tangential enlargement of the cortex was driven by changes in the patterns of cortical neurogenesis, including alterations in the proportions of distinct progenitor types. Some cortical cell populations travel to the cortex through tangential migration, others migrate radially. A number of recent studies have begun to characterize the chick, mouse, human and non-human primate cortical transcriptome to help us understand how gene expression relates to the development, and to the anatomical and functional organization of the adult neocortex. Although all mammalian forms share the basic layout of cortical areas, the areal proportions and distributions are driven by distinct evolutionary pressures acting on sensory and motor experiences during the individual ontogenies. PMID:24776993

  9. 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

  10. 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).

  11. Non-coding RNAs in mammalian sexual development.

    PubMed

    McFarlane, L; Wilhelm, D

    2009-01-01

    The present decade is witnessing a paradigm shift in our understanding of gene regulation. RNA, once relegated to an intermediary between DNA and protein, has emerged as a key contributor in the coordination of complex developmental pathways. For sexually reproducing organisms, propagation of the species is accomplished via an elaborate sexual phenotype. In mammals this consists of a highly complex cell lineage that has the capacity for intricate self-differentiation whilst maintaining the potential to generate all cell types upon fertilization. In addition, mammals possess a diverse range of somatic reproductive tissues and organs that often undergo dynamic morphological changes in response to a variety of external and internal cues. Although the protein component required to mediate these processes continues to be vigorously investigated, it is becoming increasingly apparent that an understanding of the non-coding RNA (ncRNA) component is required to develop a comprehensive picture of mammalian sexual development. PMID:20197714

  12. Two stages in the development of a mammalian retinocollicular projection.

    PubMed

    Mark, R F; Freeman, T C; Ding, Y; Marotte, L R

    1993-11-18

    The retinocollicular projection in the marsupial mammal the wallaby Macropus eugenii, has been investigated anatomically to determine the order in the developing projection and electrophysiologically to determine the time of onset of synaptic transmission by recording evoked potentials in the colliculus in response to stimulation of the optic nerve. There are two clear stages: a protracted period when retinal axons grow into the colliculus in coarse retinotopic order with no recordable electrical activity followed by the formation of terminal zones in retinotopically correct positions, the loss of more widely distributed axons and the onset of evoked potentials. The two stages are not seen in non-mammalian vertebrates where the projection is functional from the beginning. PMID:8110999

  13. Reprogenetics: Preimplantational genetics diagnosis

    PubMed Central

    Coco, Roberto

    2014-01-01

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

  14. Coculture of Preimplantation Embryos With Outgrowth Embryos Improves Embryonic Developmental Competence in Mice.

    PubMed

    Kim, Jihyun; Lee, Jaewang; Kim, Seok Hyun; Jun, Jin Hyun

    2016-07-01

    Mammalian embryonic development is an intricate succession of physiological and morphological events. Many studies have focused on optimizing in vitro culture systems for improvement in embryonic development. In this study, we established a novel coculture method with outgrowth embryos and investigated how this coculture system improves the preimplantation and peri-implantation embryonic development both in vitro and in utero. We found that outgrowth embryos secrete vesicles, as observed by time-lapse monitoring and scanning electron microscopy. Coculture with outgrowth embryos also significantly increased the percentages of morula, blastocyst, hatching, and outgrowth (P < .01). The total number of cells and inner cell mass were increased, and apoptotic index was decreased (P < .05) by upregulating Survivin and Lif messenger RNA expression levels (P < .05) in the coculture compared to the control group. Furthermore, implantation rates in utero after embryo transfer were significantly higher for cocultured embryos than those for the control group (P < .05). We conclude that embryotrophic factors secreted from outgrowth embryos could improve the developmental competence of in vitro cultured mouse preimplantation embryos. Findings of specific embryotrophic factors from outgrowth embryos might be valuable for advancing reproductive technologies in the future. PMID:26704525

  15. 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

  16. The role of Six1 in mammalian auditory system development.

    PubMed

    Zheng, Weiming; Huang, Li; Wei, Zhu-Bo; Silvius, Derek; Tang, Bihui; Xu, Pin-Xian

    2003-09-01

    The homeobox Six genes, homologues to Drosophila sine oculis (so) gene, are expressed in multiple organs during mammalian development. However, their roles during auditory system development have not been studied. We report that Six1 is required for mouse auditory system development. During inner ear development, Six1 expression was first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using recently generated Six1 mutant mice, we found that all Six1(+/-) mice showed some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1(-/-) mice displayed malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1(-/-) embryos arrested at the otic vesicle stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium. Because we previously reported that Six1 expression in the otic vesicle is Eya1 dependent, we first clarified that Eya1 expression was unaffected in Six1(-/-) otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. We also analyzed several other otic markers and found that the expression of Pax2 and Pax8 was unaffected in Six1(-/-) otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1 is required for regional

  17. 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

  18. ENU mutagenesis reveals that Notchless homolog 1 (Drosophila) affects Cdkn1a and several members of the Wnt pathway during murine pre-implantation development

    PubMed Central

    2012-01-01

    Background Our interests lie in determining the genes and genetic pathways that are important for establishing and maintaining maternal-fetal interactions during pregnancy. Mutation analysis targeted to a 34 Mb domain flanked by Trp53 and Wnt3 demonstrates that this region of mouse chromosome 11 contains a large number of essential genes. Two mutant alleles (l11Jus1 and l11Jus4), which fall into the same complementation group, survive through implantation but fail prior to gastrulation. Results Through a positional cloning strategy, we discovered that these homozygous mutant alleles contain non-conservative missense mutations in the Notchless homolog 1 (Drosophila) (Nle1) gene. NLE1 is a member of the large WD40-repeat protein family, and is thought to signal via the canonical NOTCH pathway in vertebrates. However, the phenotype of the Nle1 mutant mice is much more severe than single Notch receptor mutations or even in animals in which NOTCH signaling is blocked. To test the hypothesis that NLE1 functions in multiple signaling pathways during pre-implantation development, we examined expression of multiple Notch downstream target genes, as well as select members of the Wnt pathway in wild-type and mutant embryos. We did not detect altered expression of any primary members of the Notch pathway or in Notch downstream target genes. However, our data reveal that Cdkn1a, a NOTCH target, was upregulated in Nle1 mutants, while several members of the Wnt pathway are downregulated. In addition, we found that Nle1 mutant embryos undergo caspase-mediated apoptosis as hatched blastocysts, but not as morulae or blastocysts. Conclusions Taken together, these results uncover potential novel functions for NLE1 in the WNT and CDKN1A pathways during embryonic development in mammals. PMID:23231322

  19. 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

  20. 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.

  1. 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

  2. ANEUPLOIDY TEST DEVELOPMENT: KINETOCHORE STAINING IN MAMMALIAN SYSTEMS

    EPA Science Inventory

    The purpose of the project was to determine the feasibility of using human-derived antibodies against the chromosomal kinetochore region coupled with immunofluorescence staining as a method for evaluating the induction of aneuploidy in mammalian cells in vitro and in vivo. The te...

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

    PubMed Central

    2011-01-01

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

  4. [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

  5. Early embryonic development and preimplantation changes in the uterus of the bat Rhinopoma hardwickei hardwickei (Gray) (Rhinopomatidae).

    PubMed

    Karim, K B; Fazil, M

    1987-04-01

    Rhinopoma hardwickei hardwickei has an annual reproductive cycle. Although many of the females become inseminated from the latter half of February until about the middle of April, ovulation has not been recorded until the 11th of March. A single follicle reached full development and released one ovum from either of the ovaries with nearly equal frequency, and a single conceptus was carried in the ipsilateral uterine cornu during each cycle. The embryo descended into the uterus as an early morula and attained the bilaminar blastocyst stage before undergoing implantation. As the morula advanced in age, the embryonic surface of the zona became progressively more basophilic. Hence in advanced morulae, the inner surface of the zona pellucida took a dark stain with hematoxylin and appeared like a distinct thin membrane, while the rest of the thickness of the zona was eosinophilic. Although progestational changes commenced in both uterine cornua, they became augmented in the uterine cornu on the side of ovulation and blastocyst attachment. After blastocyst attachment, the contralateral cornu reverted to an anestrus condition. The progestational changes became less conspicuous from the cranial to the caudal end of the uterus. Evidently, there was a linear gradient in the progestational response of the uterus with the cranial end being most responsive and the caudal end least responsive. The precise mechanism which brings this about is not known. PMID:3604954

  6. 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

  7. 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.

  8. Teratological research using in vitro systems. I. Mammalian whole embryo culture.

    PubMed Central

    Flynn, T J

    1987-01-01

    Approximately 390 literature references (through spring 1986) were reviewed for mammalian whole embryo culture procedures, with particular attention to the development of those cultures as systems for teratogenicity testing. The existing procedures could be conveniently divided into three groups, which are defined by the periods of embryogenesis that they embrace: preimplantation, peri-implantation, and post-implantation culture systems. The literature on peri-implantation embryo culture was sparse, and it did not appear that this procedure is being actively developed as a teratogen screening test. The extensive literature on both preimplantation and postimplantation embryo culture suggested considerable use of these two methods in evaluating embryotoxicants. The following discussion was compiled from information gleaned from all references. However, in the interest of brevity, only representative articles are specifically cited. Because the background and methodology for each system are distinct, each system will be discussed separately. PMID:3304996

  9. 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.

  10. 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

  11. Impairment of Preimplantation Porcine Embryo Development by Histone Demethylase KDM5B Knockdown Through Disturbance of Bivalent H3K4me3-H3K27me3 Modifications1

    PubMed Central

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

    2015-01-01

    ABSTRACT KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for embryonic stem cell self-renewal, cell fate, and murine embryonic development. However, little is known about the role of KDM5B during preimplantation embryo development. Here we show that KDM5B is critical to porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired porcine embryo development to the blastocyst stage. The impairment of embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for porcine embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications. PMID:25609834

  12. 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. PMID:19399586

  13. Characterization of BRD4 during Mammalian Postmeiotic Sperm Development

    PubMed Central

    Bryant, Jessica M.; Donahue, Greg; Wang, Xiaoshi; Meyer-Ficca, Mirella; Luense, Lacey J.; Weller, Angela H.; Bartolomei, Marisa S.; Blobel, Gerd A.; Meyer, Ralph G.; Garcia, Benjamin A.

    2015-01-01

    During spermiogenesis, the postmeiotic phase of mammalian spermatogenesis, transcription is progressively repressed as nuclei of haploid spermatids are compacted through a dramatic chromatin reorganization involving hyperacetylation and replacement of most histones with protamines. Although BRDT functions in transcription and histone removal in spermatids, it is unknown whether other BET family proteins play a role. Immunofluorescence of spermatogenic cells revealed BRD4 in a ring around the nuclei of spermatids containing hyperacetylated histones. The ring lies directly adjacent to the acroplaxome, the cytoskeletal base of the acrosome, previously linked to chromatin reorganization. The BRD4 ring does not form in acrosomal mutant mice. Chromatin immunoprecipitation followed by sequencing in spermatids revealed enrichment of BRD4 and acetylated histones at the promoters of active genes. BRD4 and BRDT show distinct and synergistic binding patterns, with a pronounced enrichment of BRD4 at spermatogenesis-specific genes. Direct association of BRD4 with acetylated H4 decreases in late spermatids as acetylated histones are removed from the condensing nucleus in a wave following the progressing acrosome. These data provide evidence of a prominent transcriptional role for BRD4 and suggest a possible removal mechanism for chromatin components from the genome via the progressing acrosome as transcription is repressed and chromatin is compacted during spermiogenesis. PMID:25691659

  14. Preimplantation factor (PIF) promotes human trophoblast invasion.

    PubMed

    Moindjie, Hadia; Santos, Esther Dos; Loeuillet, Laurence; Gronier, Héloise; de Mazancourt, Philippe; Barnea, Eytan R; Vialard, François; Dieudonne, Marie-Noëlle

    2014-11-01

    Preimplantation factor (PIF) is a peptide secreted by viable mammalian embryos. Moreover, it can be detected in the circulation of pregnant women. Recently, it was shown that PIF promotes invasion in trophoblast cell lines in vitro. Successful human embryo implantation depends on a deep and highly controlled invasion of extravillous trophoblast (EVT) in the maternal endometrium. Trophoblast invasion is regulated in part by matrix metalloproteinase (MMP) activity and integrin expression. The present study demonstrates the presence of PIF in early pregnancy and characterizes its effects on primary human trophoblast invasion. At the fetomaternal interface, intense PIF labeling by immunohistochemistry was present during early gestation in villous trophoblasts and EVTs. A decrease of labeling was observed at term. Furthermore, PIF significantly promoted invasion of human EVT isolated from first-trimester placenta. The proinvasive regulatory effect of PIF in EVT was associated with 1) increased MMP9 activity and 2) reduced tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA expression. PIF also regulated alpha v and alpha 1 integrin mRNA expressions. Last, the proinvasive effect of PIF appeared to be mediated by the mitogen-activated protein kinase (MAPK), phosphoinositide-3-kinase (PI3K), and Janus-kinase signal transducer and activator of transcription (JAK-STAT) signaling pathways. In summary, this work describes the direct, positive effect of PIF on the control of human trophoblastic cell invasion by modulation of MMP/TIMP balance and integrin expression. Moreover, these results suggest that PIF is involved in pathological pregnancies characterized by insufficient or excessive trophoblast invasion. PMID:25232018

  15. [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. PMID:27183788

  16. 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.

  17. 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. PMID:26273607

  18. 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

  19. Constraints on Mammalian forelimb development: insights from developmental disparity.

    PubMed

    Ross, Darcy; Marcot, Jonathan D; Betteridge, Keith J; Nascone-Yoder, Nanette; Bailey, C Scott; Sears, Karen E

    2013-12-01

    Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark-based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo-wide constraint. PMID:24299415

  20. 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

  1. 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

  2. Preimplantation genetic diagnosis for hemoglobinopathies.

    PubMed

    Kuliev, Anver; Pakhalchuk, Tatiana; Verlinsky, Oleg; Rechitsky, Svetlana

    2011-01-01

    Hemoglobinopathies are the most frequent indications for preimplantation genetic diagnosis (PGD), allowing couples at-risk of bearing offspring with thalassemia and sickle cell disease to reproduce without fear of having an affected child. The present experience includes PGD for sickle cell disease, α- and β-thalassemia (α- and β-thal). We present here the results of the world's largest experience of over 395 PGD cycles for hemoglobin (Hb) disorders, resulting in the birth of 98 healthy, hemoglobinopathy-free children, with seven pregnancies still ongoing. One-third of these cases were performed in combination with HLA typing, allowing the birth of unaffected children who were also HLA identical to the affected siblings with hemoglobinopathies in these families, with successful or pending stem cell transplantation in a dozen of them. The results show that PGD is presently a practical approach for prevention of hemoglobinopathies, gradually also becoming a useful approach to improving access to HLA-compatible stem cell transplantation for this group of diseases. PMID:21910603

  3. HDAC1 and HDAC2 in mouse oocytes and preimplantation embryos: Specificity versus compensation.

    PubMed

    Ma, P; Schultz, R M

    2016-07-01

    Oocyte and preimplantation embryo development entail dynamic changes in chromatin structure and gene expression, which are regulated by a number of maternal and zygotic epigenetic factors. Histone deacetylases (HDACs), which tighten chromatin structure, repress transcription and gene expression by removing acetyl groups from histone or non-histone proteins. HDAC1 and HDAC2 are two highly homologous Class I HDACs and display compensatory or specific roles in different cell types or in response to different stimuli and signaling pathways. We summarize here the current knowledge about the functions of HDAC1 and HDAC2 in regulating histone modifications, transcription, DNA methylation, chromosome segregation, and cell cycle during oocyte and preimplantation embryo development. What emerges from these studies is that although HDAC1 and HDAC2 are highly homologous, HDAC2 is more critical than HDAC1 for oocyte development and reciprocally, HDAC1 is more critical than HDAC2 for preimplantation development. PMID:27082454

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

    PubMed Central

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

    2014-01-01

    Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer’s disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)–dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development. PMID:25205808

  5. 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

  6. 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

  7. 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

  8. Preimplantation genetic diagnosis in Saudi Arabia

    PubMed Central

    Abotalib, Zeinab

    2013-01-01

    Preimplantation genetic diagnosis (PGD) testing is the practice of obtaining a cellular biopsy sample from a developing human oocyte or embryo, acquired via a cycle of in vitro fertilization (IVF); evaluating the genetic composition of this sample; and using this information to determine which embryos will be optimal for subsequent uterine transfer. PGD has become an increasingly useful adjunct to IVF procedures. The ability to provide couples who are known carriers of genetic abnormalities the opportunity to deliver healthy babies has opened a new frontier in reproductive medicine. The purpose of the PGD is enables us to choose which embryos will be implanted into the mother. In the present study 137 families who had undergone IVF at Habib Medical Centre, were enrolled for the PGD analysis. The couple visited the clinic for the sex selection, recurrent fetal loss and with the recurrent IVF failure. 802 embryos were tested by the biopsy method and 512 are found to be normal and 290 were abnormal embryos. In this study only 24% of the embryos were transferred and the remaining was not transferred because of the abnormalities or undesired sex of the embryos. The structural and numerical abnormalities were found to be 16.8%. PMID:23750087

  9. Preimplantation genetic diagnosis in Saudi Arabia.

    PubMed

    Abotalib, Zeinab

    2013-01-01

    Preimplantation genetic diagnosis (PGD) testing is the practice of obtaining a cellular biopsy sample from a developing human oocyte or embryo, acquired via a cycle of in vitro fertilization (IVF); evaluating the genetic composition of this sample; and using this information to determine which embryos will be optimal for subsequent uterine transfer. PGD has become an increasingly useful adjunct to IVF procedures. The ability to provide couples who are known carriers of genetic abnormalities the opportunity to deliver healthy babies has opened a new frontier in reproductive medicine. The purpose of the PGD is enables us to choose which embryos will be implanted into the mother. In the present study 137 families who had undergone IVF at Habib Medical Centre, were enrolled for the PGD analysis. The couple visited the clinic for the sex selection, recurrent fetal loss and with the recurrent IVF failure. 802 embryos were tested by the biopsy method and 512 are found to be normal and 290 were abnormal embryos. In this study only 24% of the embryos were transferred and the remaining was not transferred because of the abnormalities or undesired sex of the embryos. The structural and numerical abnormalities were found to be 16.8%. PMID:23750087

  10. 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

  11. [Pre-implant esthetic study].

    PubMed

    Missika, P; Khayat, P

    1990-09-01

    The first dental prostheses used on Branemark implants were aesthetically disappointing both for the dentists and their patients. Therefore the authors will consider the various aesthetic problems encountered when treating loss of teeth with implant systems. The problems related to resorption are numerous: large bone losses are resolved by adapting removable acrylic, carrying out bone transplants immediately fixed by the implants, using filling materials, or complete dentures fixed with attachments supported by the implants. Periodontal surgery often provides a solution to the problem of gum visibility at the level of the maxillary anterior teeth. The problems related to the site where implants emerge can often be avoided by consultation between the surgeon and the prosthodontist and by flexing a surgical guide compiled from a pre-prosthetic analysis of the clinical situation. The aesthetic problems related to the actual implant systems are dependent on three factors: When the prosthesis is directly screwed onto the implant, the axis of the implant determines the axis of the dental prosthesis and can lead to the emergence of the screw on the buccal surface; With angulated cores, orientated screws provide the required solution. The implant material, when metallic leads to an unsightly border at the gingival level. Ceramic implants, or the "ceraming" of titanium, provide a solution to this problem. In case of diastema the use of an implant system gives the best choice in comparison to the more conventional treatments. In conclusion, the authors point out the importance of pre-implant analysis which must give an evaluation of the aesthetic result. The fragility of the aesthetic evaluation should encourage dentists to obtain the "clear and written consent" of their patients, accepting the risks run by treatment of this kind. PMID:2268774

  12. 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.

  13. DEVELOPMENT, STANDARDIZATION AND VALIDATION OF THE MAMMALIAN IN VIVO ASSAYS IN THE PROPOSED TIER I SCREENING BATTERY FOR ENDOCRINE DISRUPTORS

    EPA Science Inventory

    This research directly supports the development, standardization and validation of several Tier 1 screening mammalian in vivo assays. Through the development and use of many of these assays for testing specific hypothesis in their respective research programs, these investigato...

  14. 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

  15. Transcription-independent heritability of induced histone modifications in the mouse preimplantation embryo.

    PubMed

    VerMilyea, Matthew D; O'Neill, Laura P; Turner, Bryan M

    2009-01-01

    Enzyme-catalyzed, post-translational modifications of core histones have been implicated in the complex changes in gene expression that drive early mammalian development. However, until recently the small number of cells available from the preimplantation embryo itself has prevented quantitative analysis of histone modifications at key regulator genes. The possible involvement of histone modifications in the embryo's response to extracellular signals, or as determinants of cell fate or lineage progression, remains unclear. Here we describe the use of a recently-developed chromatin immunoprecipitation technique (CChIP) to assay histone modification levels at key regulator genes (Pou5f1, Nanog, Cdx2, Hoxb1, Hoxb9) as mouse embryos progress from 8-cell to blastocyst in culture. Only by the blastocyst stage, when the embryonic (Inner Cell Mass) and extra-embryonic (Trophoblast) lineages are compared, do we see the expected association between histone modifications previously linked to active and silent chromatin, and transcriptional state. To explore responses to an environmental signal, we exposed embryos to the histone deacetylase inhibitor, anti-epileptic and known teratogen valproic acid (VPA), during progression from 8-cell to morula stage. Such treatment increased H4 acetylation and H3 lysine 4 methylation at the promoters of Hoxb1 and Hoxb9, but not the promoters of Pou5f1, Nanog,Cdx2 or the housekeeping gene Gapdh. Despite the absence of detectable Hoxb transcription, these VPA-induced changes were heritable, following removal of the inhibitor, at least until the blastocyst stage. The selective hyperacetylation of Hoxb promoters in response to a histone deacetylase inhibitor, suggests that Hox genes have a higher turnover of histone acetates than other genes in the preimplantation embryo. To explain the heritability, through mitosis, of VPA-induced changes in histone modification at Hoxb promoters, we describe how an epigenetic feed-forward loop, based on cross

  16. 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. PMID:26975478

  17. Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

    PubMed Central

    Antony, N.; McDougall, A. R.; Mantamadiotis, T.; Cole, T. J.; Bird, A. D.

    2016-01-01

    During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1−/− mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1−/− mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1−/− mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

  18. Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms.

    PubMed

    Antony, N; McDougall, A R; Mantamadiotis, T; Cole, T J; Bird, A D

    2016-01-01

    During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1(-/-) mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1(-/-) mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1(-/-) mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. Cellular and molecular events on the development of mammalian thyroid C cells.

    PubMed

    Kameda, Yoko

    2016-03-01

    Thyroid C cells synthesize and secrete calcitonin, a serum calcium-lowering hormone. This review provides our current understanding of mammalian thyroid C cells from the molecular and morphological perspectives. Several transcription factors and signaling molecules involved in the development of C cells have been identified, and genes expressed in the pharyngeal pouch endoderm, neural crest-derived mesenchyme in the pharyngeal arches, and ultimobranchial body play critical roles for the development of C cells. It has been generally accepted, without much-supporting evidence, that mammalian C cells, as well as the avian cells, are derived from the neural crest. However, by fate mapping of neural crest cells in both Wnt1-Cre/R26R and Connexin(Cxn)43-lacZ transgenic mice, we showed that neural crest cells colonize neither the fourth pharyngeal pouch nor the ultimobranchial body. E-cadherin, an epithelial cell marker, is expressed in thyroid C cells and their precursors, the fourth pharyngeal pouch and ultimobranchial body. Furthermore, E-cadherin is colocalized with calcitonin in C cells. Recently, lineage tracing in Sox17-2A-iCre/R26R mice has clarified that the pharyngeal endoderm-derived cells give rise to C cells. Together, these findings indicate that mouse thyroid C cells are endodermal in origin. Developmental Dynamics 245:323-341, 2016. © 2015 Wiley Periodicals, Inc. PMID:26661795

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

    PubMed Central

    Son, Esther Y.; Crabtree, Gerald R.

    2015-01-01

    The BAF (mammalian SWI/SNF) complexes are a family of multi-subunit ATP-dependent chromatin remodelers that use ATP hydrolysis to alter chromatin structure. Distinct BAF complex compositions are possible through combinatorial assembly of homologous subunit families and can serve non-redundant functions. In mammalian neural development, developmental stage-specific BAF assemblies are found in ES cells, neural progenitors and postmitotic neurons. In particular, the neural progenitor-specific BAF complexes are essential for controlling the kinetics and mode of neural progenitor cell division, while neuronal BAF function is necessary for the maturation of postmitotic neuronal phenotypes as well as long-term memory formation. The microRNA-mediated mechanism for transitioning from npBAF to nBAF complexes is instructive for the neuronal fate and can even convert fibroblasts into neurons. The high frequency of BAF subunit mutations in neurological disorders underscores the rate-determining role of BAF complexes in neural development, homeostasis and plasticity. PMID:25195934

  7. Development of an Improved Mammalian Overexpression Method for Human CD62L

    PubMed Central

    Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.

    2014-01-01

    We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

  8. Development of an improved mammalian overexpression method for human CD62L.

    PubMed

    Brown, Haley A; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D

    2015-01-01

    We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5-30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402

  9. Heat Shock Memory in Preimplantation Mouse Embryos

    PubMed Central

    Jia, Yanwei; Hartshorn, Cristina; Hartung, Odelya; Wangh, Lawrence J.

    2010-01-01

    To investigate the consequences of possible physiological stress to embryos caused by the in vitro fertilization procedures, we used as a model heat shock response in preimplantation mouse embryos. A heat shock “memory” was discovered that renders cleavage-stage embryos more responsive at the transcriptional level to secondary perturbation with very low doses of heat, even several cell cycles after the initial stress has occurred. PMID:20378108

  10. An Important Role of Pumilio 1 in Regulating the Development of the Mammalian Female Germline.

    PubMed

    Mak, Winifred; Fang, Caodi; Holden, Tobias; Dratver, Milana Bockhur; Lin, Haifan

    2016-06-01

    Pumilio/FBF (PUF) proteins are a highly conserved family of translational regulators. The Drosophila PUF protein, Pumilio, is crucial for germline establishment and fertility. In mammals, primordial folliculogenesis is a key process that establishes the initial cohort of female mammalian germ cells prior to birth, and this primordial follicle pool is a prerequisite for female reproductive competence. We sought to understand whether PUF proteins have a conserved role in mammals during primordial folliculogenesis and female reproductive competency. In mammals, two homologs of Pumilio exist: Pumilio 1 (Pum1) and Pum2. Here, we report that PUMILIO (PUM) 1 plays an important role in the establishment of the primordial follicle pool, meiosis, and female reproductive competency, whereas PUM2 does not have a detectable function in these processes. Furthermore, we show that PUM1 facilitates the transition of the late meiotic prophase I oocyte from pachytene to diplotene stage by regulating SYCP1 protein. Our study reveals an important role of translational regulation in mammalian female germ cell development. PMID:27170441

  11. Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws.

    PubMed

    Hamrick, M W

    2001-01-01

    Paleontological evidence indicates that the evolutionary diversification of mammals early in the Cenozoic era was characterized by an adaptive radiation of distal limb structures. Likewise, neontological data show that morphological variation in distal limb integumentary appendages (e.g., nails, hooves, and claws) can be observed not only among distantly related mammalian taxa but also among closely related species within the same clade. Comparative analysis of nail, claw, and hoof morphogenesis reveals relatively subtle differences in mesenchymal and epithelial patterning underlying these adult differences in distal limb appendage morphology. Furthermore, studies of regulatory gene expression during vertebrate claw development demonstrate that many of the signaling molecules involved in patterning ectodermal derivatives such as teeth, hair, and feathers are also involved in organizing mammalian distal limb appendages. For example, Bmp4 signaling plays an important role during the recruitment of mesenchymal cells into the condensations forming the terminal phalanges, whereas Msx2 affects the length of nails and claws by suppressing proliferation of germinal epidermal cells. Evolutionary changes in the form of distal integumentary appendages may therefore result from changes in gene expression during formation of mesenchymal condensations (Bmp4, posterior Hox genes), induction of the claw fold and germinal matrix (shh), and/or proliferation of epidermal cells in the claw matrix (Msx1, Msx2). The prevalence of convergences and parallelisms in nail and claw structure among mammals underscores the existence of multiple morphogenetic pathways for evolutionary change in distal limb appendages. PMID:11710767

  12. 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

  13. 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

  14. Foetal and placental growth in the mouse after pre-implantation development in vitro under oxygen concentrations of 5 and 20%.

    PubMed

    Harlow, G M; Quinn, P

    1979-06-01

    Blastocysts which developed from two-cell mouse embryos in culture tubes containing an atmosphere with 20% oxygen had approximately 20% fewer blastomeres than blastocysts which developed under an oxygen concentration of 5%. When these smaller blastocysts were transferred to the uteri of pseudopregnant foster mothers, the foetuses developing were as viable as those developing from blastocysts cultured under 5% oxygen, indicating their ability to regulate for a lower blastomere number by at least day 17 of development. The transfer operation itself had no adverse effect on foetal or placental growth. However, culture of blastocysts in vitro did depress foetal though not placental growth, suggesting that the inner cell mass is more susceptible than the trophectoderm to culture in vitro. Foetal but not placental growth was lower following the transfer of blastocysts to a day-3 rather than a day-4 uterus. Four cases of placental fusion were found. In one case, the foetuses were contained within the same embryonic sac and may have been twins. PMID:508209

  15. 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

  16. Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs.

    PubMed

    Park, Bola; Lee, Hanna; Lee, Yongjin; Elahi, Fazle; Lee, Joohyeong; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Eunsong

    2016-08-01

    This study was conducted to evaluate the effects of treatment with the cAMP modulators cilostamide and/or forskolin during pre-IVM culture on meiotic progression, gap junction communication, intraoocyte cAMP level and glutathione content, embryonic development after parthenogenesis, and somatic cell nuclear transfer in pigs. Cumulus-oocyte complexes were cultured for 24 hours in unsupplemented medium or media containing 20 μM cilostamide and/or 50 μM forskolin. After pre-IVM, oocytes were cultured for 41 to 44 hours in a standard IVM medium to induce oocyte maturation. When the nuclear status of oocytes was examined after pre-IVM for 24 hours, a higher (P < 0.01) proportion of oocytes treated with forskolin (85.5%) and cilostamide + forskolin (92.6%) remained at the germinal vesicle stage compared with untreated (20.6%) and cilostamide-treated oocytes (54.7%). cAMP level in pre-IVM oocytes was significantly increased by combined treatment with cilostamide + forskolin (21.38 fmol/oocyte) relative to the no pre-IVM control, no treatment, cilostamide, and forskolin groups (2.85, 1.88, 1.74, and 8.95 fmol/oocyte, respectively). Forskolin with or without cilostamide significantly maintained open-gap junction communication relative to no treatment. Blastocyst formation in parthenogenesis was significantly (P < 0.01) improved by forskolin (65.3%) relative to other treatments (28.3% to 48.1%). Supplementation of pre-IVM with dibutyryl cAMP showed similar blastocyst formation as forskolin treatment (61.1% and 61.0%, respectively). In somatic cell nuclear transfer, simultaneous treatment with cilostamide + forskolin significantly (P < 0.05) increased embryonic development to the blastocyst stage (42.9%) relative to the no pre-IVM, control, and cilostamide groups (32.3, 28.6, and 32.8%, respectively). The glutathione contents in pre-IVM oocytes were increased by no treatment, forskolin, and cilostamide + forskolin (1.38, 1.39, and 1.27 pixels

  17. 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

  18. 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

  19. 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

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

    PubMed Central

    Brezina, Paul R.

    2013-01-01

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

  1. 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

  2. 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

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

    PubMed

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

    2015-08-01

    Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo. PMID:26241857

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

    PubMed Central

    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.

    2015-01-01

    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

  5. FURTHER DEVELOPMENT OF A MAMMALIAN DNA ALKALINE UNWINDING BIOASSAY WITH POTENTIAL APPLICATION TO HAZARD IDENTIFICATION FOR CONTAMINANTS FROM ENVIRONMENTAL SAMPLES

    EPA Science Inventory

    Recently, the authors detailed a DNA alkaline unwinding assay (DAUA) that can be used to rapidly measure chemically induced strand breaks in mammalian cells. Further developments of the assay include: studies on the relationship between DNA adducts and DNA strand breaks; evaluati...

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

    PubMed

    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

  7. Mammalian COPII coat component SEC24C is required for embryonic development in mice.

    PubMed

    Adams, Elizabeth J; Chen, Xiao-Wei; O'Shea, K Sue; Ginsburg, David

    2014-07-25

    COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution. PMID:24876386

  8. A Novel 4EHP-GIGYF2 Translational Repressor Complex Is Essential for Mammalian Development

    PubMed Central

    Morita, Masahiro; Ler, Lian Wee; Fabian, Marc R.; Siddiqui, Nadeem; Mullin, Michael; Henderson, Valerie C.; Alain, Tommy; Fonseca, Bruno D.; Karashchuk, Galina; Bennett, Christopher F.; Kabuta, Tomohiro; Higashi, Shinji; Larsson, Ola; Topisirovic, Ivan; Smith, Robert J.; Gingras, Anne-Claude

    2012-01-01

    The binding of the eukaryotic initiation factor 4E (eIF4E) to the mRNA 5′ cap structure is a rate-limiting step in mRNA translation initiation. eIF4E promotes ribosome recruitment to the mRNA. In Drosophila, the eIF4E homologous protein (d4EHP) forms a complex with binding partners to suppress the translation of distinct mRNAs by competing with eIF4E for binding the 5′ cap structure. This repression mechanism is essential for the asymmetric distribution of proteins and normal embryonic development in Drosophila. In contrast, the physiological role of the mammalian 4EHP (m4EHP) was not known. In this study, we have identified the Grb10-interacting GYF protein 2 (GIGYF2) and the zinc finger protein 598 (ZNF598) as components of the m4EHP complex. GIGYF2 directly interacts with m4EHP, and this interaction is required for stabilization of both proteins. Disruption of the m4EHP-GIGYF2 complex leads to increased translation and perinatal lethality in mice. We propose a model by which the m4EHP-GIGYF2 complex represses translation of a subset of mRNAs during embryonic development, as was previously reported for d4EHP. PMID:22751931

  9. 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

  10. 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

  11. 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

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

    PubMed Central

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

    2015-01-01

    Objective: To investigate the single blastocyst transfer in preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles. Methods: 80 PGD/PGS cycles undergoing blastocyst biopsy were studied. There were 88 warming cycles during the study period. Only one warmed blastocyst was transferred per cycle. The outcomes were followed up to the infants were born. Results: The embryo implantation rate was 54.55% (48/88). The clinical pregnancy rate was 54.55% (48/88) per transfer cycle and 60% (48/80) per initial PGD/PGS cycle. There was no multi-pregnant in this study. The live birth rate was 42.05% (37/88) per transfer cycle and 46.25% (37/80) per initial PGD/PGS cycle. Conclusion: In PGD/PGS cycles, single blastocyst transfer reduces the multiple pregnancy rate without affecting the clinical outcomes. PMID:26885112

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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. PMID:26487780

  15. 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

  16. Methionine-dependent histone methylation at developmentally important gene loci in mouse preimplantation embryos.

    PubMed

    Kudo, Mari; Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

    2015-12-01

    The involvement of specific nutrients in epigenetic gene regulation is a possible mechanism underlying nutrition-directed phenotypic alteration. However, the involvement of nutrients in gene-specific epigenetic regulation remains poorly understood. Methionine has been received attention as a possible nutrient involved in epigenetic modifications, as it is a precursor of the universal methyl donor for epigenetic methylation of DNA and histones. In the present study, the disruption of methionine metabolism by ethionine, an antimetabolite of methionine, induced abnormally higher expression of genes related to cell lineage differentiation and resulted in impaired blastocyst development of mouse preimplantation embryos in vitro. These effects were mitigated by the presence of methionine. Importantly, ethionine treatment induced lower trimethylation of histone H3 lysine 9 but did not affect methylation of DNA in the promoter regions of the examined genes. These results demonstrated that intact methionine metabolism is required for proper epigenetic histone modifications and normal expression of developmentally important genes during preimplantation development. PMID:26372092

  17. 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

  18. Distinct nuclear localization patterns of DNA methyltransferases in developing and mature mammalian retina.

    PubMed

    Nasonkin, Igor O; Lazo, Kevin; Hambright, Dustin; Brooks, Matthew; Fariss, Robert; Swaroop, Anand

    2011-07-01

    DNA methyltransferases--DNMT1, DNMT3a, and DNMT3b--produce methylation patterns that dynamically regulate chromatin remodeling and gene expression. The vertebrate retina provides an ideal model to elucidate molecular control of neurogenesis as all neuronal cell types and Müller glia are generated in a conserved order from common pools of progenitor cells. As a prelude to exploring epigenetic regulation of mammalian retinal development, we investigated the expression of Dnmt1, Dnmt3a, and Dnmt3b in the mouse retina from embryonic day (E) 10.5 to 10 months of age. High levels of transcripts for all three Dnmt genes were observed in early stages of retinal differentiation, with significantly reduced expression after birth. Although DNMT1 protein is abundant in retinal progenitors at E10.5, it becomes restricted to postmitotic cells by E15.5. Most cells in the postnatal retina show nuclear immunostaining of DNMT1; however, the photoreceptors exhibit distinctive patterns. In rods, weak expression of DNMT1 is detected in perinuclear region and in the nucleus, whereas a strong nuclear labeling is evident in cones. DNMT3a and DNMT3b show a discrete pattern in developing retina with high expression at E11.5, little or no immunostaining by E15.5, and then postnatal expression overlapping with DNMT1 in early born neurons (ganglion, amacrine and horizontal cells, and cones). Robust nuclear localization of DNMTs in cones compared to rods suggests a potential role of DNA methylation in differential remodeling of chromatin in these two specialized neurons. Our studies indicate that DNA methyltransferases contribute to the establishment and maturation of cell fates during retinal development. PMID:21452232

  19. 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

  20. Preimplantation genetic diagnosis for inherited neurological disorders.

    PubMed

    Tur-Kaspa, Ilan; Jeelani, Roohi; Doraiswamy, P Murali

    2014-07-01

    Preimplantation genetic diagnosis (PGD) is an option for couples at risk of having offspring with an inherited debilitating or fatal neurological disorder who wish to conceive a healthy child. PGD has been carried out for conditions with various modes of inheritance, including spinal muscular atrophy, Huntington disease, fragile X syndrome, and chromosomal or mitochondrial disorders, and for susceptibility genes for cancers with nervous system involvement. Most couples at risk of transmitting a genetic mutation would opt for PGD over prenatal testing and possible termination of a pregnancy. The aim of this Perspectives article is to assist neurologists in counselling and treating patients who wish to explore the option of PGD to enable conception of an unaffected child. PGD can be accomplished for most disorders in which the genetic basis is known, and we argue that it is time for clinicians and neurological societies to consider the evidence and to formulate guidelines for the responsible integration of PGD into modern preventative neurology. PMID:24866878

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

    PubMed

    King, D S

    1999-04-01

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

  2. PreImplantation factor (PIF) detection in maternal circulation in early pregnancy correlates with live birth (bovine model)

    PubMed Central

    2013-01-01

    Background Early identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome. Methods Artificially inseminated (AI) blind-coded Angus cattle (N = 21-23) serum samples (day10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N = 30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3 kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC. Results PIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins

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

    PubMed

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

    2016-08-01

    The interferon alpha-responsive gene (Ifrg15) mRNA is highly expressed in various stages during preimplantation mammalian embryo development. Unfortunately, few studies have investigated the effect of Ifrg15 in this process. In mammals, the fusion of male and female pronuclei generates a diploid zygote, and is an important step for subsequent cleavage and blastocyst formation. Here, by using RNA interference, rescue experiments, immunofluorescence staining and live cell observations, we found that preimplantation embryo development was arrested at the 1-cell stage after knocking down Ifrg15 expression. This induced DNA damage and prevented the cleavage of embryos. Furthermore, the effect of Ifrg15 deficiency in arresting preimplantation embryo development produced by specific short interfering RNA microinjection was concentration-dependent. Using transcriptome expression profiles, gene ontogeny functional annotation and enrichment analysis, we gained 197 enriched pathways based on 1445 differentially expressed genes (DEGs). Of these, 12 pathways and about one third of the DEGs were involved in DNA damage, DNA repair, cell cycle, and developmental processes. Thus, the IFRG15 protein might be an important molecule for maintaining genomic integrity and stability through upregulating or downregulating a cascade of genes to permit normal preimplantation embryo development. PMID:26911731

  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. 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.

  7. Self-correction of chromosomal abnormalities in human preimplantation embryos and embryonic stem cells.

    PubMed

    Bazrgar, Masood; Gourabi, Hamid; Valojerdi, Mojtaba Rezazadeh; Yazdi, Poopak Eftekhari; Baharvand, Hossein

    2013-09-01

    Aneuploidy is commonly seen in human preimplantation embryos, most particularly at the cleavage stage because of genome activation by third cell division. Aneuploid embryos have been used for the derivation of normal embryonic stem cell (ESC) lines and developmental modeling. This review addresses aneuploidies in human preimplantation embryos and human ESCs and the potential of self-correction of these aberrations. Diploid-aneuploid mosaicism is the most frequent abnormality observed; hence, embryos selected by preimplantation genetic diagnosis at the cleavage or blastocyst stage could be partly abnormal. Differentiation is known as the barrier for eliminating mosaic embryos by death and/or decreased division of abnormal cells. However, some mosaicisms, such as copy number variations could be compatible with live birth. Several reasons have been proposed for self-correction of aneuploidies during later stages of development, including primary misdiagnosis, allocation of the aneuploidy in the trophectoderm, cell growth advantage of diploid cells in mosaic embryos, lagging of aneuploid cell division, extrusion or duplication of an aneuploid chromosome, and the abundance of DNA repair gene products. Although more studies are needed to understand the mechanisms of self-correction as a rare phenomenon, most likely, it is related to overcoming mosaicism. PMID:23557100

  8. 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.

  9. Snail Coordinately Regulates Downstream Pathways to Control Multiple Aspects of Mammalian Neural Precursor Development

    PubMed Central

    Zander, Mark A.; Burns, Sarah E.; Yang, Guang; Kaplan, David R.

    2014-01-01

    The Snail transcription factor plays a key role in regulating diverse developmental processes but is not thought to play a role in mammalian neural precursors. Here, we have examined radial glial precursor cells of the embryonic murine cortex and demonstrate that Snail regulates their survival, self-renewal, and differentiation into intermediate progenitors and neurons via two distinct and separable target pathways. First, Snail promotes cell survival by antagonizing a p53-dependent death pathway because coincident p53 knockdown rescues survival deficits caused by Snail knockdown. Second, we show that the cell cycle phosphatase Cdc25b is regulated by Snail in radial precursors and that Cdc25b coexpression is sufficient to rescue the decreased radial precursor proliferation and differentiation observed upon Snail knockdown. Thus, Snail acts via p53 and Cdc25b to coordinately regulate multiple aspects of mammalian embryonic neural precursor biology. PMID:24719096

  10. 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.

  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. 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

  13. Mammalian Par3 regulates progenitor cell asymmetric division via Notch signaling in the developing neocortex

    PubMed Central

    Bultje, Ronald S.; Castaneda-Castellanos, David R.; Jan, Lily Yeh; Jan, Yuh-Nung; Kriegstein, Arnold R.; Shi, Song-Hai

    2009-01-01

    Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. Here we found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex. PMID:19640478

  14. Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol.

    PubMed

    Dietmair, Stefanie; Timmins, Nicholas E; Gray, Peter P; Nielsen, Lars K; Krömer, Jens O

    2010-09-15

    Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 degrees C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells. PMID:20435011

  15. Demands for carbohydrates as major energy substrates depend on the preimplantation developmental stage in pig embryos: Differential use of fructose by parthenogenetic diploids before and after the 4-cell stage in the pig

    PubMed Central

    SHIBUTANI, Mihiro; LEE, Jibak; MIYANO, Takashi; MIYAKE, Masashi

    2015-01-01

    The embryo culture technique has been improving, but the detailed demands for energy substrates such as glucose, fructose, pyruvate and lactate of preimplantation embryos are still unclear. In the present study, the demands of pig preimplantation embryos at each different developmental stage were investigated by use of parthenogenetic diploids as a model of pig preimplantation embryos. Pig parthenogenetic diploids showed different use of glucose and fructose before and after the 4-cell stage. Although glucose supported the development of pig embryos throughout the preimplantation stages and even maintained the expansion and hatching of blastocysts, it suppressed development to the blastocyst stage when glucose coexisted with pyruvate and lactate from 4 h after activation, but not after 48 h (early 4-cell stage). Since ketohexokinase that metabolizes fructose was not expressed in 2-cell and 4-cell diploids, a medium that included only fructose as a major energy substrate did not support early cleavage of pig diploids beyond the 4-cell stage, and almost no diploids developed to the morula stage just as in a medium without carbohydrates. These results may explain the different suppressive effects on pig preimplantation development between glucose and fructose when pyruvate and lactate were present in a medium. In addition, 4-cell diploids that had been cultured in a medium with pyruvate and lactate developed to the expanded blastocyst stage without any carbohydrates as a major energy substrate. These results show that the demands for carbohydrates are different depending on the developmental stage in pig preimplantation embryos. PMID:25736264

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Effect of Maternal Methionine Supplementation on the Transcriptome of Bovine Preimplantation Embryos

    PubMed Central

    Peñagaricano, Francisco; Souza, Alex H.; Carvalho, Paulo D.; Driver, Ashley M.; Gambra, Rocio; Kropp, Jenna; Hackbart, Katherine S.; Luchini, Daniel; Shaver, Randy D.; Wiltbank, Milo C.; Khatib, Hasan

    2013-01-01

    Maternal nutrition exclusively during the periconceptional period can induce remarkable effects on both oocyte maturation and early embryo development, which in turn can have lifelong consequences. The objective of this study was to evaluate the effect of maternal methionine supplementation on the transcriptome of bovine preimplantation embryos. Holstein cows were randomly assigned to one of two treatments differing in level of dietary methionine (1.89 Met vs. 2.43 Met % of metabolizable protein) from calving until embryo flushing. High quality preimplantation embryos from individual cows were pooled and then analyzed by RNA sequencing. Remarkably, a subtle difference in methionine supplementation in maternal diet was sufficient to cause significant changes in the transcriptome of the embryos. A total of 276 genes out of 10,662 showed differential expression between treatments (FDR <0.10). Interestingly, several of the most significant genes are related to embryonic development (e.g., VIM, IFI6, BCL2A1, and TBX15) and immune response (e.g., NKG7, TYROBP, SLAMF7, LCP1, and BLA-DQB). Likewise, gene set enrichment analysis revealed that several Gene Ontology terms, InterPro entries, and KEGG pathways were enriched (FDR <0.05) with differentially expressed genes involved in embryo development and immune system. The expression of most genes was decreased by maternal methionine supplementation, consistent with reduced transcription of genes with increased methylation of specific genes by increased methionine. Overall, our findings provide evidence that supplementing methionine to dams prior to conception and during the preimplantation period can modulate gene expression in bovine blastocysts. The ramifications of the observed gene expression changes for subsequent development of the pregnancy and physiology of the offspring warrant further investigation in future studies. PMID:23991086

  2. Mammalian aromatases.

    PubMed

    Conley, A; Hinshelwood, M

    2001-05-01

    Aromatase is the enzyme complex that catalyses the synthesis of oestrogens from androgens, and therefore it has unique potential to influence the physiological balance between the sex steroid hormones. Both aromatase cytochrome P450 (P450arom) and NADPH-cytochrome P450 reductase (reductase), the two essential components of the enzyme complex, are highly conserved among mammals and vertebrates. Aromatase expression occurs in the gonads and brain, and is essential for reproductive development and fertility. Of interest are the complex mechanisms involving alternative promoter utilization that have evolved to control tissue-specific expression in these tissues. In addition, in a number of species, including humans, expression of aromatase has a broader tissue distribution, including placenta, adipose and bone. The relevance of oestrogen synthesis and possibly androgen metabolism in these peripheral sites of expression is now becoming clear from studies in P450arom knockout (ArKO) mice and from genetic defects recognized recently in both men and women. Important species differences in the physiological roles of aromatase expression are also likely to emerge, despite the highly conserved nature of the enzyme system. The identification of functionally distinct, tissue-specific isozymes of P450arom in at least one mammal, pigs, and several species of fish indicates that there are additional subtle, but physiologically significant, species-specific roles for aromatase. Comparative studies of mammalian and other vertebrate aromatases will expand understanding of the role played by this ancient enzyme system in the evolution of reproduction and the adaptive influence of oestrogen synthesis on general health and well being. PMID:11427156

  3. Functional characterization of SOX2 in bovine preimplantation embryos.

    PubMed

    Goissis, Marcelo D; Cibelli, Jose B

    2014-02-01

    To date, efforts to establish pluripotent embryonic stem cells from bovine embryos have failed. The lack of reliable pluripotency markers is an important drawback when attempting to derive these cells. This study aimed to identify genes upregulated in the inner cell mass (ICM) of bovine blastocysts, and we selected SOX2 for further characterization. Spatial and temporal localization of the SOX2 protein revealed that its expression starts at the 16-cell stage and then becomes restricted to the ICMs of blastocysts. To study the role of SOX2 during the early development of bovine embryos, we designed siRNA to target SOX2. We began by injecting this siRNA into zygotes; the rate at which blastocysts developed declined compared to noninjected or scramble-injected controls. When only one blastomere of a two-cell embryo was injected with SOX2 siRNA, we observed development rates similar to those of controls. Daughter cells of the injected blastomere were tracked by TRITC fluorescence and found to contribute to the ICM, as select cells also lacked SOX2. Gene expression analysis revealed a decrease in SOX2 and NANOG gene expression in siRNA-injected embryos, but OCT4 expression remained unchanged. We conclude that SOX2 localizes exclusively in the ICM of bovine blastocysts, and its downregulation negatively impacts preimplantation development; however, it is still unclear as to why downregulation of SOX2 in one cell of a two-cell embryo does not affect the composition of the ICM. PMID:24389873

  4. Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

    PubMed

    Liu, Honglin; Kim, Jin-Moon; Aoki, Fugaku

    2004-05-01

    Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic

  5. Abscisic Acid: A Phytohormone and Mammalian Cytokine as Novel Pharmacon with Potential for Future Development into Clinical Applications.

    PubMed

    Sakthivel, Priya; Sharma, Niharika; Klahn, Philipp; Gereke, Marcus; Bruder, Dunja

    2016-01-01

    The isoprenoid stress-associated phytohormone abscisic acid (ABA) has recently been recognized to possess multifaceted biological functions in mammals and to exert potent curative effects in a number of clinically relevant human diseases. Studies with human specimens have unequivocally shown that ABA retains its stress-related functional attributes, previously identified in plants, which contribute to enhanced inflammatory defense mechanisms in mammals. Besides, studies performed in animal models revealed prominent anti-inflammatory properties of ABA as indicated by a marked reduction of immune cell infiltrates at the sites of inflammation. Thus, ABA treatment ultimately leads to the profound improvement of both non-communicable and communicable diseases which are associated with an overall alleviated course of inflammation. In addition to its action on the mammalian immune system, ABA was also shown to exert diverse physiological functions on non-immune components. One of the most remarkable features of ABA is to stimulate and expand mesenchymal stem cells, which may open a new avenue for its potential use in the field of regenerative medicine. Furthermore, ABA has been reported to play an important role in the maintenance of glycemic control. In this review, we summarize current understanding of the significance of ABA in the mammalian system, its prophylactic and therapeutic effects in various disease settings and the future directions for the development of ABA as novel drug candidate for the improved treatment of inflammatory and infectious human diseases. PMID:27048335

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

    PubMed

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

    1996-02-01

    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

  7. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro.

    PubMed

    Lane, M; Gardner, D K

    1992-04-01

    The morphology, cleavage rate and viability of preimplantation embryos from random bred Swiss mice were assessed after culture in different incubation volumes and embryo densities. Decreasing the incubation volume, from 320 to 20 microliters, significantly increased blastocyst cell number (P less than 0.01) and embryo development after transfer (P less than 0.01). Increasing the number of embryos incubated per drop from 1 to 16 significantly increased the number of two-cell embryos reaching the blastocyst stage in 5 or 320 microliters. Culturing embryos in groups significantly increased blastocyst cell numbers in all volumes employed and elevated embryo viability. Such observations are consistent with the hypothesis that the preimplantation mammalian embryo produces a factor(s) which can stimulate its own development. The results of this study have implications for clinical in-vitro fertilization, where embryos are routinely cultured individually in relatively large volumes. PMID:1522203

  8. 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. PMID:26607207

  9. 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

  10. 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

  11. Intrinsic retroviral reactivation in human preimplantation embryos and pluripotent cells.

    PubMed

    Grow, Edward J; Flynn, Ryan A; Chavez, Shawn L; Bayless, Nicholas L; Wossidlo, Mark; Wesche, Daniel J; Martin, Lance; Ware, Carol B; Blish, Catherine A; Chang, Howard Y; Pera, Renee A Reijo; Wysocka, Joanna

    2015-06-11

    Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome. The most recently acquired human ERV is HERVK(HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor. Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins. However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection. Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK 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 HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development. PMID:25896322

  12. Mammalian pheromones.

    PubMed

    Liberles, Stephen D

    2014-01-01

    Mammalian pheromones control a myriad of innate social behaviors and acutely regulate hormone levels. Responses to pheromones are highly robust, reproducible, and stereotyped and likely involve developmentally predetermined neural circuits. Here, I review several facets of pheromone transduction in mammals, including (a) chemosensory receptors and signaling components of the main olfactory epithelium and vomeronasal organ involved in pheromone detection; (b) pheromone-activated neural circuits subject to sex-specific and state-dependent modulation; and (c) the striking chemical diversity of mammalian pheromones, which range from small, volatile molecules and sulfated steroids to large families of proteins. Finally, I review (d) molecular mechanisms underlying various behavioral and endocrine responses, including modulation of puberty and estrous; control of reproduction, aggression, suckling, and parental behaviors; individual recognition; and distinguishing of own species from predators, competitors, and prey. Deconstruction of pheromone transduction mechanisms provides a critical foundation for understanding how odor response pathways generate instinctive behaviors. PMID:23988175

  13. 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

  14. [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

  15. The primate preimplantation embryo is a target for relaxin during early pregnancy

    PubMed Central

    VandeVoort, Catherine A.; Mtango, Namdori R.; Latham, Keith E.; StewartPhD, Dennis R.

    2011-01-01

    OBJECTIVE To determine if preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle. DESIGN Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/ml) for 8 days. SETTING Research laboratory. ANIMALS Rhesus monkey. INTERVENTIONS Controlled ovarian stimulation to obtain oocytes for in vitro produced embryos that were cultured with or without human recombinant relaxin. MAIN OUTCOME MEASURES The rate of blastocyst development and the percentage of blastocysts and ICM/TE ratio were measured on Day 8 of culture. The presence of relaxin receptor (RXFP1) mRNA in 8 cell embryos was observed by array hybridization. RESULTS RXFP1 receptor expression was localized to the inner cell mass of blastocysts as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/ trophectoderm cell ratio was unchanged with relaxin supplementation, however the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos. CONCLUSIONS These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development. PMID:21645893

  16. Discovery of a Novel Prolactin in Non-Mammalian Vertebrates: Evolutionary Perspectives and Its Involvement in Teleost Retina Development

    PubMed Central

    Huang, Xigui; Hui, Michelle N. Y.; Liu, Yun; Yuen, Don S. H.; Zhang, Yong; Chan, Wood Yee; Lin, Hao Ran; Cheng, Shuk Han; Cheng, Christopher H. K.

    2009-01-01

    Background The three pituitary hormones, viz. prolactin (PRL), growth hormone (GH) and somatolactin (SL), together with the mammalian placental lactogen (PL), constitute a gene family of hormones with similar gene structure and encoded protein sequences. These hormones are believed to have evolved from a common ancestral gene through several rounds of gene duplication and subsequent divergence. Principal Findings In this study, we have identified a new PRL-like gene in non-mammalian vertebrates through bioinformatics and molecular cloning means. Phylogenetic analyses showed that this novel protein is homologous to the previously identified PRL. A receptor transactivation assay further showed that this novel protein could bind to PRL receptor to trigger the downstream post-receptor event, indicating that it is biologically active. In view of its close phylogenetic relationship with PRL and also its ability to activate PRL receptor, we name it as PRL2 and the previously identified PRL as PRL1. All the newly discovered PRL2 sequences possess three conserved disulfide linkages with the exception of the shark PRL2 which has only two. In sharp contrast to the classical PRL1 which is predominantly expressed in the pituitary, PRL2 was found to be mainly expressed in the eye and brain of the zebrafish but not in the pituitary. A largely reduced inner nuclear layer of the retina was observed after morpholino knockdown of zebrafish PRL2, indicating its role on retina development in teleost. Significance The discovery of this novel PRL has revitalized our understanding on the evolution of the GH/PRL/SL/PL gene family. Its unique expression and functions in the zebrafish eye also provide a new avenue of research on the neuroendocrine control of retina development in vertebrates. PMID:19584915

  17. Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos.

    PubMed

    Lee, Jae H; Cho, Yong Ii; Choi, Sung S; Kim, Hae-Won; Min, Churl K; Lee, Sang J

    2016-01-01

    Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis. PMID:27341639

  18. 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

  19. Effects of ammonium dinitramide on preimplantation embryos in Sprague-Dawley rats.

    PubMed

    Graeter, L J; Wolfe, R E; Kinkead, E R; Flemming, C D

    1998-01-01

    Ammonium dinitramide (ADN) is a class 1.1 oxidizer that may be used in rocket propellants and explosives. Previous studies have shown that ADN is a female reproductive toxicant, causing implantation failure in Sprague-Dawley rats when it is administered during the preimplantation period of gestation. The purpose of this follow-up study was to identify the mechanism(s) associated with implantation failure following exposure to ADN. Mated female rats were treated with 2.0 grams per liter (g l-1) ADN in their drinking water for 24, 48, 72, or 96 h before preimplantation embryos were harvested from the oviducts or uterine horns. On gestation day 1 (GD-1), comparable numbers of morphologically normal two-cell embryos were harvested from the oviducts of the treatment and control groups. On GD-2, the development of the embryos harvested from the treated animals was either slowed or halted when compared to the control embryos. By GD-4, 98% of the embryos harvested from the control group had developed to the morula or blastocyst stage; these were collected from the uterine horns. On GD-4 in the treated group, 41% of the harvested embryos remained at the two- to six-cell stage and 59% were degenerate; 82% of these embryos were collected from the oviducts. These data suggest that the implantation failure seen in animals treated with ADN is due to embryolethality. PMID:9891911

  20. 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

  1. Preimplantation genetic diagnosis for cystic fibrosis: a case report.

    PubMed

    Biazotti, Maria Cristina Santoro; Pinto Junior, Walter; Albuquerque, Maria Cecília Romano Maciel de; 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

  2. 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

  3. Selection and Expression Profiles of Reference Genes in Mouse Preimplantation Embryos of Different Ploidies at Various Developmental Stages

    PubMed Central

    Gu, Yanli; Shen, Xinghui; Zhou, Dongjie; Wang, Zhendong; Zhang, Na; Shan, Zhiyan; Jin, Lianhong; Lei, Lei

    2014-01-01

    Real-time reverse transcription quantitative polymerase chain reaction (qPCR) has become the most frequently used system for studies of gene expression. Manystudies have provided reliable evidence that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. Therefore, it is critical to verify candidate reference genes to analyze gene expression accurately in both diploid and polyploid embryos. We examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, and the stability of three genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but the increasing degree were disproportionate with the ploidies. There were no significant difference in reference gene expressions among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. Ubc, Ppia, and Pgk1 were the three most stable reference genes in diploid and polyploid embryos. PMID:24927500

  4. The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis.

    PubMed

    Cimadomo, Danilo; Capalbo, Antonio; Ubaldi, Filippo Maria; Scarica, Catello; Palagiano, Antonio; Canipari, Rita; Rienzi, Laura

    2016-01-01

    Preimplantation Genetic Diagnosis and Screening (PGD/PGS) for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential. PMID:26942198

  5. 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

  6. 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

  7. 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

  8. BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

    PubMed Central

    Stottmann, Rolf W.; Choi, Murim; Mishina, Yuji; Meyers, Erik N.; Klingensmith, John

    2010-01-01

    Summary The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium. PMID:15073157

  9. From zebrafish heart jogging genes to mouse and human orthologs: using Gene Ontology to investigate mammalian heart development.

    PubMed Central

    Lovering, Ruth C

    2014-01-01

    For the majority of organs in developing vertebrate embryos, left-right asymmetry is controlled by a ciliated region; the left-right organizer node in the mouse and human, and the Kuppfer’s vesicle in the zebrafish. In the zebrafish, laterality cues from the Kuppfer’s vesicle determine asymmetry in the developing heart, the direction of ‘heart jogging’ and the direction of ‘heart looping’.  ‘Heart jogging’ is the term given to the process by which the symmetrical zebrafish heart tube is displaced relative to the dorsal midline, with a leftward ‘jog’. Heart jogging is not considered to occur in mammals, although a leftward shift of the developing mouse caudal heart does occur prior to looping, which may be analogous to zebrafish heart jogging. Previous studies have characterized 30 genes involved in zebrafish heart jogging, the majority of which have well defined orthologs in mouse and human and many of these orthologs have been associated with early mammalian heart development.    We undertook manual curation of a specific set of genes associated with heart development and we describe the use of Gene Ontology term enrichment analyses to examine the cellular processes associated with heart jogging.  We found that the human, mouse and zebrafish ‘heart jogging orthologs’ are involved in similar organ developmental processes across the three species, such as heart, kidney and nervous system development, as well as more specific cellular processes such as cilium development and function. The results of these analyses are consistent with a role for cilia in the determination of left-right asymmetry of many internal organs, in addition to their known role in zebrafish heart jogging.    This study highlights the importance of model organisms in the study of human heart development, and emphasises both the conservation and divergence of developmental processes across vertebrates, as well as the limitations of this approach. PMID:24627794

  10. 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. PMID:24627794

  11. 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

  12. 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

  13. 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

  14. The biology of mammalian parenting and its effect on offspring social development.

    PubMed

    Rilling, James K; Young, Larry J

    2014-08-15

    Parents know the transformative nature of having and caring for a child. Among many mammals, giving birth leads from an aversion to infant stimuli to irresistible attraction. Here, we review the biological mechanisms governing this shift in parental motivation in mammals. Estrogen and progesterone prepare the uterus for embryo implantation and placental development. Prolactin stimulates milk production, whereas oxytocin initiates labor and triggers milk ejection during nursing. These same molecules, interacting with dopamine, also activate specific neural pathways to motivate parents to nurture, bond with, and protect their offspring. Parenting in turn shapes the neural development of the infant social brain. Recent work suggests that many of the principles governing parental behavior and its effect on infant development are conserved from rodent to humans. PMID:25124431

  15. 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.

  16. Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos

    PubMed Central

    Kobolak, Julianna; Kiss, Katalin; Polgar, Zsuzsanna; Mamo, Solomon; Rogel-Gaillard, Claire; Tancos, Zsuzsanna; Bock, Istvan; Baji, Arpad G; Tar, Krisztina; Pirity, Melinda K; Dinnyes, Andras

    2009-01-01

    Background The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos. Results The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types. Conclusion In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This

  17. Conserved roles of fibroblast growth factor receptor 2 signaling in the regulation of inner cell mass development in bovine blastocysts.

    PubMed

    Akizawa, Hiroki; Nagatomo, Hiroaki; Odagiri, Haruka; Kohri, Nanami; Yamauchi, Nobuhiko; Yanagawa, Yojiro; Nagano, Masashi; Takahashi, Masashi; Kawahara, Manabu

    2016-06-01

    A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P < 0.05)-which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan-FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2-KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle. Mol. Reprod. Dev. 83: 516-525, 2016. © 2016 Wiley Periodicals, Inc. PMID:27060901

  18. Development of coherent neuronal activity patterns in mammalian cortical networks: common principles and local hetereogeneity.

    PubMed

    Egorov, Alexei V; Draguhn, Andreas

    2013-01-01

    Many mammals are born in a very immature state and develop their rich repertoire of behavioral and cognitive functions postnatally. This development goes in parallel with changes in the anatomical and functional organization of cortical structures which are involved in most complex activities. The emerging spatiotemporal activity patterns in multi-neuronal cortical networks may indeed form a direct neuronal correlate of systemic functions like perception, sensorimotor integration, decision making or memory formation. During recent years, several studies--mostly in rodents--have shed light on the ontogenesis of such highly organized patterns of network activity. While each local network has its own peculiar properties, some general rules can be derived. We therefore review and compare data from the developing hippocampus, neocortex and--as an intermediate region--entorhinal cortex. All cortices seem to follow a characteristic sequence starting with uncorrelated activity in uncoupled single neurons where transient activity seems to have mostly trophic effects. In rodents, before and shortly after birth, cortical networks develop weakly coordinated multineuronal discharges which have been termed synchronous plateau assemblies (SPAs). While these patterns rely mostly on electrical coupling by gap junctions, the subsequent increase in number and maturation of chemical synapses leads to the generation of large-scale coherent discharges. These patterns have been termed giant depolarizing potentials (GDPs) for predominantly GABA-induced events or early network oscillations (ENOs) for mostly glutamatergic bursts, respectively. During the third to fourth postnatal week, cortical areas reach their final activity patterns with distinct network oscillations and highly specific neuronal discharge sequences which support adult behavior. While some of the mechanisms underlying maturation of network activity have been elucidated much work remains to be done in order to fully

  19. 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

  20. 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

  1. Dosage Effects of Cohesin Regulatory Factor PDS5 on Mammalian Development: Implications for Cohesinopathies

    PubMed Central

    Zhang, Bin; Chang, Jufang; Fu, Ming; Huang, Jie; Kashyap, Rakesh; Salavaggione, Ezequiel; Jain, Sanjay; Shashikant, Kulkarni; Deardorff, Matthew A.; Uzielli, Maria L. Giovannucci; Dorsett, Dale; Beebe, David C.; Jay, Patrick Y.; Heuckeroth, Robert O.; Krantz, Ian; Milbrandt, Jeffrey

    2009-01-01

    Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion proteins, is characterized by multisystem developmental abnormalities. PDS5, a cohesion protein, is important for proper chromosome segregation in lower organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant mice have developmental abnormalities resembling CdLS; however the role of Pds5A in mammals and the association of PDS5 proteins with CdLS are unknown. To delineate genetic interactions between Pds5A and Pds5B and explore mechanisms underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously, these mice exhibit multiple abnormalities that were previously observed in Pds5B-deficient mice, including cleft palate, skeletal patterning defects, growth retardation, congenital heart defects and delayed migration of enteric neuron precursors. They also frequently display renal agenesis, an abnormality not observed in Pds5B−/− mice. While Pds5A−/− and Pds5B−/− mice die at birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most likely of heart failure, indicating that total Pds5 gene dosage is critical for normal development. In addition, characterization of these compound homozygous-heterozygous mice revealed a severe abnormality in lens formation that does not occur in either Pds5A−/− or Pds5B−/− mice. We further identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding domain in a familial case of CdLS, in which affected individuals also develop megacolon. This study shows that PDS5A and PDS5B functions other than those involving chromosomal dynamics are important for normal development, highlights the sensitivity of key developmental processes on PDS5 signaling, and provides mechanistic insights into how PDS5 mutations may lead to CdLS. PMID:19412548

  2. Dosage effects of cohesin regulatory factor PDS5 on mammalian development: implications for cohesinopathies.

    PubMed

    Zhang, Bin; Chang, Jufang; Fu, Ming; Huang, Jie; Kashyap, Rakesh; Salavaggione, Ezequiel; Jain, Sanjay; Kulkarni, Shashikant; Shashikant, Kulkarni; Deardorff, Matthew A; Uzielli, Maria L Giovannucci; Dorsett, Dale; Beebe, David C; Jay, Patrick Y; Heuckeroth, Robert O; Krantz, Ian; Milbrandt, Jeffrey

    2009-01-01

    Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion proteins, is characterized by multisystem developmental abnormalities. PDS5, a cohesion protein, is important for proper chromosome segregation in lower organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant mice have developmental abnormalities resembling CdLS; however the role of Pds5A in mammals and the association of PDS5 proteins with CdLS are unknown. To delineate genetic interactions between Pds5A and Pds5B and explore mechanisms underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously, these mice exhibit multiple abnormalities that were previously observed in Pds5B-deficient mice, including cleft palate, skeletal patterning defects, growth retardation, congenital heart defects and delayed migration of enteric neuron precursors. They also frequently display renal agenesis, an abnormality not observed in Pds5B(-/-) mice. While Pds5A(-/-) and Pds5B(-/-) mice die at birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most likely of heart failure, indicating that total Pds5 gene dosage is critical for normal development. In addition, characterization of these compound homozygous-heterozygous mice revealed a severe abnormality in lens formation that does not occur in either Pds5A(-/-) or Pds5B(-/-) mice. We further identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding domain in a familial case of CdLS, in which affected individuals also develop megacolon. This study shows that PDS5A and PDS5B functions other than those involving chromosomal dynamics are important for normal development, highlights the sensitivity of key developmental processes on PDS5 signaling, and provides mechanistic insights into how PDS5 mutations may lead to CdLS. PMID:19412548

  3. 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

  4. 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.

  5. Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development.

    PubMed

    Xue, Li; Chen, Xiaoyong; Chang, Yanjie; Bieker, James J

    2004-06-01

    Erythroid Krüppel-like factor (EKLF) plays an essential role in enabling beta-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose. PMID:14764531

  6. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.

    PubMed

    Petropoulos, Sophie; Edsgärd, Daniel; Reinius, Björn; Deng, Qiaolin; Panula, Sarita Pauliina; Codeluppi, Simone; Plaza Reyes, Alvaro; Linnarsson, Sten; Sandberg, Rickard; Lanner, Fredrik

    2016-05-01

    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

  7. 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

  8. Rabbit antiserum to mouse embryonic stem cells delays compaction of mouse preimplantation embryos

    PubMed Central

    Cong, Yingli; Cui, Lifang; Zhang, Zhenhong; Xi, Jianzhong; Wang, Mianjuan

    2014-01-01

    Background: Mouse embryonic stem (ES) cells are derived from the inner cell mass (ICM) of the preimplantation blastocysts. So it is suggested that ES and ICM cells should have similar cellular surface molecules and antiserum to ES cells can inhibit ICM development. Objective: The objective of this study was to evaluate the effect of rabbit antiserum to ES cells on mouse preimplantation embryo development and chimera production. Materials and Methods: Mouse 4-cell embryos were matured in vitro at 37.5oC, in humidified 5% CO2 atmosphere for 12-36 h. The embryos were cultured in KSOM medium with or without antiserum for 12-36 h. The ratios of in vitro embryo development of the blastocysts, cell division, attachment potential, alkaline phosphatase activity, post-implantation development, and chimera production were assessed and compared with the control group. P<0.05 was considered as significant. Results: The rabbit antiserum to mouse ES cells showed delay in embryo compaction and induced decompaction at 8-cell stage. The development of 4-cell embryos in the presence of the antiserum for 36h did not lead to a reduced or absent ICM. These embryos still displayed positive alkaline phosphatase activity, normal cell division, embryo attachment, outgrowth formation, implantation and post-implantation development. In addition, decompaction induced by antiserum did not increase production and germline transmission of chimeric mice. Conclusion: The results showed that antiserum to ES cells delayed embryo compaction and did not affect post-implantation development and chimera production. PMID:24799859

  9. Gene-Chemical Interactions in the Developing Mammalian Nervous System: Effects on Proliferation, Neurogenesis and Differentiation

    PubMed Central

    Fox, Donald A.; Opanashuk, Lisa; Zharkovsky, Aleksander; Weiss, Bernie

    2010-01-01

    The orderly formation of the nervous system requires a multitude of complex, integrated and simultaneously occurring processes. Neural progenitor cells expand through proliferation, commit to different cell fates, exit the cell cycle, generate different neuronal and glial cell types, and new neurons migrate to specified areas and establish synaptic connections. Gestational and perinatal exposure to environmental toxicants, pharmacological agents and drugs of abuse produce immediate, persistent or late-onset alterations in behavioral, cognitive, sensory and/or motor functions. These alterations reflect the disruption of the underlying processes of CNS formation and development. To determine the neurotoxic mechanisms that underlie these deficits it is necessary to analyze and dissect the complex molecular processes that occur during the proliferation, neurogenesis and differentiation of cells. This symposium will provide a framework for understanding the orchestrated events of neurogenesis, the coordination of proliferation and cell fate specification by selected genes, and the effects of well-known neurotoxicants on neurogenesis in the retina, hippocampus and cerebellum. These three tissues share common developmental profiles, mediate diverse neuronal activities and function, and thus provide important substrates for analysis. This paper summarizes four invited talks that were presented at the 12th International Neurotoxicology Association meeting held in Jerusalem, Israel during the summer of 2009. Donald A. Fox described the structural and functional alterations following low-level gestational lead exposure in children and rodents that produced a supernormal electroretinogram and selective increases in neurogenesis and cell proliferation of late-born retinal neurons (rod photoreceptors and bipolar cells), but not Müller glia cells, in mice. Lisa Opanashuk discussed how dioxin [TCDD] binding to the arylhydrocarbon receptor [AhR], a transcription factor that

  10. Fliih, a Gelsolin-Related Cytoskeletal Regulator Essential for Early Mammalian Embryonic Development

    PubMed Central

    Campbell, Hugh D.; Fountain, Shelley; McLennan, Ian S.; Berven, Leise A.; Crouch, Michael F.; Davy, Deborah A.; Hooper, Jane A.; Waterford, Kynan; Chen, Ken-Shiung; Lupski, James R.; Ledermann, Birgit; Young, Ian G.; Matthaei, Klaus I.

    2002-01-01

    The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation. PMID:11971982

  11. 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

  12. 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

  13. Skeletal development in sloths and the evolution of mammalian vertebral patterning

    PubMed Central

    Hautier, Lionel; Weisbecker, Vera; Sánchez-Villagra, Marcelo R.; Goswami, Anjali; Asher, Robert J.

    2010-01-01

    Mammals show a very low level of variation in vertebral count, particularly in the neck. Phenotypes exhibited at various stages during the development of the axial skeleton may play a key role in testing mechanisms recently proposed to explain this conservatism. Here, we provide osteogenetic data that identify developmental criteria with which to recognize cervical vs. noncervical vertebrae in mammals. Except for sloths, all mammals show the late ossification of the caudal-most centra in the neck after other centra and neural arches. In sloths with 8–10 ribless neck vertebrae, the caudal-most neck centra ossify early, matching the pattern observed in cranial thoracic vertebrae of other mammals. Accordingly, we interpret the ribless neck vertebrae of three-toed sloths caudal to V7 as thoracic based on our developmental criterion. Applied to the unusual vertebral phenotype of long-necked sloths, these data support the interpretation that elements of the axial skeleton with origins from distinct mesodermal tissues have repatterned over the course of evolution. PMID:20956304

  14. 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.

  15. First Evidence of DAAM1 Localization During the Post-Natal Development of Rat Testis and in Mammalian Sperm.

    PubMed

    Pariante, Paolo; Dotolo, Raffaele; Venditti, Massimo; Ferrara, Diana; Donizetti, Aldo; Aniello, Francesco; Minucci, Sergio

    2016-10-01

    Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a formin-family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt-Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement, and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood-testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. J. Cell. Physiol. 231: 2172-2184, 2016. © 2016 Wiley Periodicals, Inc. PMID:26831620

  16. The Past, Present, and Future of Preimplantation Genetic Testing.

    PubMed

    Imudia, Anthony N; Plosker, Shayne

    2016-06-01

    Preimplantation genetic testing (PGT) of oocytes and embryos is the earliest form of prenatal testing. PGT requires in vitro fertilization for embryo creation. In the past 25 years, the use of PGT has increased dramatically. The indications of PGT include identification of embryos harboring single-gene disorders, chromosomal structural abnormalities, chromosomal numeric abnormalities, and mitochondrial disorders; gender selection; and identifying unaffected, HLA-matched embryos to permit the creation of a savior sibling. PGT is not without risks, limitations, or ethical controversies. This review discusses the techniques and clinical applications of different forms of PGT and the debate surrounding its associated uncertainty and expanded use. PMID:27235919

  17. 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

  18. 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

  19. Isotope Labeling in Mammalian Cells

    PubMed Central

    Dutta, Arpana; Saxena, Krishna; Klein-Seetharaman, Judith

    2011-01-01

    Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. Here, we provide an overview of some of the recent developments in expression strategies for mammalian expression systems in view of NMR investigations. PMID:22167668

  20. 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

  1. High Frequency of Imprinted Methylation Errors in Human Preimplantation Embryos

    PubMed Central

    White, Carlee R.; Denomme, Michelle M.; Tekpetey, Francis R.; Feyles, Valter; Power, Stephen G. A.; Mann, Mellissa R. W.

    2015-01-01

    Assisted reproductive technologies (ARTs) represent the best chance for infertile couples to conceive, although increased risks for morbidities exist, including imprinting disorders. This increased risk could arise from ARTs disrupting genomic imprints during gametogenesis or preimplantation. The few studies examining ART effects on genomic imprinting primarily assessed poor quality human embryos. Here, we examined day 3 and blastocyst stage, good to high quality, donated human embryos for imprinted SNRPN, KCNQ1OT1 and H19 methylation. Seventy-six percent day 3 embryos and 50% blastocysts exhibited perturbed imprinted methylation, demonstrating that extended culture did not pose greater risk for imprinting errors than short culture. Comparison of embryos with normal and abnormal methylation didn’t reveal any confounding factors. Notably, two embryos from male factor infertility patients using donor sperm harboured aberrant methylation, suggesting errors in these embryos cannot be explained by infertility alone. Overall, these results indicate that ART human preimplantation embryos possess a high frequency of imprinted methylation errors. PMID:26626153

  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. PMID:27272931

  3. 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.

  4. 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

  5. Development of a model system to study leukotriene-induced modification of radiation sensitivity in mammalian cells

    SciTech Connect

    Walden, T.L.; Holahan, E.V.; Catravas, G.N.

    1986-01-01

    Leukotrienes (LT) are an important class of biological mediators, for which no information exists concerning their synthesis following a radiation insult, or on their ability to modify cellular response to a subsequent radiation exposure. LT are derived from arachidonic acid, as are prostaglandins, although by a separate enzyme system. Prostaglandins are able to modify radiosensitivity of mammalian cells in vivo and in vitro. In addition, the cytoprotective effect induced by prostaglandins may have significance in cancer therapy since certain breast cancers which secrete elevated levels of prostaglandins are more resistant to therapy than similar tumors without the prostaglandin elevation. The objective of this study was to define a model system in which the metabolic fate of the LT could be monitored, and the effort of LT on the ionizing radiation sensitivity of mammalian cells in vitro could also be characterized.

  6. Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development

    PubMed Central

    Zhou, Hao; Clapham, David E.

    2009-01-01

    Magnesium (Mg2+) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg2+ transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg2+ influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg2+. Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg2+ concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg2+ or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg2+ transport system. PMID:19717468

  7. 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. PMID:24835332

  8. [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. PMID:18330104

  9. 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

  10. 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

  11. 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. PMID:27153491

  12. Midgestational abnormalities associated with in vitro preimplantation N-methyl-N-nitrosourea exposure with subsequent transfer to surrogate mothers.

    PubMed Central

    Bossert, N L; Iannaccone, P M

    1985-01-01

    Mouse blastocyst functions have been shown to be disrupted by in vitro exposure to N-methyl-N-nitrosourea (MeNU). After exposure, the chemically treated blastocysts were transferred to the uteri of pseudopregnant surrogate mothers. Implantation rate and birth rate have been shown previously to decrease in a concentration-dependent manner. Because of the large progressive decrease in the 50% effective concentration (EC50) for cytotoxicity, implantation rate, and live birth rate, we have investigated the midgestational effects of preimplantation exposure to MeNU after the transfer of treated embryos to surrogate mothers. A concentration-dependent decrease in normal implantation and a concurrent concentration-dependent increase in resorption number was observed in surrogates sacrificed at gestational age day 12 or day 15. Gross malformations were significantly increased by preimplantation exposure, in vitro, to MeNU. Fetal body length did not differ between fetuses developed from solvent-treated blastocysts and those that developed from natural pregnancies (nontransferred control) at either gestational age examined. Fetal body length was significantly shorter in fetuses developed from MeNU-treated blastocysts. Images PMID:3866250

  13. Mammalian Endogenous Retroviruses.

    PubMed

    Mager, Dixie L; Stoye, Jonathan P

    2015-02-01

    Over 40% of mammalian genomes comprise the products of reverse transcription. Among such retrotransposed sequences are those characterized by the presence of long terminal repeats (LTRs), including the endogenous retroviruses (ERVs), which are inherited genetic elements closely resembling the proviruses formed following exogenous retrovirus infection. Sequences derived from ERVs make up at least 8 to 10% of the human and mouse genomes and range from ancient sequences that predate mammalian divergence to elements that are currently still active. In this chapter we describe the discovery, classification and origins of ERVs in mammals and consider cellular mechanisms that have evolved to control their expression. We also discuss the negative effects of ERVs as agents of genetic disease and cancer and review examples of ERV protein domestication to serve host functions, as in placental development. Finally, we address growing evidence that the gene regulatory potential of ERV LTRs has been exploited multiple times during evolution to regulate genes and gene networks. Thus, although recently endogenized retroviral elements are often pathogenic, those that survive the forces of negative selection become neutral components of the host genome or can be harnessed to serve beneficial roles. PMID:26104559

  14. 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.

  15. 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.

  16. [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

  17. 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. PMID:25953344

  18. 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

  19. Developmental onset of mixed-function oxidase activity in preimplantation mouse embryos

    SciTech Connect

    Filler, R.; Lew, K.J.

    1981-11-01

    Two-cell embryos, obtained from the C57BL/6N and DBA/2N strains, were cultured in media that supported in vitro differentiation and that contained (/sup 3/H)benzo(a)pyrene. High-pressure liquid chromatography of the activated intermediates formed during in vitro early embryonic development indicated that the onset of polynuclear aromatic hydrocarbon activation coincided with blastocyst formation. Comparison of individual oxygenated intermediates metabolically formed from embryos genetically ''responsive'' or ''nonresponsive'' to aromatic hydrocarbons revealed significant quantitative differences in the production of dihydrodiol, quinone, and phenolic derivatives. In addition to exhibiting basal mixed-function oxidase activity, blastocysts were also responsive to enzymatic induction when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. The presence of operative metabolite-detoxifying pathways was also assayed. Enzymatic treatment of water-soluble metabolites with ..beta..-glucuronidase or arylsulfatase revealed that neither glucuronic acid conjugates nor sulfate ester derivatives were present. These data, therefore, provide direct evidence that late preimplanation mouse embryos (day 3 1/2 of gestation) are similar to later developmental stages in having the enzymatic capability for xenobiotic activation and enzyme induction but are dissimilar with respect to their detoxification mechanisms(s). Moreover, the ability of preimplantation embryos to activate directly polynuclear aromatic hydrocarbon to bioreactive intermediates may be of importance in assessing the ontological susceptibility of the developing embryo to carcinogenic or teratogenic chemicals.

  20. 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

  1. 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

  2. Simultaneous preimplantation genetic diagnosis for Tay-Sachs and Gaucher disease.

    PubMed

    Altarescu, Gheona; Brooks, Barry; Margalioth, Ehud; Eldar Geva, Talia; Levy-Lahad, Ephrat; Renbaum, Paul

    2007-07-01

    Preimplantation genetic diagnosis (PGD) for single gene defects is described for a family in which each parent is a carrier of both Tay-Sachs (TS) and Gaucher disease (GD). A multiplex fluorescent polymerase chain reaction protocol was developed that simultaneously amplified all four familial mutations and 10 informative microsatellite markers. In one PGD cycle, seven blastomeres were analysed, reaching a conclusive diagnosis in six out of seven embryos for TS and in five out of seven embryos for GD. Of the six diagnosed embryos, one was wild type for both TS and GD, and three were wild type for GD and carriers of TS. Two remaining embryos were compound heterozygotes for TS. Two transferable embryos developed into blastocysts (wt/wt and wt GD/carrier TS) and both were transferred on day 5. This single cycle of PGD resulted in a healthy live child. Allele drop-out (ADO) was observed in three of 34 reactions, yielding an 8% ADO rate. The occurrence of ADO in single cell analysis and undetected recombination events are primary causes of misdiagnosis in PGD and emphasize the need to use multiple polymorphic markers. So far as is known, this is the first report of concomitant PGD for two frequent Ashkenazi Jewish recessive disorders. PMID:17623543

  3. 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

  4. 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

  5. Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos.

    PubMed

    Wang, Li; Cram, David S; Shen, Jiandong; Wang, Xiaohong; Zhang, Jianguang; Song, Zhuo; Xu, Genming; Li, Na; Fan, Junmei; Wang, Shufang; Luo, Yaning; Wang, Jun; Yu, Li; Liu, Jiayin; Yao, Yuanqing

    2014-08-01

    Chromosome aneuploidies commonly arise in embryos produced by assisted reproductive technologies and represent a major cause of implantation failure and miscarriage. Currently, preimplantation genetic diagnosis (PGD) is performed by array-based methods to identify euploid embryos for transfer to the patient. We speculated that a combination of next-generation sequencing technologies and sophisticated bioinformatics would deliver a more comprehensive and accurate methodology to improve the overall efficacy of embryo testing. To meet this challenge, we developed a high-resolution copy number variation (CNV) sequencing pipeline suitable for single-cell analysis. In validation studies, we showed that CNV-Seq was highly sensitive and specific for detection of euploidy, aneuploidy, and segmental imbalances in 24 whole genome amplification samples from PGD embryos that were originally diagnosed by gold standard array comparative genomic hybridization. In addition, CNV-Seq was capable of detecting, mapping, and accurately quantifying terminal chromosome imbalances down to 1 Mb in size originating from abnormal segregation of translocation chromosomes. These validation studies indicate that CNV-Seq displays the hallmarks of an accurate and reliable embryo test with the potential to further improve the overall efficacy of PGD. PMID:24966395

  6. Medium-Based Noninvasive Preimplantation Genetic Diagnosis for Human α-Thalassemias-SEA

    PubMed Central

    Wu, Haitao; Ding, Chenhui; Shen, Xiaoting; Wang, Jing; Li, Rong; Cai, Bing; Xu, Yanwen; Zhong, Yiping; Zhou, Canquan

    2015-01-01

    Abstract To develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for α-thalassemias-SEA. The embryos of α-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of α-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium. The diagnosis efficiency of medium-based α-thalassemias–SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based α-thalassemias–SEA detection is Day 5 (D5) following IVF. Medium-based α-thalassemias–SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD. PMID:25816038

  7. 'The BRCA clock is ticking!': negotiating medical concerns and reproductive goals in preimplantation genetic diagnosis.

    PubMed

    Rubin, Lisa R; Werner-Lin, Allison; Sagi, Michal; Cholst, Ina; Stern, Rikki; Lilienthal, Debra; Hurley, Karen

    2014-09-01

    Despite research on BRCA1/2 mutation carriers attitudes towards preimplantation genetic diagnosis (PGD), considerably less is known about individuals' experience with its use. Through case reports of BRCA1/2 mutation carriers' thoughts on, and use of, PGD, this paper highlights how the option of PGD is experienced and negotiated in the context of reproductive and life-course goals. Drawing on qualitative interviews with 38 BRCA1/2 mutation carriers, this article focuses on a subsample of 10 interviewees who sought consultation for, and/or attempted, PGD, with in-depth reports of 3 cases and summary decisions of the remaining 7. Three couples decided against PGD, and one was deciding at the time of the interview. Interviewees discuss key aspects of their experience prior to, and going through, PGD for BRCA1/2, including potential challenges of becoming pregnant through PGD and of heightened pressure to achieve their reproductive goals more quickly. Despite considerable focus on ethical issues in screening embryos for mutations associated with adult-onset cancer risk, less attention has been paid to the technical, logistical, and related psychosocial issues. Narrative case reports may help individuals develop appropriate expectations of PGD for BRCA prepare for possibly challenging decisions and outcomes, and ultimately determine whether it is compatible with their reproductive goals. PMID:25105219

  8. 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

  9. 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

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

    PubMed Central

    Schulz, Laura C.; Roberts, R. Michael

    2011-01-01

    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 blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased towards a particular fate as early as the 2-cell stage. Here, 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. Presence or absence of leptin polarization was not linked to differences in ability of embryos to develop normally to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm but upon blastocoel expansion it was lost from 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. PMID:21444625

  11. 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

  12. Low-dose agrochemicals and lawn-care pesticides induce developmental toxicity in murine preimplantation embryos.

    PubMed Central

    Greenlee, Anne R; Ellis, Tammy M; Berg, Richard L

    2004-01-01

    Occupational exposures to pesticides may increase parental risk of infertility and adverse pregnancy outcomes such as spontaneous abortion, preterm delivery, and congenital anomalies. Less is known about residential use of pesticides and the risks they pose to reproduction and development. In the present study we evaluate environmentally relevant, low-dose exposures to agrochemicals and lawn-care pesticides for their direct effects on mouse preimplantation embryo development, a period corresponding to the first 5-7 days after human conception. Agents tested were those commonly used in the upper midwestern United States, including six herbicides [atrazine, dicamba, metolachlor, 2,4-dichlorophenoxyacetic acid (2,4-D)], pendimethalin, and mecoprop), three insecticides (chlorpyrifos, terbufos, and permethrin), two fungicides (chlorothalonil and mancozeb), a desiccant (diquat), and a fertilizer (ammonium nitrate). Groups of 20-25 embryos were incubated 96 hr in vitro with either individual chemicals or mixtures of chemicals simulating exposures encountered by handling pesticides, inhaling drift, or ingesting contaminated groundwater. Incubating embryos with individual pesticides increased the percentage of apoptosis (cell death) for 11 of 13 chemicals (p development to blastocyst and mean cell number per embryo for 3 of 13 agents (p development to blastocyst and mean cell number per embryo (p development, with a variety of agents, and at concentrations assumed to be without adverse health consequences for humans. PMID:15121514

  13. Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle.

    PubMed

    Mullen, Michael P; Elia, Giuliano; Hilliard, Mark; Parr, Mervyn H; Diskin, Michael G; Evans, Alex C O; Crowe, Mark A

    2012-05-01

    Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy. PMID:22463384

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  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. DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry

    PubMed Central

    Okamoto, Yoshinori; Yoshida, Naoko; Suzuki, Toru; Shimozawa, Nobuhiro; Asami, Maki; Matsuda, Tomonari; Kojima, Nakao; Perry, Anthony C. F.; Takada, Tatsuyuki

    2016-01-01

    Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes. PMID:26750605

  1. 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

  2. DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

    PubMed

    Okamoto, Yoshinori; Yoshida, Naoko; Suzuki, Toru; Shimozawa, Nobuhiro; Asami, Maki; Matsuda, Tomonari; Kojima, Nakao; Perry, Anthony C F; Takada, Tatsuyuki

    2016-01-01

    Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10-48 h after fertilization, implying that any passive 'demethylation' following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes. PMID:26750605

  3. 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. PMID:21799744

  4. Requirement of Leukemia Inhibitory Factor or Epidermal Growth Factor for Pre-Implantation Embryogenesis via JAK/STAT3 Signaling Pathways

    PubMed Central

    Lee, Tsung-Hsein; Huang, Chun-Chia; Chen, Chung-I; Huang, Lii-Sheng; Lee, Maw-Sheng

    2016-01-01

    Leukemia inhibitory factor (LIF) plays a key role in the survivability of mouse embryos during pre-implantation. In this study, we verified the role of LIF by detecting gene expression in morula stage embryos through DNA microarray. Our results showed that LIF knockdown affected expression of 369 genes. After LIF supplementation, the epidermal growth factor (EGF) is most affected by LIF expression. To observe the correlation between LIF and EGF, the LIF knockdown embryos were supplemented with various growth factors, including LIF, EGF, GM-CSF, TGF, and IGF II. Only LIF and EGF caused the rate of blastocyst development to recover significantly from 52% of control to 83% and 93%, respectively. All of the variables, including the diameter of blastocysts, the number of blastomeres, and cells in ICM and TE, were almost restored. Moreover, EGF knockdown also impaired blastocyst development, which was reversed by LIF or EGF supplementation. The treatment with various signaling suppressors revealed that both EGF and LIF promoted embryonic development through the JAK/STAT3 signaling pathway. These data suggest that the EGF and LIF can be compensatory to each other during early embryonic development, and at least one of them is necessary for sustaining the normal development of pre-implantation embryos. PMID:27096934

  5. Identification and expression patterns of novel long non-coding RNAs in neural progenitors of the developing mammalian cortex

    PubMed Central

    Aprea, Julieta; Lesche, Mathias; Massalini, Simone; Prenninger, Silvia; Alexopoulou, Dimitra; Dahl, Andreas; Hiller, Michael; Calegari, Federico

    2015-01-01

    Long non-coding (lnc)RNAs play key roles in many biological processes. Elucidating the function of lncRNAs in cell type specification during organ development requires knowledge about their expression in individual progenitor types rather than in whole tissues. To achieve this during cortical development, we used a dual-reporter mouse line to isolate coexisting proliferating neural stem cells, differentiating neurogenic progenitors and newborn neurons and assessed the expression of lncRNAs by paired-end, high-throughput sequencing. We identified 379 genomic loci encoding novel lncRNAs and performed a comprehensive assessment of cell-specific expression patterns for all, annotated and novel, lncRNAs described to date. Our study provides a powerful new resource for studying these elusive transcripts during stem cell commitment and neurogenesis.

  6. Single cells get together: High-resolution approaches to study the dynamics of early mouse development.

    PubMed

    Saiz, Néstor; Plusa, Berenika; Hadjantonakis, Anna-Katerina

    2015-12-01

    Embryonic development is a complex and highly dynamic process during which individual cells interact with one another, adopt different identities and organize themselves in three-dimensional space to generate an entire organism. Recent technical developments in genomics and high-resolution quantitative imaging are making it possible to study cellular populations at single-cell resolution and begin to integrate different inputs, for example genetic, physical and chemical factors, that affect cell differentiation over spatial and temporal scales. The preimplantation mouse embryo allows the analysis of cell fate decisions in vivo with high spatiotemporal resolution. In this review we highlight how the application of live imaging and single-cell resolution analysis pipelines is providing an unprecedented level of insight on the processes that shape the earliest stages of mammalian development. PMID:26183190

  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. 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

  10. BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.

    PubMed

    Vieux-Rochas, Maxence; Bouhali, Kamal; Mantero, Stefano; Garaffo, Giulia; Provero, Paolo; Astigiano, Simonetta; Barbieri, Ottavia; Caratozzolo, Mariano F; Tullo, Apollonia; Guerrini, Luisa; Lallemand, Yvan; Robert, Benoît; Levi, Giovanni; Merlo, Giorgio R

    2013-01-01

    The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations. PMID:23382810

  11. Mammalian cardiolipin biosynthesis.

    PubMed

    Mejia, Edgard M; Nguyen, Hieu; Hatch, Grant M

    2014-04-01

    Cardiolipin is a major phospholipid in mitochondria and is involved in the generation of cellular energy in the form of ATP. In mammalian and eukaryotic cells it is synthesized via the cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol phosphate pathway. This brief review will describe some of the more recent studies on mammalian cardiolipin biosynthesis and provide an overview of regulation of cardiolipin biosynthesis. In addition, the important role that this key phospholipid plays in disease processes including heart failure, diabetes, thyroid hormone disease and the genetic disease Barth Syndrome will be discussed. PMID:24144810

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

    PubMed Central

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

    2008-01-01

    SUMMARY 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 is 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. PMID:18682239

  13. 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. PMID:18682239

  14. 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

  15. Toward predictive models of mammalian cells.

    PubMed

    Ma'ayan, Avi; Blitzer, Robert D; Iyengar, Ravi

    2005-01-01

    Progress in experimental and theoretical biology is likely to provide us with the opportunity to assemble detailed predictive models of mammalian cells. Using a functional format to describe the organization of mammalian cells, we describe current approaches for developing qualitative and quantitative models using data from a variety of experimental sources. Recent developments and applications of graph theory to biological networks are reviewed. The use of these qualitative models to identify the topology of regulatory motifs and functional modules is discussed. Cellular homeostasis and plasticity are interpreted within the framework of balance between regulatory motifs and interactions between modules. From this analysis we identify the need for detailed quantitative models on the basis of the representation of the chemistry underlying the cellular process. The use of deterministic, stochastic, and hybrid models to represent cellular processes is reviewed, and an initial integrated approach for the development of large-scale predictive models of a mammalian cell is presented. PMID:15869393

  16. 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.

  17. 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

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

    PubMed Central

    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

  19. Wnt signalling pathway parameters for mammalian cells.

    PubMed

    Tan, Chin Wee; Gardiner, Bruce S; Hirokawa, Yumiko; Layton, Meredith J; Smith, David W; Burgess, Antony W

    2012-01-01

    Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters

  20. Mammalian Septins Nomenclature

    PubMed Central

    Macara, Ian G.; Baldarelli, Richard; Field, Christine M.; Glotzer, Michael; Hayashi, Yasuhide; Hsu, Shu-Chan; Kennedy, Mary B.; Kinoshita, Makoto; Longtine, Mark; Low, Claudia; Maltais, Lois J.; McKenzie, Louise; Mitchison, Timothy J.; Nishikawa, Toru; Noda, Makoto; Petty, Elizabeth M.; Peifer, Mark; Pringle, John R.; Robinson, Phillip J.; Roth, Dagmar; Russell, S.E. Hilary; Stuhlmann, Heidi; Tanaka, Manami; Tanaka, Tomoo; Trimble, William S.; Ware, Jerry; Zeleznik-Le, Nancy J.; Zieger, Barbara

    2002-01-01

    There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1–SEPT10 and Sept1–Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase “v” and a number, e.g., SEPT4_v1. PMID:12475938

  1. Mammalian sweet taste receptors.

    PubMed

    Nelson, G; Hoon, M A; Chandrashekar, J; Zhang, Y; Ryba, N J; Zuker, C S

    2001-08-10

    The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery. PMID:11509186

  2. 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

  3. Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea.

    PubMed

    Roux, Isabelle; Wu, Jingjing Sherry; McIntosh, J Michael; Glowatzki, Elisabeth

    2016-08-01

    Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the "muscle-type" nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870-5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651-2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (-)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs. PMID:27098031

  4. Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation.

    PubMed

    Guignot, F; Reigner, F; Perreau, C; Tartarin, P; Babilliot, J M; Bed'hom, B; Vidament, M; Mermillod, P; Duchamp, G

    2015-11-01

    Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 μm in

  5. Development of a model describing regulation of casein synthesis by the mammalian target of rapamycin (mTOR) signaling pathway in response to insulin, amino acids, and acetate.

    PubMed

    Castro, J J; Arriola Apelo, S I; Appuhamy, J A D R N; Hanigan, M D

    2016-08-01

    To improve dietary protein use efficiency in lactating cows, mammary protein synthesis responses to AA, energy substrates, and hormones must be better understood. These entities exert their effects through stimulation of mRNA translation via control of initiation and elongation rates at the cellular level. A central protein kinase of this phenomenon is the mammalian target of rapamycin (mTOR), which transfers the nutritional and hormonal stimuli onto a series of proteins downstream through a cascade of phosphorylation reactions that ultimately affect protein synthesis. The objective of this work was to further develop an existing mechanistic model of mTOR phosphorylation responses to insulin and total essential AA to include the effects of specific essential AA and acetate mediated by signaling proteins including protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK), and mTOR and to add a representation of milk protein synthesis. Data from 6 experiments in MAC-T cells and mammary tissue slices previously conducted in our laboratory were assembled and used to parameterize the dynamic system of differential equations representing Akt, AMPK, and mTOR in their phosphorylated and dephosphorylated states and the resulting regulation of milk protein synthesis. The model predicted phosphorylated Akt, mTOR, AMPK, and casein synthesis rates with root mean square prediction errors of 16.8, 28.4, 33.0, and 54.9%, respectively. All other dependent variables were free of mean and slope bias, indicating an adequate representation of the data. Whereas mTOR was not very sensitive to changes in insulin or acetate levels, it was highly sensitive to leucine and isoleucine, and this signal appeared to be effectively transduced to casein synthesis. Although prior work had observed a relationship with additional essential AA, and data supporting those conclusions were present in the data set, we were unable to derive significant relationships with any essential

  6. [Dynamics of ultrastructural morphology of the nucleolar apparatus in bovine preimplantation embryos collected in an area of chronic irradiation].

    PubMed

    Pivko, J; Baran, V; Grafenau, P; Kopecný, V; Pelechatyj, N S; Bondarcuk, V N; Kozuch, A J; Kovalcik, L M

    1997-02-01

    Ultrastructural morphology and immunoelectron microscopy of the nucleus and nucleologenesis in early preimplantation cow embryos were applied in an attempt to demonstrate a possible radiation injury to that early stage of development due to chronical irradiation of the animals in the Tchernobyl area. Mostly eight cell embryos as well as morulae were collected from superovulated cows which were previously constantly kept in zones of different levels of radioactive irradiation. In addition to the normometric status of reproductive organs in no case was it possible to detect an apparent deviation in the nuclear morphology or in the process of nucleologenesis as compared to the physiological situation (Kopecný et al., 1989b, 1991, 1996). This observation was supported by an immunoelectron microscope study of DNA association and penetration in the differentiated nucleolus in the late 8-cell stage. These observations show that the otherwise demonstrated radiation injury localized in the genome does not probably influence markedly the early events of the developing embryo and that the aberrant cytoplasmic command of the nuclear events known in other types of oocyte/early cow embryo impairment (review Kopecný and Nicmann, 1993; Kanka et al. 1991; Pavlok et al., 1993) is not seen in early embryos collected from chronically irradiated animals. PMID:9148569

  7. Capacitation-Associated Glycocomponents of Mammalian Sperm.

    PubMed

    Liu, Min

    2016-05-01

    Mammalian fertilization is a series of events that are mostly carbohydrate mediated. The male gamete glycocomponents are extensively synthesized and modified during sperm development and sperm transport in the reproductive tracts. Freshly ejaculated mammalian sperm are required to undergo capacitation, which takes place in the female reproductive system, in order to become fully fertilizable. Several lines of evidence reveal changes in glycosylated sperm constituents during capacitation. Although the contributions of these molecular changes to capacitation are not completely understood, the presence, rearrangement, and/or modification of these sperm glycocomponents have been demonstrated to be important for fertilization. The following review summarizes mammalian sperm glycoconstituents, with emphasis on their molecular changes during capacitation. PMID:26363036

  8. 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.

  9. 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

  10. The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.

    PubMed

    Girardet, Anne; Viart, Victoria; Plaza, Stéphanie; Daina, Gemma; De Rycke, Martine; Des Georges, Marie; Fiorentino, Francesco; Harton, Gary; Ishmukhametova, Aliya; Navarro, Joaquima; Raynal, Caroline; Renwick, Pamela; Saguet, Florielle; Schwarz, Martin; SenGupta, Sioban; Tzetis, Maria; Roux, Anne-Françoise; Claustres, Mireille

    2016-04-01

    Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented. PMID:26014425

  11. The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus

    PubMed Central

    Girardet, Anne; Viart, Victoria; Plaza, Stéphanie; Daina, Gemma; De Rycke, Martine; Des Georges, Marie; Fiorentino, Francesco; Harton, Gary; Ishmukhametova, Aliya; Navarro, Joaquima; Raynal, Caroline; Renwick, Pamela; Saguet, Florielle; Schwarz, Martin; SenGupta, Sioban; Tzetis, Maria; Roux, Anne-Françoise; Claustres, Mireille

    2016-01-01

    Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented. PMID:26014425

  12. 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

  13. [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

  14. 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.

  15. Bach to the future: response to: Extending preimplantation genetic diagnosis: medical and non-medical uses.

    PubMed

    Ashcroft, R

    2003-08-01

    Professor Robertson sketches an elegant framework for policy evaluation and regulation of the use of preimplantation genetic diagnosis for various medical, medical related, and non-medical purposes. In criticism of his position, I argue that the distinction between policy and ethics upon which his argument relies is highly unstable, and the approach taken to ethical evaluation of particular parental interests leaves open many issues which the policy approach would hope to exclude. In conclusion I argue that while his position ultimately fails, the onus is on his critics to come up with a viable and satisfying alternative. PMID:12930853

  16. 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.

  17. Crossroads between Bacterial and Mammalian Glycosyltransferases

    PubMed Central

    Brockhausen, Inka

    2014-01-01

    Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

  18. Mammalian phospholipase C.

    PubMed

    Kadamur, Ganesh; Ross, Elliott M

    2013-01-01

    Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale. PMID:23140367

  19. Successful haematopoietic stem cell transplantation in 44 children from healthy siblings conceived after preimplantation HLA matching.

    PubMed

    Kahraman, Semra; Beyazyurek, Cagri; Yesilipek, Mehmet Akif; Ozturk, Gulyuz; Ertem, Mehmet; Anak, Sema; Kansoy, Savas; Aksoylar, Serap; Kuşkonmaz, Barış; Oniz, Haldun; Slavin, Shimon; Karakas, Zeynep; Tac, Huseyin Avni; Gulum, Nese; Ekmekci, Gokhan Cumhur

    2014-09-01

    Haematopoietic stem cell transplantation (HSCT) remains the best therapeutic option for many acquired and inherited paediatric haematological disorders. Unfortunately, the probability of finding an HLA matched donor is limited. An alternative technique is PGD combined with HLA matching, which offers the possibility of selecting unaffected embryos that are HLA compatible with the sick child, with the aim of possible use of stem cells from the resulting baby in future. Since the first successful report for Fanconi anaemia a decade ago, the therapeutic success of this technique was reported in a few cases and for a limited number of disorders. Here, we report full recovery of 44 sick children who received HSCT from healthy infants conceived after pre-implantation HLA matching for the following 10 indications; beta-thalassaemia, Wiskott-Aldrich syndrome, Fanconi anaemia, sickle cell anaemia, acute myeloid leukaemia, acute lymphoblastic leukaemia, Glanzmann's thrombasthaenia, Diamond-Blackfan anaemia, X-linked adrenoleukodystrophy and mucopolysaccharidosis type I. No serious complications were observed among recipients and donors. Graft failure occurred in four children with beta-thalassaemia where a second HSCT was planned. Preimplantation HLA matching is a reliable technique and provides a realistic option for couples seeking treatment for an affected child when no HLA-matched donor is available. PMID:25066893

  20. 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

  1. 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

  2. Developmental neuropsychological assessment of 4- to 5-year-old children born following Preimplantation Genetic Diagnosis (PGD): A pilot study.

    PubMed

    Sacks, Gilat Chaya; Altarescu, Gheona; Guedalia, Judith; Varshaver, Irit; Gilboa, Tal; Levy-Lahad, Ephrat; Eldar-Geva, Talia

    2016-01-01

    The purpose of this pilot study was to evaluate developmental neuropsychological profiles of 4- to 5-year-old children born after Preimplantation Genetic Diagnosis (PGD). Twenty-seven participants received a neurological examination and a battery of neuropsychological assessments including Wechsler Preschool & Primary Scale of Intelligence - Third Edition (WPPSI-III; cognitive development), Preschool Language Scale, Fourth Edition (PLS-4; language development), Wide Range Assessment of Visual Motor Abilities (visual motor abilities), Childhood Autism Rating Scales II (a screening test for autistic spectrum disorders), and the Miles ABC Test (ocular dominance). Parental questionnaires included the Behavior Rating Inventory of Executive Function Preschool Version (BRIEF-P; executive function), Child Behavior Checklist (CBCL) and the Carey Temperament Scales Behavioral Style Questionnaire (socioemotional development and temperament), and the Vineland Adaptive Behavior Scales, Interview Edition, Second Edition (general adaptive behavior). Subjects' tests results were compared to each test's norms. Children born after PGD demonstrated scores within the normal or above-normal ranges for all developmental outcomes (mean ± SD): WPPSI-III-VIQ 107.4 ± 14.4 (p = .013), PLS-4-Total 113.2 ± 12.4, p < .001), CBCL-Total 41.1 ± 8.6 (p < .001), BRIEF-P-Global Executive Composite 44.8 ± 9.5 (p = .009). Twelve (44%) of the PGD children had a significant difference between their VIQ and PIQ scores (compared to 27% in the general population). One subject was found to show possible signs of autistic spectrum disorder, although a family history of autism was noted. In conclusion, in this pilot study, children assessed at age 4-5 years and conceived after PGD displayed developmental neuropsychological outcomes within normal limits as compared to their chronologic peers. A larger study is needed to evaluate and follow the neuropsychological development of children born after PGD. PMID

  3. A generic, flexible protocol for preimplantation human leukocyte antigen typing alone or in combination with a monogenic disease, for rapid case work-up and application.

    PubMed

    Kakourou, Georgia; Destouni, Aspasia; Vrettou, Christina; Traeger-Synodinos, Jan; Kanavakis, Emmanuel

    2014-01-01

    Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for β-thalassemia (β-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases. PMID:24131134

  4. 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

  5. Structure of mammalian metallothionein

    SciTech Connect

    Kaegi, J.H.R.; Vasak, M.; Lerch, K.; Gilg, D.E.O.; Hunziker, P.; Bernhard, W.R.; Good, M.

    1984-03-01

    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 Me/sub 7/(Cys/sup -/)/sub 20/ stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. 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 surrounded by properly folded segments of the chain providing the ligands. /sup 1/H-NMR data and infrared absorption measurements are consistent with a tightly folded structure rich in ..beta..-type conformation. 79 references, 11 figures, 4 tables.

  6. Mammalian Sirtuins and Energy Metabolism

    PubMed Central

    Li, Xiaoling; Kazgan, Nevzat

    2011-01-01

    Sirtuins are highly conserved NAD+-dependent protein deacetylases and/or ADP-ribosyltransferases that can extend the lifespan of several lower model organisms including yeast, worms and flies. The seven mammalian sirtuins, SIRT1 to SIRT7, have emerged as key metabolic sensors that directly link environmental signals to mammalian metabolic homeostasis and stress response. Recent studies have shed light on the critical roles of sirtuins in mammalian energy metabolism in response to nutrient signals. This review focuses on the involvement of two nuclear sirtuins, SIRT1 and SIRT6, and three mitochondrial sirtuins, SIRT3, SIRT4, and SIRT5, in regulation of diverse metabolic processes. PMID:21614150

  7. Preimplantation Mouse Embryo Selection Guided by Light-Induced Dielectrophoresis

    PubMed Central

    Valley, Justin K.; Swinton, Paul; Boscardin, W. John; Lue, Tom F.; Rinaudo, Paolo F.; Wu, Ming C.; Garcia, Maurice M.

    2010-01-01

    Selection of optimal quality embryos for in vitro fertilization (IVF) transfer is critical to successful live birth outcomes. Currently, embryos are chosen based on subjective assessment of morphologic developmental maturity. A non-invasive means to quantitatively measure an embryo's developmental maturity would reduce the variability introduced by the current standard. We present a method that exploits the scaling electrical properties of pre-transfer embryos to quantitatively discern embryo developmental maturity using light-induced dielectrophoresis (DEP). We show that an embryo's DEP response is highly correlated with its developmental stage. Uniquely, this technique allows one to select, in sequence and under blinded conditions, the most developmentally mature embryos among a mixed cohort of morphologically indistinguishable embryos cultured in optimized and sub-optimal culture media. Following assay, embryos continue to develop normally in vitro. Light-induced dielectrophoresis provides a non-invasive, quantitative, and reproducible means to select embryos for applications including IVF transfer and embryonic stem cell harvest. PMID:20405021

  8. 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

  9. 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.

  10. Mammalian Interphase Cdks

    PubMed Central

    2012-01-01

    Cyclin-dependent kinases (Cdks) drive cell cycle progression in all eukaryotes. Yeasts have a single major Cdk that mediates distinct cell cycle transitions via association with different cyclins. The closest homolog in mammals, Cdk1, drives mitosis. Mammals have additional Cdks—Cdk2, Cdk4, and Cdk6—that represent the major Cdks activated during interphase (iCdks). A large body of evidence has accrued that suggests that activation of iCdks dictates progression though interphase. In apparent contradiction, deficiency in each individual iCdk, respectively, in knockout mice proved to be compatible with live birth and in some instances fertility. Moreover, murine embryos could be derived with Cdk1 as the only functional Cdk. Thus, none of the iCdks is strictly essential for mammalian cell cycle progression, raising the possibility that Cdk1 is the dominant regulator in interphase. However, an absence of iCdks has been accompanied by major shifts in cyclin association to Cdk1, suggesting gain in function. After considerable tweaking, a chemical genetic approach has recently been able to examine the impact of acute inhibition of Cdk2 activity without marked distortion of cyclin/Cdk complex formation. The results suggest that, when expressed at its normal levels, Cdk2 performs essential roles in driving human cells into S phase and maintaining genomic stability. These new findings appear to have restored order to the cell cycle field, bringing it full circle to the view that iCdks indeed play important roles. They also underscore the caveat in knockdown and knockout approaches that protein underexpression can significantly perturb a protein interaction network. We discuss the implications of the new synthesis for future cell cycle studies and anti–Cdk-based therapy of cancer and other diseases. PMID:23634250

  11. 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.

  12. Birth Defects Associated with Perturbations in Pre-implantation, Gastrulation & Axis Extension: from Conjoined Twinning to Caudal Dysgenesis

    PubMed Central

    Ferrer-Vaquer, Anna; Hadjantonakis, Anna-Katerina

    2012-01-01

    Congenital malformations represent approximately 3 in 100 live births within the human population. Understanding their pathogenesis and ultimately formulating effective treatments is underpinned by knowledge of the events and factors that regulate normal embryonic development. Studies in model organisms, primarily in the mouse, the most prominent genetically tractable mammalian model, have equipped us with a rudimentary understanding of mammalian development from early lineage commitment to morphogenetic processes. In this way information provided by studies in the mouse can, in some cases, be used to draw parallels with other mammals, including human. Here we provide an overview of our current understanding of the general sequence of developmental events from early cell cleavages to gastrulation and axis extension occurring in human embryos. We will also review some of the rare birth defects occurring at these stages, in particular those resulting in conjoined twinning or caudal dysgenesis. PMID:24014416

  13. Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment.

    PubMed Central

    Goldstein, A L; McCusker, J H

    2001-01-01

    Saccharomyces cerevisiae, a close relative of the pathogenic Candida species, is an emerging opportunistic pathogen. An isogenic series of S. cerevisiae strains, derived from a human clinical isolate, were used to examine the role of evolutionarily conserved pathways in fungal survival in a mouse host. As is the case for the corresponding Candida albicans and Cryptococcus neoformans mutants, S. cerevisiae purine and pyrimidine auxotrophs were severely deficient in survival, consistent with there being evolutionary conservation of survival traits. Resistance to the antifungal drug 5-fluorocytosine was not deleterious and appeared to be slightly advantageous in vivo. Of mutants in three amino acid biosynthetic pathways, only leu2 mutants were severely deficient in vivo. Unlike the glyoxylate cycle, respiration was very important for survival; however, the mitochondrial genome made a respiration-independent contribution to survival. Mutants deficient in pseudohyphal formation were tested in vivo; flo11Delta mutants were phenotypically neutral while flo8Delta, tec1Delta, and flo8Delta tec1Delta mutants were slightly deficient. Because of its ease of genetic manipulation and the immense S. cerevisiae database, which includes the best annotated eukaryotic genome sequence, S. cerevisiae is a superb model system for the identification of gene products important for fungal survival in the mammalian host environment. PMID:11606528

  14. 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

  15. [Introduction in Switzerland of preimplantation genetic testing: progress or downward spiral?].

    PubMed

    Irion, Nicole Fournet; Irion, Olivier

    2016-01-13

    The Swiss law on Assisted Reproductive Techniques (LPMA) has been modified in order to authorize preimplantation genetic diagnosis (PGD). PGD has been performed for 20 years. Switzerland is one of the last european countries where it is still prohibited. As a result, couples carrying a severe inherited disease and infertile couples with recurrent implantation failure or miscarriage have to cross the borders in order to have access to the appropriate treatments. Despite the recent popular approval to change the Constitution, the new LPMA cannot be implemented as the opponants have launched a referendum in order to obtain a more restrictive law. If they succeed, the affected couples will be left with a scientifically obsolete law that will not allow them to have access to an effective and compassionate treatment. PMID:26946702

  16. The first successful live birth following preimplantation genetic diagnosis using PCR for type 1 citrullinemia

    PubMed Central

    Cho, Jae-Hyun; Lee, Kyung-Hee; Jeon, Il-Kyung; Kim, Jae-Min; Kang, Byung-Moon

    2014-01-01

    Type 1 citrullinemia (CTLN1) is an autosomal recessive inherited metabolic disorder caused by anargininosuccinicnate synthetase deficiency. The patient was a 38-year-old Korean woman who is a carrier for CTLN1 and her first baby was diagnosed with CTLN1. Preimplantation genetic diagnosis (PGD) for CTLN1 in day 3 embryos using polymerase chain reaction was performed for live birth of healthy baby who is no affected with CTLN1. One unaffected blastocyst was transferred. This resulted in a clinical pregnancy and the live birth of healthy male twin. They were confirmed to be unaffected with CTNL1 by post natal diagnosis. This is the first case report of the use of PGD for CTNL1. PMID:24883299

  17. 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

  18. A simple method for counting nuclei in the preimplantation mouse embryo.

    PubMed

    Ebert, K M; Hammer, R E; Papaioannou, V E

    1985-09-15

    An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed. PMID:2412884

  19. Selecting barrenness: the use of preimplantation genetic diagnosis by congenitally infertile women to select for infertility.

    PubMed

    Shah, Kavita R

    2010-01-01

    Congenitally infertile women such as those with Turner syndrome or Mayer Rokitansky-Kuster-Hauser syndrome have available the technologies of oocyte harvesting, cryropreservation, in-vitro fertilization, and gestational surrogacy in order to have genetically related offspring. Since congenital infertility results in a variety of experiences that impacts on nearly every aspect of a person's life, in the future it is possible that these women might desire a congenitally infertile child through the use of preimplantation genetic diagnosis so as to share this common bond. While infertility results in a relatively normal quality of life, it is morally wrong to necessitate the future use of infertility services with its variable success rate on a child. Also, whereas the woman has fundamental reproductive autonomy, she lacks the substantive autonomy regarding the specific characteristics of her child. Finally, the infertile community does exhibit a strong presence, but it lacks characteristics that define it as a culture. PMID:21644427

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

    PubMed

    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

  1. Choosing between possible lives: legal and ethical issues in preimplantation genetic diagnosis.

    PubMed

    Scott, Rosamund

    2006-01-01

    This article critically appraises the current legal scope of the principal applications of preimplantation genetic diagnosis (PGD). This relatively new technique, which is available to some parents undergoing in vitro fertilization (IVF) treatment, aims to ensure that a child is not born with a seemingly undesirable genetic condition. The question addressed here is whether there should be serious reasons to test for genetic conditions in embryos in order to be able to select between them. The Human Fertilisation and Embryology Authority and the Human Genetics Commission have decided that there should be such reasons by broadly aligning the criteria for PGD with those for selective abortion. This stance is critically explored, as are its implications for the possible use of PGD to select either against or for marginal features or for significant traits. The government is currently reviewing the legal scope and regulation of PGD. PMID:17340769

  2. 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

  3. Fate Mapping Mammalian Corneal Epithelia.

    PubMed

    Richardson, Alexander; Wakefield, Denis; Di Girolamo, Nick

    2016-04-01

    The anterior aspect of the cornea consists of a stratified squamous epithelium, thought to be maintained by a rare population of stem cells (SCs) that reside in the limbal transition zone. Although migration of cells that replenish the corneal epithelium has been studied for over a century, the process is still poorly understood and not well characterized. Numerous techniques have been employed to examine corneal epithelial dynamics, including visualization by light microscopy, the incorporation of vital dyes and DNA labels, and transplantation of genetically marked cells that have acted as cell and lineage beacons. Modern-day lineage tracing utilizes molecular methods to determine the fate of a specific cell and its progeny over time. Classically employed in developmental biology, lineage tracing has been used more recently to track the progeny of adult SCs in a number of organs to pin-point their location and understand their movement and influence on tissue regeneration. This review highlights key discoveries that have led researchers to develop cutting-edge genetic tools to effectively and more accurately monitor turnover and displacement of cells within the mammalian corneal epithelium. Collating information on the basic biology of SCs will have clinical ramifications in furthering our knowledge of the processes that govern their role in homeostasis, wound-healing, transplantation, and how we can improve current unsatisfactory SC-based therapies for patients suffering blinding corneal disease. PMID:26774909

  4. Aneuploidy in mammalian somatic cells in vivo.

    PubMed

    Cimino, M C; Tice, R R; Liang, J C

    1986-01-01

    Aneuploidy is an important potential source of human disease and of reproductive failure. Nevertheless, the ability of chemical agents to induce aneuploidy has been investigated only sporadically in intact (whole-animal) mammalian systems. A search of the available literature from the EMCT Aneuploidy File (for years 1970-1983) provided 112 papers that dealt with aneuploidy in mammalian somatic cells in vivo. 59 of these papers did not meet minimal criteria for analysis and were rejected from subsequent review. Of the remaining 53 papers that dealt with aneuploidy induction by chemical agents in mammalian somatic cells in vivo, only 3 (6%) contained data that were considered to be supported conclusively by adequate study designs, execution, and reporting. These 3 papers dealt with 2 chemicals, one of which, mercury, was negative for aneuploidy induction in humans, and the other, pyrimethamine, was positive in an experimental rodent study. The majority of papers (94%) were considered inconclusive for a variety of reasons. The most common reasons for calling a study inconclusive were (a) combining data on hyperploidy with those on hypoploidy and/or polyploidy, (b) an inadequate or unspecified number of animals and/or cells per animal scored per treatment group, and (c) poor data presentation such that animal-to-animal variability could not be assessed. Suggestions for protocol development are made, and the future directions of research into aneuploidy induction are discussed. PMID:3941670

  5. Structure and function of mammalian aldehyde oxidases.

    PubMed

    Terao, Mineko; Romão, Maria João; Leimkühler, Silke; Bolis, Marco; Fratelli, Maddalena; Coelho, Catarina; Santos-Silva, Teresa; Garattini, Enrico

    2016-04-01

    Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX. PMID:26920149

  6. Sirtuins: Guardians of Mammalian Healthspan

    PubMed Central

    Giblin, William; Skinner, Mary E.; Lombard, David B.

    2014-01-01

    The first link between sirtuins and longevity was made 15 years ago in yeast. These initial studies sparked efforts by many laboratories working in diverse model organisms to elucidate the relationships between sirtuins, lifespan, and age-associated dysfunction. Here we discuss the current understanding of how sirtuins relate to aging. We focus primarily on mammalian sirtuins SIRT1, SIRT3, and SIRT6, the three sirtuins for which the most relevant data are available. Strikingly, a large body of evidence now indicates that these and other mammalian sirtuins suppress a variety of age-related pathologies and promote healthspan. Moreover, increased expression of SIRT1 or SIRT6 extends mouse lifespan. Overall, these data point to important roles for sirtuins in promoting mammalian health, and perhaps in modulating the aging process. PMID:24877878

  7. 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.

  8. Reverse genetics for mammalian reovirus.

    PubMed

    Boehme, Karl W; Ikizler, Miné; Kobayashi, Takeshi; Dermody, Terence S

    2011-10-01

    Mammalian orthoreoviruses (reoviruses) are highly tractable models for studies of viral replication and pathogenesis. The versatility of reovirus as an experimental model has been enhanced by development of a plasmid-based reverse genetics system. Infectious reovirus can be recovered from cells transfected with plasmids encoding cDNAs of each reovirus gene segment using a strategy that does not require helper virus and is independent of selection. In this system, transcription of each gene segment is driven by bacteriophage T7 RNA polymerase, which can be supplied transiently by recombinant vaccinia virus (rDIs-T7pol) or by cells that constitutively express the enzyme. Reverse genetics systems have been developed for two prototype reovirus strains, type 1 Lang (T1L) and type 3 Dearing (T3D). Each reovirus cDNA was encoded on an independent plasmid for the first-generation rescue system. The efficiency of virus recovery was enhanced in a second-generation system by combining the cDNAs for multiple reovirus gene segments onto single plasmids to reduce the number of plasmids from 10 to 4. The reduction in plasmid number and the use of baby hamster kidney cells that express T7 RNA polymerase increased the efficiency of viral rescue, reduced the incubation time required to recover infectious virus, and eliminated potential biosafety concerns associated with the use of recombinant vaccinia virus. Reovirus reverse genetics has been used to introduce mutations into viral capsid and nonstructural components to study viral protein-structure activity relationships and can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications. PMID:21798351

  9. Pre-implant right ventricular function might be an important predictor of the response to cardiac resynchronization therapy

    PubMed Central

    2011-01-01

    Objective Cardiac resynchronization therapy is proven efficacious in patients with heart failure (HF). Presence of biventricular HF is associated with a worse prognosis than having only left ventricular (LV) HF and pacing might deteriorate heart function. The aim of the study was to assess a possible significance of right ventricular (RV) pre-implant systolic function to predict response to CRT. Design We studied 22 HF-patients aged 72 ± 11 years, QRS-duration 155 ± 20 ms and with an LV ejection fraction (EF) of 26 ± 6% before and four weeks after receiving a CRT-device. Results There were no changes in LV diameters or end systolic volume (ESV) during the study. However, end diastolic volume (EDV) decreased from 226 ± 71 to 211 ± 64 ml (p = 0.02) and systolic maximal velocities (SMV) increased from 2.2 ± 0.4 to 2.6 ± 0.9 cm/s (p = 0.04). Pre-implant RV-SMV (6.2 ± 2.6 cm/s) predicted postoperative increase in LV contractility, p = 0.032. Conclusions Pre-implant decreased RV systolic function might be an important way to predict a poor response to CRT implicating that other treatments should be considered. Furthermore we found that 3D- echocardiography and Tissue Doppler Imaging were feasible to detect short-term changes in LV function. PMID:22029550

  10. 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

  11. 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

  12. 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

  13. 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

  14. PreImplantation factor (PIF*) promotes embryotrophic and neuroprotective decidual genes: effect negated by epidermal growth factor

    PubMed Central

    2014-01-01

    Background Intimate embryo-maternal interaction is paramount for pregnancy success post-implantation. The embryo follows a specific developmental timeline starting with neural system, dependent on endogenous and decidual factors. Beyond altered genetics/epigenetics, post-natal diseases may initiate at prenatal/neonatal, post-natal period, or through a continuum. Preimplantation factor (PIF) secreted by viable embryos promotes implantation and trophoblast invasion. Synthetic PIF reverses neuroinflammation in non-pregnant models. PIF targets embryo proteins that protect against oxidative stress and protein misfolding. We report of PIF’s embryotrophic role and potential to prevent developmental disorders by regulating uterine milieu at implantation and first trimester. Methods PIF’s effect on human implantation (human endometrial stromal cells (HESC)) and first-trimester decidua cultures (FTDC) was examined, by global gene expression (Affymetrix), disease-biomarkers ranking (GeneGo), neuro-specific genes (Ingenuity) and proteins (mass-spectrometry). PIF co-cultured epidermal growth factor (EGF) in both HESC and FTDC (Affymetrix) was evaluated. Results In HESC, PIF promotes neural differentiation and transmission genes (TLX2, EPHA10) while inhibiting retinoic acid receptor gene, which arrests growth. PIF promotes axon guidance and downregulates EGF-dependent neuroregulin signaling. In FTDC, PIF promotes bone morphogenetic protein pathway (SMAD1, 53-fold) and axonal guidance genes (EPH5) while inhibiting PPP2R2C, negative cell-growth regulator, involved in Alzheimer’s and amyotrophic lateral sclerosis. In HESC, PIF affects angiotensin via beta-arrestin, transforming growth factor-beta (TGF-β), notch, BMP, and wingless-int (WNT) signaling pathways that promote neurogenesis involved in childhood neurodevelopmental diseases—autism and also affected epithelial-mesenchymal transition involved in neuromuscular disorders. In FTDC, PIF upregulates neural development

  15. 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

  16. 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

  17. Breast Cancer, BRCA Mutations, and Attitudes Regarding Pregnancy and Preimplantation Genetic Diagnosis

    PubMed Central

    Woodson, Ashley H.; Muse, Kimberly I.; Lin, Heather; Jackson, Michelle; Mattair, Danielle N.; Schover, Leslie; Woodard, Terri; McKenzie, Laurie; Theriault, Richard L.; Hortobágyi, Gabriel N.; Arun, Banu; Peterson, Susan K.; Profato, Jessica

    2014-01-01

    Background. Women with premenopausal breast cancer may face treatment-related infertility and have a higher likelihood of a BRCA mutation, which may affect their attitudes toward future childbearing. Methods. Premenopausal women were invited to participate in a questionnaire study administered before and after BRCA genetic testing. We used the Impact of Event Scale (IES) to evaluate the pre- and post-testing impact of cancer or carrying a BRCA mutation on attitudes toward future childbearing. The likelihood of pursuing prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD) was also assessed in this setting. Univariate analyses determined factors contributing to attitudes toward future childbearing and likelihood of PND or PGD. Results. One hundred forty-eight pretesting and 114 post-testing questionnaires were completed. Women with a personal history of breast cancer had less change in IES than those with no history of breast cancer (p = .003). The 18 BRCA-positive women had a greater change in IES than the BRCA-negative women (p = .005). After testing, 31% and 24% of women would use PND and PGD, respectively. BRCA results did not significantly affect attitudes toward PND/PGD. Conclusion. BRCA results and history of breast cancer affect the psychological impact on future childbearing. Intentions to undergo PND or PGD do not appear to change after disclosure of BRCA results. Additional counseling for patients who have undergone BRCA testing may be warranted to educate patients about available fertility preservation options. PMID:24951607

  18. 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

  19. Preimplantation genetic screening- the required RCT that has not yet been carried out.

    PubMed

    Orvieto, Raoul

    2016-01-01

    The utilization of trophectoderm biopsy combined with comprehensive chromosome screening (CCS) tests for embryonic aneuploidy was recently suggested to improve IVF outcome, however, not without criticisms. The ongoing discussion on the unrestricted clinical adoption of preimplantation genetic screening (PGS) has called for a proper randomized controlled trial (RCT), aiming to further evaluate the cumulative live birth rates (LBRs) following a single oocyte retrieval, utilizing all fresh and frozen embryos. Since this study seems not to appear for various reasons, we present herewith, the hypothetical required RCT based on the hitherto published literature.After implementing data from the hitherto published literature on blastulation and aneuploidy rates, the rate of mosaicism and technical errors and implantation rates/LBRs of non-PGS day-3 and blastocyst and PGS blastocyst, we could clearly demonstrate the superiority of non-PGS embryo (day-3 and blastocyst) transfer over PGS blastocyst transfer, in terms of cumulative LBR (18.2-50 % vs 7.6-12.6 %, respectively).We therefore believe that until the proper, non-hypothetical RCT on the efficacy of this procedure will appear, PGS should be offered only under study conditions, and with appropriate informed consents. PMID:27342051

  20. Evaluation of exclusion prenatal and exclusion preimplantation genetic diagnosis for Huntington's disease in the Netherlands.

    PubMed

    van Rij, M C; de Die-Smulders, C E M; Bijlsma, E K; de Wert, G M W R; Geraedts, J P; Roos, R A C; Tibben, A

    2013-02-01

    Individuals at 50% risk of Huntington's disease (HD) who prefer not to know their carrier status, might opt for exclusion prenatal diagnosis (ePND) or exclusion preimplantation genetic diagnosis (ePGD). This study aims to provide a better understanding of couples' motives for choosing ePND or ePND, and surveys couples' experiences in order to make recommendations for the improvement of counselling for exclusion testing. This qualitative retrospective interview study focussed on couples who underwent ePND or ePGD for HD in the period 1996-2010. Seventeen couples were included of which 13 had experienced ePND and 6 ePGD. Mean time-interval since exclusion-testing was 3.9 years. Couples' moral reservations regarding termination of pregnancy (TOP) or discarding healthy embryos were counterbalanced by the wish to protect their future child against HD. Seven couples had terminated a total of 11 pregnancies with a 50% HD risk, none showed regret. ePGD was used by couples who wanted to avoid (another) TOP. ePND and ePGD are acceptable reproductive options for a specific group of counsellees. To guarantee sound standards of care, it is imperative that candidate couples be given in-depth non-directive counselling about all possible scenarios, and adequate professional and psychological support prior to, during and after ePND/ePGD. PMID:23137131

  1. ESHRE task force on ethics and Law22: preimplantation genetic diagnosis.

    PubMed

    De Wert, G; Dondorp, W; Shenfield, F; Devroey, P; Tarlatzis, B; Barri, P; Diedrich, K; Provoost, V; Pennings, G

    2014-08-01

    This Task Force document discusses some relatively unexplored ethical issues involved in preimplantation genetic diagnosis (PGD). The document starts from the wide consensus that PGD is ethically acceptable if aimed at helping at-risk couples to avoid having a child with a serious disorder. However, if understood as a limit to acceptable indications for PGD, this 'medical model' may turn out too restrictive. The document discusses a range of possible requests for PGD that for different reasons fall outwith the accepted model and argues that instead of rejecting those requests out of hand, they need to be independently assessed in the light of ethical criteria. Whereas, for instance, there is no good reason for rejecting PGD in order to avoid health problems in a third generation (where the second generation would be healthy but faced with burdensome reproductive choices if wanting to have children), using PGD to make sure that one's child will have the same disorder or handicap as its parents, is ethically unacceptable. PMID:24927929

  2. The Effect of Preimplantation Genetic Screening on Implantation Rate in Women over 35 Years of Age

    PubMed Central

    Moayeri, Mina; Saeidi, Hojatolah; Modarresi, Mohammad Hossein; Hashemi, Mehrdad

    2016-01-01

    Objective Advanced maternal age (AMA) is an important factor in decreasing success of assisted reproductive technology by having a negative effect on the success rate of intra-cytoplasmic sperm injection (ICSI), particularly by increasing the rate of embryo aneuploidy. It has been suggested that the transfer of euploid embryos increases the implantation and pregnancy rates, and decreases the abortion rate. Preimplantation genetic screening (PGS) is a method for selection of euploid embryos. Past studies, however, have reported different results on the success of pregnancy after PGS in AMA. Investigating the pregnancy rate of ICSI with and without PGS in female partners over 35 years of age referred to infertility centers in Tehran. Materials and Methods In this randomized controlled trial, 150 couples with the female partner over age of 35 were included. Fifty couples underwent PGS and the remaining were used as the control group. PGS was carried out using fluorescent in situ hybridization (FISH) for chromosomes 13, 18, 21, X and Y. Results of embryo transfer following PGS were evaluated and compared with those in the control group. Results Implantation rates obtained in the PGS and control groups were 30 and 32% respectively and not significantly different (P>0.05). Conclusion PGS for chromosomes 13, 18, 21, X and Y does not increase implantation rate in women over 35 years of age and therefore the regular use of PGS in AMA is not recommended. PMID:27054114

  3. Non-Invasive Prenatal Diagnosis in the Management of Preimplantation Genetic Diagnosis Pregnancies

    PubMed Central

    Bustamante-Aragones †, Ana; Perlado-Marina †, Sara; Trujillo-Tiebas, Maria José; Gallego-Merlo, Jesús; Lorda-Sanchez, Isabel; Rodríguez-Ramirez, Luz; Linares, Concepcion; Hernandez, Corazón; Rodriguez de Alba, Marta

    2014-01-01

    Prenatal diagnosis (PD) is recommended in pregnancies after a Preimplantation Genetic Diagnosis (PGD). However, conventional PD entails a risk of fetal loss which makes PGD patients reluctant to undergo obstetric invasive procedures. The presence of circulating fetal DNA in maternal blood allows performing a non-invasive prenatal diagnosis (NIPD) without risk for the pregnancy outcome. This work shows the introduction of NIPD for confirmation of PGD results in eight pregnancies. In those pregnancies referred to PGD for an X-linked disorder (six out of eight), fetal sex determination in maternal blood was performed to confirm fetal sex. One pregnancy referred to PGD for Marfan syndrome and one referred for Huntington disease (HD) were also analyzed. In seven out of eight cases, PGD results were confirmed by NIPD in maternal blood. No results were obtained in the HD pregnancy. NIPD in PGD pregnancies can be a reliable alternative for couples that after a long process feel reluctant to undergo PD due to the risk of pregnancy loss. PMID:26237485

  4. Preimplantation genetic diagnosis for inherited breast cancer: first clinical application and live birth in Spain.

    PubMed

    Ramón Y Cajal, Teresa; Polo, Ana; Martínez, Olga; Giménez, Carles; Arjona, César; Llort, Gemma; Bassas, Lluís; Viscasillas, Pere; Calaf, Joaquin

    2012-06-01

    Carriers of a mutation in BRCA1/2 genes confront a high lifetime risk of breast and ovarian cancer and fifty percent probability of passing the mutation to their offspring. Current options for risk management influence childbearing decisions. The indications for preimplantation genetic diagnosis (PGD) have now been expanded to include predisposition for single-gene, late-onset cancer but few cases have been reported to date despite the favorable opinion among professionals and carriers. A 28-year-old BRCA1 mutation carrier (5273G>A in exon 19) with a strong maternal history of breast cancer and 2 years of infertility decided to pursue PGD to have a healthy descendent after an accurate assessment of her reproductive options. The procedure was approved by the national regulation authority and a PGD cycle was initiated. Four out of 6 embryos harbored the mutation. The two unaffected embryos were implanted in the uterus. A singleton pregnancy was achieved and a male baby was delivered at term. Consented umbilical cord blood testing confirmed the accuracy of the technique. Individualized PGD for inherited breast predisposition is feasible in the context of a multidisciplinary team. PMID:22179695

  5. On the relation between moral, legal and evaluative justifications of pre-implantation genetic diagnosis (PGD).

    PubMed

    Lohmann, Georg

    2003-01-01

    In Germany the question whether to uphold or repeal the judicial prohibition on Pre-implantation Genetic Diagnosis (PGD) is being debated from quite different standpoints. This paper differentiates the major arguments according to their reasons as a) moral, b) evaluative (i.e. cultural/religious), and c) legal. The arguments for and against PGD can be divided by content into three groups: arguments relating to the status of the embryo, focusing on individual actions in the implementation of PGD, and relating to the foreseeable or probable consequences of PGD. In Germany, from a legal perspective, the status of the embryo does not permit the intervention of PGD; from a purely moral perspective, a prohibition on PGD does not appear defensible. It remains an open question, however, whether the moral argument permitting PGD should be restricted for evaluative (cultural) reasons. The paper discusses the species-ethical reasons, for which Jurgen Habermas sees worrisome consequences in the wake of PGD to the extent that we comprehend it as the forerunner of a 'positive eugenics'. It would so disrupt the natural preconditions of our universal morality. The question of whether to prohibit or allow PGD is not merely a question of simple moral and/or legal arguments, but demands a choice between evaluative, moral and (still to be specified) species-ethical arguments, and the question remains open. PMID:16206459

  6. BRD4 regulates Nanog expression in mouse embryonic stem cells and preimplantation embryos

    PubMed Central

    Liu, W; Stein, P; Cheng, X; Yang, W; Shao, N-Y; Morrisey, E E; Schultz, R M; You, J

    2014-01-01

    Bromodomain-containing protein 4 (BRD4) is an important epigenetic reader implicated in the pathogenesis of a number of different cancers and other diseases. Brd4-null mouse embryos die shortly after implantation and are compromised in their ability to maintain the inner cell mass, which gives rise to embryonic stem cells (ESCs). Here we report that BRD4 regulates expression of the pluripotency factor Nanog in mouse ESCs and preimplantation embryos, as well as in human ESCs and embryonic cancer stem cells. Inhibition of BRD4 function using a chemical inhibitor, small interfering RNAs, or a dominant-negative approach suppresses Nanog expression, and abolishes the self-renewal ability of ESCs. We also find that BRD4 associates with BRG1 (brahma-related gene 1, aka Smarca4 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4)), a key regulator of ESC self-renewal and pluripotency, in the Nanog regulatory regions to regulate Nanog expression. Our study identifies Nanog as a novel BRD4 target gene, providing new insights for the biological function of BRD4 in stem cells and mouse embryos. Knowledge gained from these non-cancerous systems will facilitate future investigations of how Brd4 dysfunction leads to cancers. PMID:25146928

  7. Preimplantation genetic diagnosis (PGD) for Huntington's disease: the experience of three European centres

    PubMed Central

    Van Rij, Maartje C; De Rademaeker, Marjan; Moutou, Céline; Dreesen, Jos CFM; De Rycke, Martine; Liebaers, Inge; Geraedts, Joep PM; De Die-Smulders, Christine EM; Viville, Stéphane

    2012-01-01

    This study provides an overview of 13 years of experience of preimplantation genetic diagnosis (PGD) for Huntington's disease (HD) at three European PGD centres in Brussels, Maastricht and Strasbourg. Information on all 331 PGD intakes for HD, couples' reproductive history, PGD approach, treatment cycles and outcomes between 1995 and 2008 were collected prospectively. Of 331 couples for intake, 68% requested direct testing and 32% exclusion testing (with a preponderance of French couples). At the time of PGD intake, 39% of women had experienced one or more pregnancies. A history of pregnancy termination after prenatal diagnosis was observed more frequently in the direct testing group (25%) than in the exclusion group (10% P=0.0027). PGD workup was based on two approaches: (1) direct testing of the CAG-triplet repeat and (2) linkage analysis using intragenic or flanking microsatellite markers of the HTT gene. In total, 257 couples had started workup and 174 couples (70% direct testing, 30% exclusion testing) completed at least one PGD cycle. In total, 389 cycles continued to oocyte retrieval (OR). The delivery rates per OR were 19.8%, and per embryo transfer 24.8%, resulting in 77 deliveries and the birth of 90 children. We conclude that PGD is a valuable and safe reproductive option for HD carriers and couples at risk of transmitting HD. PMID:22071896

  8. Short tandem repeats haplotyping of the HLA region in preimplantation HLA matching.

    PubMed

    Fiorentino, Francesco; Kahraman, Semra; Karadayi, Hüseyin; Biricik, Anil; Sertyel, Semra; Karlikaya, Güvenc; Saglam, Yaman; Podini, Daniele; Nuccitelli, Andrea; Baldi, Marina

    2005-08-01

    Recently, preimplantation genetic diagnosis (PGD) has been considered for several indications beyond its original purpose, not only to test embryos for genetic disease but also to select embryos for a nondisease trait, such as specific human leukocyte antigen (HLA) genotypes, related to immune compatibility with an existing affected child in need of a haematopoetic stem cell (HSC) transplant. We have optimized an indirect single-cell HLA typing protocol based on a multiplex fluorescent polymerase chain reaction (PCR) of short tandem repeat (STR) markers scattered throughout the HLA complex. The assay was clinically applied in 60 cycles from 45 couples. A conclusive HLA-matching diagnosis was achieved in 483/530 (91.1%) of the embryos tested. In total, 74 (15.3%) embryos revealed an HLA match with the affected siblings, 55 (11.4%) of which resulted unaffected and 46 (9.5%) have been transferred to the patients. Nine pregnancies were achieved, five healthy HLA-matched children have already been delivered and cord blood HSCs, were transplanted to three affected siblings, resulting in a successful haematopoietic reconstruction. PMID:15886713

  9. Non-Invasive Prenatal Diagnosis in the Management of Preimplantation Genetic Diagnosis Pregnancies.

    PubMed

    Bustamante-Aragones, Ana; Perlado-Marina, Sara; Trujillo-Tiebas, Maria José; Gallego-Merlo, Jesús; Lorda-Sanchez, Isabel; Rodríguez-Ramirez, Luz; Linares, Concepcion; Hernandez, Corazón; de Alba, Marta Rodriguez

    2014-01-01

    Prenatal diagnosis (PD) is recommended in pregnancies after a Preimplantation Genetic Diagnosis (PGD). However, conventional PD entails a risk of fetal loss which makes PGD patients reluctant to undergo obstetric invasive procedures. The presence of circulating fetal DNA in maternal blood allows performing a non-invasive prenatal diagnosis (NIPD) without risk for the pregnancy outcome. This work shows the introduction of NIPD for confirmation of PGD results in eight pregnancies. In those pregnancies referred to PGD for an X-linked disorder (six out of eight), fetal sex determination in maternal blood was performed to confirm fetal sex. One pregnancy referred to PGD for Marfan syndrome and one referred for Huntington disease (HD) were also analyzed. In seven out of eight cases, PGD results were confirmed by NIPD in maternal blood. No results were obtained in the HD pregnancy. NIPD in PGD pregnancies can be a reliable alternative for couples that after a long process feel reluctant to undergo PD due to the risk of pregnancy loss. PMID:26237485

  10. Killing of preimplantation mouse embryos by main ingredients of cleansers AS and LAS.

    PubMed

    Nomura, T; Hata, S; Shibata, K; Kusafuka, T

    1987-01-01

    When main ingredients of cleansers, alcohol sulfate (AS) and linear alkylbenzene sulfonate (LAS), were applied to the dorsal skin of pregnant JCL:ICR mice during preimplantation period (days 0-2), significant numbers of embryos collected from the oviducts and uteri on day 3 showed severe deformity or remained at the morula stage. Most of abnormal embryos were fragmented or remained at the 1-8 cell stages, and they were either dead or dying. Even when these abnormal embryos were cultivated in the detergent-free medium, they were not recovered, while most growth-retarded embryos (morula) could grow and hatch with one or two days lag by the further in vitro cultivation. Similar results were observed with commercially obtained kitchen detergent and hair shampoo, although such embryocidal effects were not detected with natural soap and distilled water. Fertilized eggs may be specifically sensitive to synthetic detergents. Very low doses of X-rays also induced significant yields of abnormal embryos. Major difference between X-rays and detergents was that X-ray-induced abnormality appeared at the morula or blastocyst stage, while detergent-induced one did at the earlier stages. PMID:3796668

  11. Successful birth of South India's first twins after preimplantation genetic screening of embryos

    PubMed Central

    Selvaraj, Priya; Selvaraj, Kamala; Srinivasan, Kalaichelvi; Sivakumar, Mahalakshmi

    2016-01-01

    We report the first documented successful birth of twins following preimplantation genetic screening (PGS) of cleavage stage embryos by array comparative genomic hybridization (CGH) technology, in South India. The case was a 28-year-old woman with the previous history of preclinical pregnancy and a miscarriage in two attempted in vitro fertilization cycles. Day 3 cleavage stage embryos were generated by conventional long protocol with the use of a gonadotropin-releasing hormone analog and a combination of recombinant folliculotropins and human menopausal gonadotropins. Intracytoplasmic sperm injection of oocytes thus obtained was performed, and 10 selected embryos underwent PGS using the array CGH technique. Two normal blastocysts were transferred to the patient, and she conceived twins. She delivered at 35 weeks of gestation by elective cesarean on November 19, 2014. She delivered a healthy male and female baby weighing 2.19 kg and 2.26 kg, respectively. Postnatal evaluation of babies was also normal, and the hospital course was uneventful. PGS has a definitive indication in assisted reproductive technology programs and can be utilized to improve pregnancy rates significantly. PMID:27382239

  12. The use of preimplantation genetic diagnosis in sex selection for family balancing in India.

    PubMed

    Malpani, A; Malpani, A; Modi, D

    2002-01-01

    This paper describes the use of preimplantation genetic diagnosis (PGD) in sexing embryos for family balancing in a private IVF clinic in India from April 1999 to 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, 14 clinical pregnancies and nine live births have been achieved so far, with five ongoing pregnancies. The benefits of delayed transfer 24-48 h after the embryo biopsy are that PGD centres could use the extra time available to confirm the diagnosis or introduce additional diagnostic tests for the same embryo. The selection of blastocysts for transfer should also permit the transfer of fewer embryos, thus reducing the risk of multiple gestations and increasing the pregnancy rate as a consequence of the expected higher implantation rate. This is the first report of the use of PGD in 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 briefly discussed. PMID:12470347

  13. 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

  14. 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

  15. Nanoelectrochemistry of mammalian cells

    PubMed Central

    Sun, Peng; Laforge, François O.; Abeyweera, Thushara P.; Rotenberg, Susan A.; Carpino, James; Mirkin, Michael V.

    2008-01-01

    There is a significant current interest in development of new techniques for direct characterization of the intracellular redox state and high-resolution imaging of living cells. We used nanometer-sized amperometric probes in combination with the scanning electrochemical microscope (SECM) to carry out spatially resolved electrochemical experiments in cultured human breast cells. With the tip radius ≈1,000 times smaller than that of a cell, an electrochemical probe can penetrate a cell and travel inside it without apparent damage to the membrane. The data demonstrate the possibility of measuring the rate of transmembrane charge transport and membrane potential and probing redox properties at the subcellular level. The same experimental setup was used for nanoscale electrochemical imaging of the cell surface. PMID:18178616

  16. Nanoelectrochemistry of mammalian cells.

    PubMed

    Sun, Peng; Laforge, François O; Abeyweera, Thushara P; Rotenberg, Susan A; Carpino, James; Mirkin, Michael V

    2008-01-15

    There is a significant current interest in development of new techniques for direct characterization of the intracellular redox state and high-resolution imaging of living cells. We used nanometer-sized amperometric probes in combination with the scanning electrochemical microscope (SECM) to carry out spatially resolved electrochemical experiments in cultured human breast cells. With the tip radius approximately 1,000 times smaller than that of a cell, an electrochemical probe can penetrate a cell and travel inside it without apparent damage to the membrane. The data demonstrate the possibility of measuring the rate of transmembrane charge transport and membrane potential and probing redox properties at the subcellular level. The same experimental setup was used for nanoscale electrochemical imaging of the cell surface. PMID:18178616

  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. Reprogramming mammalian somatic cells.

    PubMed

    Rodriguez-Osorio, N; Urrego, R; Cibelli, J B; Eilertsen, K; Memili, E

    2012-12-01

    Somatic cell nuclear transfer (SCNT), the technique commonly known as cloning, permits transformation of a somatic cell into an undifferentiated zygote with the potential to develop into a newborn animal (i.e., a clone). In somatic cells, chromatin is programmed to repress most genes and express some, depending on the tissue. It is evident that the enucleated oocyte provides the environment in which embryonic genes in a somatic cell can be expressed. This process is controlled by a series of epigenetic modifications, generally referred to as "nuclear reprogramming," which are thought to involve the removal of reversible epigenetic changes acquired during cell differentiation. A similar process is thought to occur by overexpression of key transcription factors to generate induced pluripotent stem cells (iPSCs), bypassing the need for SCNT. Despite its obvious scientific and medical importance, and the great number of studies addressing the subject, the molecular basis of reprogramming in both reprogramming strategies is largely unknown. The present review focuses on the cellular and molecular events that occur during nuclear reprogramming in the context of SCNT and the various approaches currently being used to improve nuclear reprogramming. A better understanding of the reprogramming mechanism will have a direct impact on the efficiency of current SCNT procedures, as well as iPSC derivation. PMID:22979962

  19. Mechanisms of mammalian iron homeostasis

    PubMed Central

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

    2012-01-01

    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 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. PMID:22703180

  20. An overview of mammalian pluripotency.

    PubMed

    Wu, Jun; Yamauchi, Takayoshi; Izpisua Belmonte, Juan Carlos

    2016-05-15

    Mammalian pluripotency is the ability to give rise to all somatic cells as well as the germ cells of an adult mammal. It is a unique feature of embryonic epiblast cells, existing only transiently, as cells pass through early developmental stages. By contrast, pluripotency can be captured and stabilized indefinitely in cell culture and can also be reactivated in differentiated cells via nuclear reprogramming. Pluripotent stem cells (PSCs) are the in vitro carriers of pluripotency and they can inhabit discrete pluripotent states depending on the stage at which they were derived and their culture conditions. Here, and in the accompanying poster, we provide a summary of mammalian pluripotency both in vivo and in vitro, and highlight recent and future applications of PSCs for basic and translational research. PMID:27190034

  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. 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

  3. Changes in expression of CWC15 during preimplantation development in the bovine embryo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently, a nonsense mutation in CWC15 was identified as a likely causative mutation affecting fertility in Jersey cattle. Although the carrier frequency in the population is relatively high (23.4%), no homozygous animals have been identified, leading to the conclusion that this mutation is associat...

  4. CRISPR/Cas9 as Tool for Functional Study of Genes Involved in Preimplantation Embryo Development

    PubMed Central

    Kwon, Jeongwoo; Namgoong, Suk; Kim, Nam-Hyung

    2015-01-01

    The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knockin. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig. PMID:25775469

  5. Towards understanding the roles of position and geometry on cell fate decisions during preimplantation development

    PubMed Central

    Biggins, John S.; Royer, Christophe; Watanabe, Tomoko; Srinivas, Shankar

    2015-01-01

    The first lineage segregation event in mouse embryos produces two separate cell populations: inner cell mass and trophectoderm. This is understood to be brought about by cells sensing their position within the embryo and differentiating accordingly. The cellular and molecular underpinnings of this process remain under investigation and have variously been considered to be completely stochastic or alternately, subject to some predisposition set up at fertilisation or before. Here, we consider these views in light of recent publications, discuss the possible role of cell geometry and mechanical forces in this process and describe how modelling could contribute in addressing this issue. PMID:26349030

  6. Molecular identification of ancient and modern mammalian magnesium transporters.

    PubMed

    Quamme, Gary A

    2010-03-01

    A large number of mammalian Mg(2+) transporters have been hypothesized on the basis of physiological data, but few have been investigated at the molecular level. The recent identification of a number of novel proteins that mediate Mg(2+) transport has enhanced our understanding of how Mg(2+) is translocated across mammalian membranes. Some of these transporters have some similarity to those found in prokaryocytes and yeast cells. Human Mrs2, a mitochondrial Mg(2+) channel, shares many of the properties of the bacterial CorA and yeast Alr1 proteins. The SLC41 family of mammalian Mg(2+) transporters has a similarity with some regions of the bacterial MgtE transporters. The mammalian ancient conserved domain protein (ACDP) Mg(2+) transporters are found in prokaryotes, suggesting an ancient origin. However, other newly identified mammalian transporters, including TRPM6/7, MagT, NIPA, MMgT, and HIP14 families, are not represented in prokaryotic genomes, suggesting more recent development. MagT, NIPA, MMgT, and HIP14 transporters were identified by differential gene expression using microarray analysis. These proteins, which are found in many different tissues and subcellular organelles, demonstrate a diversity of structural properties and biophysical functions. The mammalian Mg(2+) transporters have no obvious amino acid similarities, indicating that there are many ways to transport Mg(2+) across membranes. Most of these proteins transport a number of divalent cations across membranes. Only MagT1 and NIPA2 are selective for Mg(2+). Many of the identified mammalian Mg(2+) transporters are associated with a number of congenital disorders encompassing a wide range of tissues, including intestine, kidney, brain, nervous system, and skin. It is anticipated that future research will identify other novel Mg(2+) transporters and reveal other diseases. PMID:19940067

  7. Rapid, modular and reliable construction of complex mammalian gene circuits

    PubMed Central

    Guye, Patrick; Li, Yinqing; Wroblewska, Liliana; Duportet, Xavier; Weiss, Ron

    2013-01-01

    We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays. PMID:23847100

  8. A new mammalian host cell with enhanced survival enables completely serum-free development of high-level protein production cell lines.

    PubMed

    Rossi, Diane L; Rossi, Edmund A; Goldenberg, David M; Chang, Chien-Hsing

    2011-01-01

    With over 25 monoclonal antibodies (mAbs) currently approved and many more in development, there is considerable interest in gaining improved productivity by increasing cell density and enhancing cell survival of production cell lines. In addition, high costs and growing safety concerns with use of animal products have made the availability of serum-free cell lines more appealing. We elected to transfect the myeloma cell line Sp2/0-Ag14 with Bcl2-EEE, the constitutively active phosphomimetic mutant of Bcl2, for extended cell survival. After adaptation of the initial transfectants to serum-independent growth, a clone with superior growth properties, referred to as SpESF, was isolated and further subjected to iterative rounds of stressful growth over a period of 4 months. The effort resulted in the selection of a promising clone, designated SpESFX-10, which was shown to exhibit robust growth and resist apoptosis induced by sodium butyrate or glutamine deprivation. The advantage of SpESFX-10 as a host for generating mAb-production cell lines was demonstrated by its increased transfection efficiency, culture longevity, and mAb productivity, as well as by the feasibility of accomplishing the entire cell line development process, including transfection, subcloning, and cryopreservation, in the complete absence of serum. PMID:21473000

  9. Involvement of Cl−/HCO3− exchanger SLC26A3 and SLC26A6 in preimplantation embryo cleavage

    PubMed Central

    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. Monoclonal antibody BM89 recognizes a novel cell surface glycoprotein of the L2/HNK-1 family in the developing mammalian nervous system.

    PubMed

    Merkouri, E; Matsas, R

    1992-09-01

    A monoclonal antibody, BM89, obtained with Triton X-114-treated pig synaptic membranes as an immunogen, recognizes a neuronal antigen in the newborn porcine nervous system. By immunohistochemistry, BM89 staining was observed within the neuropil of all areas of the forebrain and spinal cord tested. In addition, BM89 labeled the cell bodies and proximal dendrites of spinal cord neurons. In the peripheral nervous system, BM89 immunoreactivity was present in a subpopulation of dorsal root ganglion neurons and was predominantly associated with non-myelinated axons in peripheral nerves. Initial biochemical characterization of the antigen in pig brain showed that it is an integral membrane glycoprotein with a molecular weight of 41,000. Moreover, it cross-reacts with the L2/HNK-1 carbohydrate epitope expressed by members of a large family of glycoproteins. Homologous antigens with molecular weights of 41,000-43,000 were identified in the rat, rabbit and fetal human brain. Immunoblotting and immunohistochemistry revealed that the epitope recognized by BM89 is developmentally regulated in the rat nervous system. In cryostat sections from rat cerebellum, spinal cord and dorsal root ganglia, an age-dependent decline of BM89 immunoreactivity was observed during postnatal development. In the cerebellum, the BM89 epitope was very abundant in cells of the external and the internal granular layers between postnatal days 5 and 15. During this period some staining was also identified in the developing molecular layer and the prospective white matter. Subsequently, and in the adult, overall staining was greatly reduced and remaining immunoreactivity was associated only with the internal granular layer. In the spinal cord and dorsal root ganglia, staining was very prominent at postnatal day 5; it decreased considerably thereafter and was barely detectable in the adult. Immunostaining of rat brain and dorsal root ganglion cultures revealed that the BM89 antigen is a cell surface

  11. Effects of rumen-protected methionine and choline supplementation on the preimplantation embryo in Holstein cows.

    PubMed

    Acosta, D A V; Denicol, A C; Tribulo, P; Rivelli, M I; Skenandore, C; Zhou, Z; Luchini, D; Corrêa, M N; Hansen, P J; Cardoso, F C

    2016-06-01

    Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87

  12. Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

    PubMed Central

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

    2013-01-01

    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. PMID:23754951

  13. 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

  14. Biology of Preimplantation Conceptus at the Onset of Elongation in Dairy Cows.

    PubMed

    Ribeiro, Eduardo S; Greco, Leandro F; Bisinotto, Rafael S; Lima, Fábio S; Thatcher, William W; Santos, José E

    2016-04-01

    The objectives of this study were to characterize changes in transcriptome of preimplantation conceptuses at the onset of elongation and associated changes in uterine histotroph composition and endometrial physiology. Lactating dairy cows (n = 160) had their ovulation synchronized by artificial insemination (study Day 0). On Day 15, uteri were flushed and endometrium tissue collected. Recovered conceptuses were classified based on morphology/length as ovoid (1-4 mm), tubular (5-19 mm), and filamentous (20-60 mm). Total RNA (n = 48) was subjected to transcriptome analysis. The uterine fluid (n = 30) was evaluated by mass spectrophotometry. Transcriptome analyses revealed drastic changes in the transition from ovoid to tubular and from tubular to filamentous. Differentially expressed genes were associated with cellular movement, cell-to-cell signaling, cellular assembly and organization, lipid metabolism, small molecule biochemistry, and molecular transport. Specific changes included reorganization of cytoskeleton and cell migration, arginine metabolism, growth factors signaling, and lipid metabolism. Functional analysis revealed fatty acids and peroxisome proliferator activated receptor gamma as upstream regulators of transcriptome changes. Expression of PPARG increased 17-fold during the onset of elongation and was highly correlated with genes involved in lipid metabolism. The histotroph was rich in amino acids, lipids, saccharides, and other intermediate metabolites, and important changes in composition occurred in the presence of a conceptus. Pregnancy had a major impact on the concentrations of important lipids in the uterine fluid and expression of genes in the endometrium. Collectively, conceptus elongation involves remarkable changes in transcriptome, composition of the histotroph, and endometrial physiology, which help elucidate important events in uterine and conceptus biology at the onset of elongation. PMID:26935601

  15. Preimplantation genetic diagnosis for mitochondrial DNA disorders: ethical guidance for clinical practice

    PubMed Central

    Bredenoord, Annelien; Dondorp, Wybo; Pennings, Guido; de Die-Smulders, Christine; Smeets, Bert; de Wert, Guido

    2009-01-01

    Although morally acceptable in theory, preimplantation genetic diagnosis (PGD) for mitochondrial DNA (mtDNA) disorders raises several ethical questions in clinical practice. This paper discusses the major conditions for good clinical practice. Our starting point is that PGD for mtDNA mutations should as far as possible be embedded in a scientific research protocol. For every clinical application of PGD for mtDNA disorders, it is not only important to avoid a ‘high risk of serious harm' to the future child, but also to consider to what extent it would be possible, desirable and proportional to try to reduce the health risks and minimize harm. The first issue we discuss is oocyte sampling, which may point out whether PGD is feasible for a specific couple. The second issue is whether one blastomere represents the genetic composition of the embryo as a whole – and how this could (or should) be investigated. The third issue regards the cutoff points below which embryos are considered to be eligible for transfer. We scrutinize how to determine these cutoff points and how to use these cutoff points in clinical practice – for example, when parents ask to take more or less risks. The fourth issue regards the number of cycles that can (or should) justifiably be carried out to find the best possible embryo. Fifth, we discuss whether follow-up studies should be conducted, particularly the genetic testing of children born after IVF/PGD. Finally, we offer the main information that is required to obtain a truly informed consent. PMID:19471315

  16. Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos

    PubMed Central

    Hirate, Yoshikazu; Hirahara, Shino; Inoue, Ken-ichi; Suzuki, Atsushi; Alarcon, Vernadeth B.; Akimoto, Kazunori; Hirai, Takaaki; Hara, Takeshi; Adachi, Makoto; Chida, Kazuhiro; Ohno, Shigeo; Marikawa, Yusuke; Nakao, Kazuki; Shimono, Akihiko; Sasaki, Hiroshi

    2013-01-01

    Summary Background In preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown. Results We showed that a combination of cell polarity and cell–cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and Amotl2 are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs) and cell–cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, Ser176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot–Lats interaction to activate the Hippo pathway. Conclusion We propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell–cell adhesion by sequestering Amot from AJ. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates. PMID:23791731

  17. Ballistic transfection of mammalian cells in vivo

    SciTech Connect

    Kolesnikov, V.A.; Zelenin, A.V.; Zelenina, I.A.

    1995-11-01

    The method of ballistic transfection initially proposed for genetic transformation of plants was used for animal cells in vitro and in situ. The method consists in bombarding the transfected cells with microparticles of heavy metals carrying foreign DNA. Penetrating the cell nucleus, the microparticles transport the introduced gene. Successful genetic transformation of the cultured mouse cells and fish embryos was realized, and this allowed the study of mammalian cells in situ. The performed studies allowed us to demonstrate expression of the reporter genes of chloramphenicol acetyltransferase, galactosidase, and neomycin phosphotransferase in the mouse liver, mammary gland and kidney explants, in the liver and cross-striated muscle of mouse and rat in situ, and in developing mouse embryos at the stages of two-cell embryo, morula, and blastocyst. All these genes were introduced by ballistic transfection. In the liver and cross-striated muscle the transgene activity was detected within two to three months after transfection. Thus, the ballistic introduction of the foreign genes in the cells in situ was demonstrated, and this opens possibilities for the use of this method in gene therapy. Methodical aspects of the bombarding and transfection are considered in detail, and the published data on transfection and genetic transformation of mammalian cells are discussed. 41 refs., 13 figs., 1 tab.

  18. 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

  19. Systems approaches for synthetic biology: a pathway toward mammalian design

    PubMed Central

    Rekhi, Rahul; Qutub, Amina A.

    2013-01-01

    We review methods of understanding cellular interactions through computation in order to guide the synthetic design of mammalian cells for translational applications, such as regenerative medicine and cancer therapies. In doing so, we argue that the challenges of engineering mammalian cells provide a prime opportunity to leverage advances in computational systems biology. We support this claim systematically, by addressing each of the principal challenges to existing synthetic bioengineering approaches—stochasticity, complexity, and scale—with specific methods and paradigms in systems biology. Moreover, we characterize a key set of diverse computational techniques, including agent-based modeling, Bayesian network analysis, graph theory, and Gillespie simulations, with specific utility toward synthetic biology. Lastly, we examine the mammalian applications of synthetic biology for medicine and health, and how computational systems biology can aid in the continued development of these applications. PMID:24130532

  20. Systems approaches for synthetic biology: a pathway toward mammalian design.

    PubMed

    Rekhi, Rahul; Qutub, Amina A

    2013-01-01

    We review methods of understanding cellular interactions through computation in order to guide the synthetic design of mammalian cells for translational applications, such as regenerative medicine and cancer therapies. In doing so, we argue that the challenges of engineering mammalian cells provide a prime opportunity to leverage advances in computational systems biology. We support this claim systematically, by addressing each of the principal challenges to existing synthetic bioengineering approaches-stochasticity, complexity, and scale-with specific methods and paradigms in systems biology. Moreover, we characterize a key set of diverse computational techniques, including agent-based modeling, Bayesian network analysis, graph theory, and Gillespie simulations, with specific utility toward synthetic biology. Lastly, we examine the mammalian applications of synthetic biology for medicine and health, and how computational systems biology can aid in the continued development of these applications. PMID:24130532

  1. Metabolism throughout follicle and oocyte development in mammals.

    PubMed

    Collado-Fernandez, Esther; Picton, Helen M; Dumollard, Rémi

    2012-01-01

    Metabolic studies of mammalian embryos started with the development of in vitro culture systems more than 40 years ago. More recently, metabolic studies have begun to shed light on the requirements of growing oocytes/follicles from the earliest stages of folliculogenesis. While growing oocytes preferentially metabolise pyruvate over glucose, the somatic compartment of ovarian follicles is more glycolytic. The metabolic preferences of the oocyte are reflected in the early zygote, which becomes increasingly dependent on glycolytic energy production as development progresses to the blastocyst stage. Furthermore, the intricate metabolic relationship between each oocyte and its somatic surroundings is critical for oocyte growth and developmental competence. Measurements of amino acid turnover in bovine oocytes indicate that glutamine, arginine and leucine are consistently depleted, while alanine is produced, showing similarities with amino acid turnover in preimplantation embryos. Amino acid profiling is a good predictor of embryo quality and might also turn out to be a predictor of oocyte developmental competence. Finally, recent studies have uncovered lipid metabolism in oocytes and early embryos, suggesting that endogenous fatty acids might be used for energy production. Together, metabolic studies have revealed the multiplicity of energetic substrates used by oocytes and early embryos, and suggest that the versatility of the metabolic pathways available for energy production is key for high developmental potential. Metabolic studies of early embryos are now being applied to follicle culture, and the goal of describing the metabolome of the growing oocyte in its follicle is now very attainable. PMID:23417402

  2. Mammalian EGF receptor activation by the rhomboid protease RHBDL2.

    PubMed

    Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

    2011-05-01

    The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines. PMID:21494248

  3. Mammalian EGF receptor activation by the rhomboid protease RHBDL2

    PubMed Central

    Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

    2011-01-01

    The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines. PMID:21494248

  4. Role of ortho-retronasal olfaction in mammalian cortical evolution.

    PubMed

    Rowe, Timothy B; Shepherd, Gordon M

    2016-02-15

    Fossils of mammals and their extinct relatives among cynodonts give evidence of correlated transformations affecting olfaction as well as mastication, head movement, and ventilation, and suggest evolutionary coupling of these seemingly separate anatomical regions into a larger integrated system of ortho-retronasal olfaction. Evidence from paleontology and physiology suggests that ortho-retronasal olfaction played a critical role at three stages of mammalian cortical evolution: early mammalian brain development was driven in part by ortho-retronasal olfaction; the bauplan for neocortex had higher-level association functions derived from olfactory cortex; and human cortical evolution was enhanced by ortho-retronasal smell. PMID:25975561

  5. Fate and Metabolism of PBDEs in Mammalian Systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polybrominated diphenyl ethers (PBDEs) belong to an emerging class of persistent organic pollutants (POPs). Although the toxicology of PBDEs is not well developed, they are persistent and bioaccumulative, and therefore, of growing environmental concern. The metabolism of PBDEs in mammalian systems h...

  6. Mast cells in mammalian brain.

    PubMed

    Dropp, J J

    1976-01-01

    Mast cells, which had until recently been believed to be not present in the mammalian brain, were studied in the brains of 29 mammalian species. Although there was considerable intraspecific and interspecific variation, mast cells were most numerous within the leptomeninges (especially in those overlying the cerebrum and the dorsal thalamus - most rodents, most carnivores, chimpanzees, squirrel monkeys and elephant), the cerebral cortex (most rodents, tiger, fox, chimpanzee, tarsier, and elephant) and in many nuclei of the dorsal thalamus (most rodents, tiger, lion, and fox). In some mammals, mast cells were also numerous in the stroma of the telencephalic choroid plexuses (chimpanzee, squirrel monkey), the putamen and the claustrum (chimpanzee), the subfornical organ (pack rat, tiger, chimpanzee), the olfactory peduncles (hooded rat, albino rat), the stroma of the diencephalic choroid plexus (lion, chimpanzee, squirrel monkey), the pineal organ (chimpanzee, squirrel monkey), some nuclei of the hypothalamus (tiger), the infundibulum (hooded rat, tiger, fox) the area postrema (pack rat, chinchilla, lion, spider monkey, chimpanzee, fox) and some nuclei and tracts of the metencephalon and the myelencephalon (tiger). Neither the sex of the animal nor electrolytic lesions made in the brains of some of the animals at various times prior to sacrifice appeared to effect the number and the distribution of mast cells. Age-related changes in mast cell number and distribution were detected in the albino rat. PMID:961335

  7. Preimplantation genetic diagnosis for couples at high risk of Down syndrome pregnancy owing to parental translocation or mosaicism

    PubMed Central

    Conn, C.; Cozzi, J.; Harper, J.; Winston, R.; Delhanty, J.

    1999-01-01

    The population risk for trisomy 21 is 1 in 700 births but some couples are at a much higher risk owing to parental translocation or mosaicism. We report on the first attempt to carry out preimplantation genetic diagnosis for two such couples using cleavage stage embryo biopsy and dual colour FISH analysis. Each couple underwent two treatment cycles. Couple 1 (suspected gonadal mosaicism for trisomy 21) had two embryos normal for chromosome 21 transferred, but no pregnancy resulted; 64% (7/11) unfertilised oocytes/embryos showed chromosome 21 aneuploidy. Couple 2 (46,XX,t(6;21)(q13;q22.3)) had a single embryo transferred resulting in a biochemical pregnancy; 91% (10/11) oocytes/embryos showed chromosome 21 imbalance, most resulting from 3:1 segregation of this translocation at gametogenesis. The opportunity to test embryos before implantation enables the outcome of female meiosis to be studied for the first time and the recurrence risk for a Down syndrome pregnancy to be assessed.


Keywords: preimplantation genetic diagnosis; Down syndrome; reciprocal translocation; gonadal mosaicism PMID:9950365

  8. Effect of pre-implanted helium on void swelling evolution in self-ion irradiated HT9

    NASA Astrophysics Data System (ADS)

    Getto, E.; Jiao, Z.; Monterrosa, A. M.; Sun, K.; Was, G. S.

    2015-07-01

    Void evolution in Fe++-irradiated ferritic-martensitic alloy HT9 was characterized in the temperature range of 400-480 °C between doses of 25 and 375 displacements per atom (dpa) with pre-implanted helium levels of 0-100 appm. A systematic study using depth profiling in cross-section samples was conducted to determine a valid region of analysis between 300 and 700 nm from the surface, which excluded effects due to the injected interstitial and the surface. Pre-implanted helium was found to promote void swelling at low doses by shortening the nucleation regime and to retard void growth at doses in the transient regime by enhancement of nucleation of small voids. Swelling was found to peak at a temperature of 460 °C. The primary effect of temperature was on the nucleation regime; nucleation regime was the shortest at 460 °C compared to that at 440 and 480 °C. The growth rate of voids was temperature-invariant. Steady state swelling was reached at 460 °C between 188 and 375 dpa at a rate of 0.02%/dpa.

  9. Hypothesis on the dual origin of the Mammalian subplate.

    PubMed

    Montiel, Juan F; Wang, Wei Zhi; Oeschger, Franziska M; Hoerder-Suabedissen, Anna; Tung, Wan Ling; García-Moreno, Fernando; Holm, Ida Elizabeth; Villalón, Aldo; Molnár, Zoltán

    2011-01-01

    The development of the mammalian neocortex relies heavily on subplate. The proportion of this cell population varies considerably in different mammalian species. Subplate is almost undetectable in marsupials, forms a thin, but distinct layer in mouse and rat, a larger layer in carnivores and big-brained mammals as pig, and a highly developed embryonic structure in human and non-human primates. The evolutionary origin of subplate neurons is the subject of current debate. Some hypothesize that subplate represents the ancestral cortex of sauropsids, while others consider it to be an increasingly complex phylogenetic novelty of the mammalian neocortex. Here we review recent work on expression of several genes that were originally identified in rodent as highly and differentially expressed in subplate. We relate these observations to cellular morphology, birthdating, and hodology in the dorsal cortex/dorsal pallium of several amniote species. Based on this reviewed evidence we argue for a third hypothesis according to which subplate contains both ancestral and newly derived cell populations. We propose that the mammalian subplate originally derived from a phylogenetically ancient structure in the dorsal pallium of stem amniotes, but subsequently expanded with additional cell populations in the synapsid lineage to support an increasingly complex cortical plate development. Further understanding of the detailed molecular taxonomy, somatodendritic morphology, and connectivity of subplate in a comparative context should contribute to the identification of the ancestral and newly evolved populations of subplate neurons. PMID:21519390

  10. A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

    PubMed Central

    Zhou, K; Takegawa, K; Emr, S D; Firtel, R A

    1995-01-01

    Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5

  11. Crystal structure of mammalian acid sphingomyelinase.

    PubMed

    Gorelik, Alexei; Illes, Katalin; Heinz, Leonhard X; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann-Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann-Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase. PMID:27435900

  12. Crystal structure of mammalian acid sphingomyelinase

    PubMed Central

    Gorelik, Alexei; Illes, Katalin; Heinz, Leonhard X.; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann–Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann–Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase. PMID:27435900

  13. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  14. Report of the NASA Mammalian Developmental Biology Working Group

    NASA Technical Reports Server (NTRS)

    Keefe, J. R.

    1985-01-01

    Development is considered to encompass all aspects of the mammalian life span from initial initial germ cell production through the complete life cycle to death of the organism. Thus, gamete production, fertilization, embryogenesis, implantation, fetogenesis, birth, peri- and postnatal maturation, and aging were all considered as stages of a development continuum relevant to problems of Space Biology. Deliberations thus far have been limited to stages of the development cycle from fertilization to early postnatal life. The deliberations are detailed.

  15. How difficult is inference of mammalian causal gene regulatory networks?

    PubMed

    Djordjevic, Djordje; Yang, Andrian; Zadoorian, Armella; Rungrugeecharoen, Kevin; Ho, Joshua W K

    2014-01-01

    Gene regulatory networks (GRNs) play a central role in systems biology, especially in the study of mammalian organ development. One key question remains largely unanswered: Is it possible to infer mammalian causal GRNs using observable gene co-expression patterns alone? We assembled two mouse GRN datasets (embryonic tooth and heart) and matching microarray gene expression profiles to systematically investigate the difficulties of mammalian causal GRN inference. The GRNs were assembled based on > 2,000 pieces of experimental genetic perturbation evidence from manually reading > 150 primary research articles. Each piece of perturbation evidence records the qualitative change of the expression of one gene following knock-down or over-expression of another gene. Our data have thorough annotation of tissue types and embryonic stages, as well as the type of regulation (activation, inhibition and no effect), which uniquely allows us to estimate both sensitivity and specificity of the inference of tissue specific causal GRN edges. Using these unprecedented datasets, we found that gene co-expression does not reliably distinguish true positive from false positive interactions, making inference of GRN in mammalian development very difficult. Nonetheless, if we have expression profiling data from genetic or molecular perturbation experiments, such as gene knock-out or signalling stimulation, it is possible to use the set of differentially expressed genes to recover causal regulatory relationships with good sensitivity and specificity. Our result supports the importance of using perturbation experimental data in causal network reconstruction. Furthermore, we showed that causal gene regulatory relationship can be highly cell type or developmental stage specific, suggesting the importance of employing expression profiles from homogeneous cell populations. This study provides essential datasets and empirical evidence to guide the development of new GRN inference methods for

  16. Body Size in Mammalian Paleobiology

    NASA Astrophysics Data System (ADS)

    Damuth, John; MacFadden, Bruce J.

    1990-11-01

    This valuable collection of essays presents and evaluates techniques of body-mass estimation and reviews current and potential applications of body-size estimates in paleobiology. Papers discuss explicitly the errors and biases of various regression techniques and predictor variables, and the identification of functionally similar groups of species for improving the accuracy of estimates. At the same time other chapters review and discuss the physiological, ecological, and behavioral correlates of body size in extant mammals; the significance of body-mass distributions in mammalian faunas; and the ecology and evolution of body size in particular paleofaunas. Coverage is particularly detailed for carnivores, primates, and ungulates, but information is also presented on marsupials, rodents, and proboscideans.

  17. Determinants of Mammalian Nucleolar Architecture

    PubMed Central

    Farley, Katherine I.; Surovtseva, Yulia; Merkel, Janie; Baserga, Susan J.

    2015-01-01

    The nucleolus is responsible for the production of ribosomes, essential machines which synthesize all proteins needed by the cell. The structure of human nucleoli is highly dynamic and is directly related to its functions in ribosome biogenesis. Despite the importance of this organelle, the intricate relationship between nucleolar structure and function remains largely unexplored. How do cells control nucleolar formation and function? What are the minimal requirements for making a functional nucleolus? Here we review what is currently known regarding mammalian nucleolar formation at nucleolar organizer regions (NORs), which can be studied by observing the dissolution and reformation of the nucleolus during each cell division. Additionally, the nucleolus can be examined by analyzing how alterations in nucleolar function manifest in differences in nucleolar architecture. Furthermore, changes in nucleolar structure and function are correlated with cancer, highlighting the importance of studying the determinants of nucleolar formation. PMID:25670395

  18. Signal transduction in mammalian oocytes during fertilization.

    PubMed

    Machaty, Zoltan

    2016-01-01

    Mammalian embryo development begins when the fertilizing sperm triggers a series of elevations in the oocyte's intracellular free Ca(2+) concentration. The elevations are the result of repeated release and re-uptake of Ca(2+) stored in the smooth endoplasmic reticulum. Ca(2+) release is primarily mediated by the phosphoinositide signaling system of the oocyte. The system is stimulated when the sperm causes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); IP3 then binds its receptor on the surface of the endoplasmic reticulum that induces Ca(2+) release. The manner in which the sperm generates IP3, the Ca(2+) mobilizing second messenger, has been the subject of extensive research for a long time. The sperm factor hypothesis has eventually gained general acceptance, according to which it is a molecule from the sperm that diffuses into the ooplasm and stimulates the phosphoinositide cascade. Much evidence now indicates that the sperm-derived factor is phospholipase C-zeta (PLCζ) that cleaves PIP2 and generates IP3, eventually leading to oocyte activation. A recent addition to the candidate sperm factor list is the post-acrosomal sheath WW domain-binding protein (PAWP), whose role at fertilization is currently under debate. Ca(2+) influx across the plasma membrane is also important as, in the absence of extracellular Ca(2+), the oscillations run down prematurely. In pig oocytes, the influx that sustains the oscillations seems to be regulated by the filling status of the stores, whereas in the mouse other mechanisms might be involved. This work summarizes the current understanding of Ca(2+) signaling in mammalian oocytes. PMID:26453398

  19. Engineered Trehalose Permeable to Mammalian Cells

    PubMed Central

    Abazari, Alireza; Meimetis, Labros G.; Budin, Ghyslain; Bale, Shyam Sundhar; Weissleder, Ralph; Toner, Mehmet

    2015-01-01

    Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies. PMID:26115179

  20. Mechanoaccumulative Elements of the Mammalian Actin Cytoskeleton.

    PubMed

    Schiffhauer, Eric S; Luo, Tianzhi; Mohan, Krithika; Srivastava, Vasudha; Qian, Xuyu; Griffis, Eric R; Iglesias, Pablo A; Robinson, Douglas N

    2016-06-01

    To change shape, divide, form junctions, and migrate, cells reorganize their cytoskeletons in response to changing mechanical environments [1-4]. Actin cytoskeletal elements, including myosin II motors and actin crosslinkers, structurally remodel and activate signaling pathways in response to imposed stresses [5-9]. Recent studies demonstrate the importance of force-dependent structural rearrangement of α-catenin in adherens junctions [10] and vinculin's molecular clutch mechanism in focal adhesions [11]. However, the complete landscape of cytoskeletal mechanoresponsive proteins and the mechanisms by which these elements sense and respond to force remain to be elucidated. To find mechanosensitive elements in mammalian cells, we examined protein relocalization in response to controlled external stresses applied to individual cells. Here, we show that non-muscle myosin II, α-actinin, and filamin accumulate to mechanically stressed regions in cells from diverse lineages. Using reaction-diffusion models for force-sensitive binding, we successfully predicted which mammalian α-actinin and filamin paralogs would be mechanoaccumulative. Furthermore, a "Goldilocks zone" must exist for each protein where the actin-binding affinity must be optimal for accumulation. In addition, we leveraged genetic mutants to gain a molecular understanding of the mechanisms of α-actinin and filamin catch-bonding behavior. Two distinct modes of mechanoaccumulation can be observed: a fast, diffusion-based accumulation and a slower, myosin II-dependent cortical flow phase that acts on proteins with specific binding lifetimes. Finally, we uncovered cell-type- and cell-cycle-stage-specific control of the mechanosensation of myosin IIB, but not myosin IIA or IIC. Overall, these mechanoaccumulative mechanisms drive the cell's response to physical perturbation during proper tissue development and disease. PMID:27185555

  1. Tick-induced allergies: mammalian meat allergy, tick anaphylaxis and their significance

    PubMed Central

    2015-01-01

    Serious tick-induced allergies comprise mammalian meat allergy following tick bites and tick anaphylaxis. Mammalian meat allergy is an emergent allergy, increasingly prevalent in tick-endemic areas of Australia and the United States, occurring worldwide where ticks are endemic. Sensitisation to galactose-α-1,3-galactose (α-Gal) has been shown to be the mechanism of allergic reaction in mammalian meat allergy following tick bite. Whilst other carbohydrate allergens have been identified, this allergen is unique amongst carbohydrate food allergens in provoking anaphylaxis. Treatment of mammalian meat anaphylaxis involves avoidance of mammalian meat and mammalian derived products in those who also react to gelatine and mammalian milks. Before initiating treatment with certain therapeutic agents (e.g., cetuximab, gelatine-containing substances), a careful assessment of the risk of anaphylaxis, including serological analysis for α-Gal specific-IgE, should be undertaken in any individual who works, lives, volunteers or recreates in a tick endemic area. Prevention of tick bites may ameliorate mammalian meat allergy. Tick anaphylaxis is rare in countries other than Australia. Tick anaphylaxis is secondarily preventable by prevention and appropriate management of tick bites. Analysis of tick removal techniques in tick anaphylaxis sufferers offers insights into primary prevention of both tick and mammalian meat anaphylaxis. Recognition of the association between mammalian meat allergy and tick bites has established a novel cause and effect relationship between an environmental exposure and subsequent development of a food allergy, directing us towards examining environmental exposures as provoking factors pivotal to the development of other food allergies and refocusing our attention upon causation of allergy in general. PMID:25653915

  2. Mammalian reproduction: an ecological perspective.

    PubMed

    Bronson, F H

    1985-02-01

    The objectives of this paper are to organize our concepts about the environmental regulation of reproduction in mammals and to delineate important gaps in our knowledge of this subject. The environmental factors of major importance for mammalian reproduction are food availability, ambient temperature, rainfall, the day/night cycle and a variety of social cues. The synthesis offered here uses as its core the bioenergetic control of reproduction. Thus, for example, annual patterns of breeding are viewed as reflecting primarily the caloric costs of the female's reproductive effort as they relate to the energetic costs and gains associated with her foraging effort. Body size of the female is an important consideration since it is correlated with both potential fat reserves and life span. Variation in nutrient availability may or may not be an important consideration. The evolutionary forces that have shaped the breeding success of males usually are fundamentally different from those acting on females and, by implication, the environmental controls governing reproduction probably also often differ either qualitatively or quantitatively in the two sexes. Mammals often live in habitats where energetic and nutrient challenges vary seasonally, even in the tropics. When seasonal breeding is required, a mammal may use a predictor such as photoperiod or a secondary plant compound to prepare metabolically for reproduction. A reasonable argument can be made, however, that opportunistic breeding, unenforced by a predictor, may be the most prevalent strategy extant among today's mammals. Social cues can have potent modulating actions. They can act either via discrete neural and endocrine pathways to alter specific processes such as ovulation, or they can induce nonspecific emotional states that secondarily affect reproduction. Many major gaps remain in our knowledge about the environmental regulation of mammalian reproduction. For one, we have a paucity of information about the

  3. Supraadditive formation of micronuclei in preimplantation mouse embryos in vitro after combined treatment with X-rays and caffeine

    SciTech Connect

    Mueller, W.U.S.; Streffer, C.; Wurm, R.

    1985-01-01

    The influence of caffeine (0.1 or 2 mM), X-rays (0.24 Gy or 0.94 Gy, or of a combination of both on the formation of micronuclei in early stages of preimplantation mouse embryos in vitro was studied. X-rays as well as caffeine induced micronuclei. The dose-effect curve after irradiation was linear for the dose range measured. Caffeine did not induce micronuclei if the concentration was 1 mM or less; between 1 mM and 7 mM, however, there was a linear increase in the number of micronuclei. A considerable enhancement of the number of radiation-induced micronuclei was observed when irradiation of the embryos was followed by a treatment with caffeine. Not only was the sum of the single effects exceeded by the combination effects, but the combination results even lay in the range of supraadditivity of the envelope of additivity.

  4. An overview of the available methods for morphological scoring of pre-implantation embryos in in vitro fertilization.

    PubMed

    Nasiri, Nahid; Eftekhari-Yazdi, Poopak

    2015-01-01

    Assessment of embryo quality in order to choose the embryos that most likely result in pregnancy is the critical goal in assisted reproductive technologies (ART). The current trend in human in vitro fertilization/embryo transfer (IVF/ET) protocols is to decrease the rate of multiple pregnancies after multiple embryo transfer with maintaining the pregnancy rate at admissible levels (according to laboratory standards). Assessment of morphological feathers as a reliable non-invasive method that provides valuable information in prediction of IVF/intra cytoplasmic sperm injection (ICSI) outcome has been frequently proposed in recent years. This article describes the current status of morphological embryo evaluation at different pre-implantation stages. PMID:25685730

  5. An Overview of The Available Methods for Morphological Scoring of Pre-Implantation Embryos in In Vitro Fertilization

    PubMed Central

    Nasiri, Nahid; Eftekhari-Yazdi, Poopak

    2015-01-01

    Assessment of embryo quality in order to choose the embryos that most likely result in pregnancy is the critical goal in assisted reproductive technologies (ART). The current trend in human in vitro fertilization/embryo transfer (IVF/ET) protocols is to decrease the rate of multiple pregnancies after multiple embryo transfer with maintaining the pregnancy rate at admissible levels (according to laboratory standards). Assessment of morphological feathers as a reliable non-invasive method that provides valuable information in prediction of IVF/intra cytoplasmic sperm injection (ICSI) outcome has been frequently proposed in recent years. This article describes the current status of morphological embryo evaluation at different pre-implantation stages. PMID:25685730

  6. Mammalian Carboxylesterase 5: Comparative Biochemistry and Genomics

    PubMed Central

    Holmes, Roger S; Cox, Laura A; VandeBerg, John L

    2008-01-01

    Carboxylesterase 5 (CES5) (also called cauxin or CES7) is one of at least five mammalian CES gene families encoding enzymes of broad substrate specificity and catalysing hydrolytic and transesterification reactions. In silico methods were used to predict the amino acid sequences, secondary structures and gene locations for CES5 genes and gene products. Amino acid sequence alignments of mammalian CES5 enzymes enabled identification of key CES sequences previously reported for human CES1, as well as other sequences that are specific to the CES5 gene family, which were consistent with being monomeric in subunit structure and available for secretion into body fluids. Predicted secondary structures for mammalian CES5 demonstrated significant conservation with human CES1 as well as distinctive mammalian CES5 like structures. Mammalian CES5 genes are located in tandem with the CES1 gene(s), are transcribed on the reverse strand and contained 13 exons. CES5 has been previously reported in high concentrations in the urine (cauxin) of adult male cats, and within a protein complex of mammalian male epididymal fluids. Roles for CES5 may include regulating urinary levels of male cat pheromones; catalysing lipid transfer reactions within mammalian male reproductive fluids; and protecting neural tissue from drugs and xenobiotics. PMID:19727319

  7. In vivo and in vitro environmental effects on mammalian oocyte quality.

    PubMed

    Krisher, Rebecca L

    2013-01-01

    The oocyte is at the center of the equation that results in female fertility. Many factors influence oocyte quality, including external factors such as maternal nutrition, stress, and environmental exposures, as well as ovarian factors such as steroids, intercellular communication, antral follicle count, and follicular fluid composition. These influences are interconnected; changes in the external environment of the female translate into ovarian changes that affect the oocyte. The lengthy period during which the oocyte remains arrested in the ovary provides ample time and opportunity for environmental factors to take their toll. An appropriate environment for growth and maturation of the oocyte, in vivo and in vitro, is critical to ensure optimal oocyte quality, which determines the success of fertilization and preimplantation embryo development, and has long-term implications for implantation, fetal growth, and offspring health. PMID:25387025

  8. Assessing the accuracy of software predictions of mammalian and microbial metabolites

    EPA Science Inventory

    New chemical development and hazard assessments benefit from accurate predictions of mammalian and microbial metabolites. Fourteen biotransformation libraries encoded in eight software packages that predict metabolite structures were assessed for their sensitivity (proportion of ...

  9. Comparative Mammalian Cell Toxicity of N-DBPs and C-DBPs

    EPA Science Inventory

    In order to generate a quantitative, direct comparison amongst classes of drinking water disinfection by-products (DBPs), we developed and calibrated in vitro mammalian cell cytotoxicity and genotoxicity assays to integrate the analytical biology with the analytical chemistry of ...

  10. Lipidome signatures in early bovine embryo development.

    PubMed

    Sudano, Mateus J; Rascado, Tatiana D S; Tata, Alessandra; Belaz, Katia R A; Santos, Vanessa G; Valente, Roniele S; Mesquita, Fernando S; Ferreira, Christina R; Araújo, João P; Eberlin, Marcos N; Landim-Alvarenga, Fernanda D C

    2016-07-15

    Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development. PMID:27107972

  11. Mammalian Tribbles Homologs at the Crossroads of Endoplasmic Reticulum Stress and Mammalian Target of Rapamycin Pathways

    PubMed Central

    Cunard, Robyn

    2013-01-01

    In 2000, investigators discovered Tribbles, a Drosophila protein that coordinates morphogenesis by inhibiting mitosis. Further work has delineated Xenopus (Xtrb2), Nematode (Nipi-3), and mammalian homologs of Drosophila tribbles, which include TRB1, TRB2, and TRB3. The sequences of tribbles homologs are highly conserved, and despite their protein kinase structure, to date they have not been shown to have kinase activity. TRB family members play a role in the differentiation of macrophages, lymphocytes, muscle cells, adipocytes, and osteoblasts. TRB isoforms also coordinate a number of critical cellular processes including glucose and lipid metabolism, inflammation, cellular stress, survival, apoptosis, and tumorigenesis. TRB family members modulate multiple complex signaling networks including mitogen activated protein kinase cascades, protein kinase B/AKT signaling, mammalian target of rapamycin, and inflammatory pathways. The following review will discuss metazoan homologs of Drosophila tribbles, their structure, expression patterns, and functions. In particular, we will focus on TRB3 function in the kidney in podocytes. This review will also discuss the key signaling pathways with which tribbles proteins interact and provide a rationale for developing novel therapeutics that exploit these interactions to provide better treatment options for both acute and chronic kidney disease. PMID:24490110

  12. Possible mechanisms of mammalian immunocontraception.

    PubMed

    Barber, M R; Fayrer-Hosken, R A

    2000-03-01

    Ecological and conservation programs in ecosystems around the world have experienced varied success in population management. One of the greatest problems is that human expansion has led to the shrinking of wildlife habitat and, as a result, the overpopulation of many different species has occurred. The pressures exerted by the increased number of animals has caused environmental damage. The humane and practical control of these populations has solicited the scientific community to arrive at a safe, effective, and cost-efficient means of population control. Immunocontraception using zona pellucida antigens, specifically porcine zona pellucida (pZP), has become one of the most promising population control tools in the world today, with notable successes in horses and elephants. A conundrum has risen where pZP, a single vaccine, successfully induces an immunocontraceptive effect in multiple species of mammals. This review describes the most current data pertaining to the mammalian zona pellucida and immunocontraception, and from these studies, we suggest several potential mechanisms of immunocontraception. PMID:10706942

  13. Mammalian mitochondrial beta-oxidation.

    PubMed Central

    Eaton, S; Bartlett, K; Pourfarzam, M

    1996-01-01

    The enzymic stages of mammalian mitochondrial beta-oxidation were elucidated some 30-40 years ago. However, the discovery of a membrane-associated multifunctional enzyme of beta-oxidation, a membrane-associated acyl-CoA dehydrogenase and characterization of the carnitine palmitoyl transferase system at the protein and at the genetic level has demonstrated that the enzymes of the system itself are incompletely understood. Deficiencies of many of the enzymes have been recognized as important causes of disease. In addition, the study of these disorders has led to a greater understanding of the molecular mechanism of beta-oxidation and the import, processing and assembly of the beta-oxidation enzymes within the mitochondrion. The tissue-specific regulation, intramitochondrial control and supramolecular organization of the pathway is becoming better understood as sensitive analytical and molecular techniques are applied. This review aims to cover enzymological and organizational aspects of mitochondrial beta-oxidation together with the biochemical aspects of inherited disorders of beta-oxidation and the intrinsic control of beta-oxidation. PMID:8973539

  14. Ghrelin Receptors in Non-Mammalian Vertebrates

    PubMed Central

    Kaiya, Hiroyuki; Kangawa, Kenji; Miyazato, Mikiya

    2012-01-01

    The growth hormone secretagogue-receptor (GHS-R) was discovered in humans and pigs in 1996. The endogenous ligand, ghrelin, was discovered 3 years later, in 1999, and our understanding of the physiological significance of the ghrelin system in vertebrates has grown steadily since then. Although the ghrelin system in non-mammalian vertebrates is a subject of great interest, protein sequence data for the receptor in non-mammalian vertebrates has been limited until recently, and related biological information has not been well organized. In this review, we summarize current information related to the ghrelin receptor in non-mammalian vertebrates. PMID:23882259

  15. RNAi microarray analysis in cultured mammalian cells.

    PubMed

    Mousses, Spyro; Caplen, Natasha J; Cornelison, Robert; Weaver, Don; Basik, Mark; Hautaniemi, Sampsa; Elkahloun, Abdel G; Lotufo, Roberto A; Choudary, Ashish; Dougherty, Edward R; Suh, Ed; Kallioniemi, Olli

    2003-10-01

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time- and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function. PMID:14525932

  16. Enhancer Evolution across 20 Mammalian Species

    PubMed Central

    Villar, Diego; Berthelot, Camille; Aldridge, Sarah; Rayner, Tim F.; Lukk, Margus; Pignatelli, Miguel; Park, Thomas J.; Deaville, Robert; Erichsen, Jonathan T.; Jasinska, Anna J.; Turner, James M.A.; Bertelsen, Mads F.; Murchison, Elizabeth P.; Flicek, Paul; Odom, Duncan T.

    2015-01-01

    Summary The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution. PMID:25635462

  17. Mammalian synthetic biology: emerging medical applications

    PubMed Central

    Kis, Zoltán; Pereira, Hugo Sant'Ana; Homma, Takayuki; Pedrigi, Ryan M.; Krams, Rob

    2015-01-01

    In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes. PMID:25808341

  18. Mammalian Response to Cenozoic Climatic Change

    NASA Astrophysics Data System (ADS)

    Blois, Jessica L.; Hadly, Elizabeth A.

    2009-05-01

    Multiple episodes of rapid and gradual climatic changes influenced the evolution and ecology of mammalian species and communities throughout the Cenozoic. Climatic change influenced the abundance, genetic diversity, morphology, and geographic ranges of individual species. Within communities these responses interacted to catalyze immigration, speciation, and extinction. Combined they affected long-term patterns of community stability, functional turnover, biotic turnover, and diversity. Although the relative influence of climate on particular evolutionary processes is oft debated, an understanding of processes at the root of biotic change yields important insights into the complexity of mammalian response. Ultimately, all responses trace to events experienced by populations. However, many such processes emerge as patterns above the species level, where shared life history traits and evolutionary history allow us to generalize about mammalian response to climatic change. These generalizations provide the greatest power to understand and predict mammalian responses to current and future global change.

  19. Bats and Rodents Shape Mammalian Retroviral Phylogeny

    PubMed Central

    Cui, Jie; Tachedjian, Gilda; Wang, Lin-Fa

    2015-01-01

    Endogenous retroviruses (ERVs) represent past retroviral infections and accordingly can provide an ideal framework to infer virus-host interaction over their evolutionary history. In this study, we target high quality Pol sequences from 7,994 Class I and 8,119 Class II ERVs from 69 mammalian genomes and surprisingly find that retroviruses harbored by bats and rodents combined occupy the major phylogenetic diversity of both classes. By analyzing transmission patterns of 30 well-defined ERV clades, we corroborate the previously published observation that rodents are more competent as originators of mammalian retroviruses and reveal that bats are more capable of receiving retroviruses from non-bat mammalian origins. The powerful retroviral hosting ability of bats is further supported by a detailed analysis revealing that the novel bat gammaretrovirus, Rhinolophus ferrumequinum retrovirus, likely originated from tree shrews. Taken together, this study advances our understanding of host-shaped mammalian retroviral evolution in general. PMID:26548564