DOCSLIB.ORG

This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Expression and Characterisation of a Novel Poly(A)-Binding Protein, PABP5 Ross C. Anderson PhD The University of Edinburgh 2010 Abstract The poly(A)-binding proteins (PABPs) are a family of eukaryotic RNA-binding proteins with key roles in mRNA translation and stability. The molecular function of PABPs have been largely revealed through study of the prototypical cytoplasmic poly(A)-binding protein, PABP1. Thus, little is known regarding other PABP family members. PABP5 contains four RNA-recognition motifs characteristic of the cytoplasmic PABPs yet is structurally distinct as it lacks a portion of the C-terminus. This region contains a proline-rich section linked to a globular domain that facilitates a number of protein-protein interactions. To date, little information has been presented regarding the expression of PABP5 and there is no data pertaining to the function of this protein, despite being mapped to a region of the X-chromosome associated with human pathological conditions. In this thesis, I present the first data documenting the expression of PABP5 within mouse tissues, and find it to be expressed at the highest levels within the brain, ovary, and testis. The limited data available suggests that gonads may be the only tissue to contain all PABPs therefore I additionally describe the expression of PABP1 and PABP4 to ascertain their cellular distribution within these tissues. This revealed that PABPs have overlapping yet distinct expression patterns in mouse gonads. The distinct structure of PABP5 suggested that its function may vary from PABP1. Characterisation of its activities in translational regulation was therefore investigated. When tethered to a reporter mRNA PABP5 had limited translational stimulatory activity, and in addition could not be isolated via m7G cap chromatography and failed to interact with translation initiation factors including eIF4G and PAIP-1. These factors interact with PABP1 to positively promote translation, implying that PABP5 function in translational regulation differs from other PABPs investigated. ii Examining why PABP5 failed to display translational stimulatory activity also revealed an interaction with the negative regulator of translation, PAIP-2. In summary, I present the first description of PABP5 cellular localisation, and have gone some way towards elucidating the molecular function of this uncharacterised protein. iii Declaration I declare that the work presented in this PhD is my own and has not been submitted for any other degree. Ross C. Anderson iv Acknowledgements The last 4 years have been the source of much stress, pain and anxiety, filled with many periods where I wondered if I would ever finish, and yet seeing the following 300 pages come together as a coherent thesis is incredibly satisfying. There are so many people to thank I don’t know where to start – special thanks to Niki, who after 7 years of working together I would prefer to call a friend rather than a boss. I am forever indebted as I know this thesis caused you no small amount of trauma! Thank you to Matt for the lab supervision and opening my eyes to the world of curry (maybe one day I will be able to eat a chilli without fainting). Thanks to Hannah and Pedro for sharing the PhD pain, and to Barbara for keeping me in line! Richard – has been an excellent drinking buddy and a source of infinite wisdom in the lab. Finally, William (aka the Beef from Dalkeith), - it’s your round mate. Outside the lab, I wanted to say thanks to Niall and Euan for the distracting weekends of drinking which have been excellent fun. I’d like to say a huge thank you to Claire – you have been amazing and I‘ll never forget the support you have given me. This thesis would not have been finished and handed in without your help. Finally, I’d like to save the biggest thank you for Babs and Stu – I know I don’t pick up the phone or jump in the car often enough (unless of course I need money or food!) but you have supported me through thick and thin and I wish there was some way I could repay you. I love you both very much and I want to dedicate this thesis to you both. To my parents. Cheers! v Abbreviations µg.............................. microgram µl............................... microlitre µM............................ micromolar µm................. .......... micrometre 3’-UTR.......... ........... 3’ untranslated region 4E-BP............ ........... eukaryotic initiation factor 4E binding protein 5’-UTR.......... ........... 5’ untranslated region A................................ adenosine aa-tRNA................... aminoacyl-transfer ribonucleic acid ActD.............. .......... actinomycin D AD................. .......... activation domain Ago........................... argonaute protein ARE............... ........... adenosine/uracil-rich element ARE-BP.................... adenosine/uracil-rich element binding protein ARS.......................... autoregulatory sequence A-site........................ aminoacyl site ATP........................... adenosine triphosphate BD............................. binding domain C............................... cytosine cDNA........................ complementary DNA vi Co-IP........................ protein complex immunoprecipitation CPE.......................... cytoplasmic polyadenylation element CPEB........................ cytoplasmic polyadenylation element binding protein CrPV......................... cricket paralysis virus CT............................. threshold cycle C-terminus............... carboxyl terminus CTP........................... cytosine triphosphate DAB.......................... diaminobenzidinetrahydrochloride DAP5........................ death associated protein 5 DAPI........................ 4’, 6-diamidino-2-phenylindole DCP1........................ decapping protein 1 DCP2........................ decapping protein 2 Dcps.......................... scavenger decapping protein ddH2O...................... double distilled water dH2O........................ distilled water DMEM..................... Dulbecco’s minimal essential medium DNA......................... deoxyribonucleic acid DNase....................... deoxyribonucleic acid nuclease dsRNA...................... double stranded ribonucleic acid E. coli........................ Escherichia coli EDTA....................... ethylene diamine tetraacetic acid eEF........................... eukaryotic elongation factor vii EGFP........................ enhanced green fluorescent protein eIF............................. eukaryotic initiation factor EJC........................... exon junction complex EMCV...................... encephalomyocarditis virus EMSA....................... electrophoresis mobility shift assay ePABP...................... embryonic polyadenylate binding protein ePABP2.................... embryonic polyadenylate binding protein 2 ER............................ endoplasmic reticulum eRF........................... eukaryotic release factor E-site........................ exit site FCS........................... fetal calf serum FMDV...................... foot and mouth disease virus G............................... guanosine g................................ gram GAC......................... guanosine triphosphatase associated centre GAP.......................... guanosine triphosphatase activating protein GCN4....................... general control nonderepressible 4 GEF.......................... guanine nucleotide exchange factor GST.......................... glutathione S-transferase GTP.......................... guanosine triphosphate GTPase..................... guanosine triphosphatase H2O.......................... water viii HCV......................... hepatitis C virus HeLa cell................... Henrietta Lacks cell His............................ histidine hnRNP...................... heterogeneous nuclear ribonucleoprotein HRI........................... heme-regulated eukaryotic initiation factor 2 alpha kinase HRP.......................... horse radish peroxidase iPABP....................... inducible polyadenylate binding protein IPTG........................ isopropyl β-D-1-thiogalactopyranoside IRE........................... iron regulatory element IRES......................... internal ribosome entry site IRP........................... iron regulatory protein ISH........................... in-situ hybridisation ITAF........................ internal ribosome entry site trans-acting factor ITC........................... isothermal titration calorimetry Kb............................. kilobase Kd............................. dissociation constant kDa........................... kilodalton L................................ litre
Load more

Recommended publications
  • Stage of the Meiotic Prophase (Dictyate Stage) from Embryonic Life
    In vivo change in the germinal vesicle of the sow oocyte during the follicular phase before the ovulatory LH surge Marie-Christine DAGUET Université Pierre et Marie Curie (Paris VI) and 1. N. R. A. 78350 Jouy-en-Josas, France. Summary. This study describes a morphological change in the germinal vesicle (GV) of the sow oocyte during the follicular phase preceding the ovulatory LH surge. Estrus synchronization obtained with methallibure treatment provided a reference point for dating the onset of the follicular phase of each sow in the experiment. The oocyte GV’s of preovula- tory follicles having a diameter of less than 2 mm (during the first 2 to 3 days of the follicular phase) showed uniformly dispersed chromatin with no condensation, while the GV’s of oocytes from preovulatory follicles of more than 2 mm diameter (from days 2 and 3 of the follicular phase to day 5) presented condensed chromatin in the shape of a crown or horse- shoe surrounding the nucleolus, and irregular chromatin clusters in the nucleoplasm ; these clusters were usually found lying against the nuclear membrane. Introduction. The oocyte nucleus, called the germinal vesicle (GV), remains at the diplotene stage of the meiotic prophase (dictyate stage) from embryonic life to the time of the ovulatory LH surge, permitting resumption of meiosis at each cycle in oocytes within suitable follicles. The literature contains many references to GV breakdown and change resulting from the ovulatory LH surge, i. e. the first stages of the resumption of meiosis (rats : Odor, 1955 ; Mandl, 1963 ; mice : Edwards and Gates, 1959 ; Donahue, 1968 ; sows : Spalding et at., 1955 ; Hunter and Polge, 1966 ; McGaughey and Polge, 1971 ; Motlik and Fulka, 1976), but little has been published on the development of the GV of oocytes from preovulatory follicles before the ovulatory LH surge.
    [Show full text]
  • The Role of Cyclin B3 in Mammalian Meiosis
    THE ROLE OF CYCLIN B3 IN MAMMALIAN MEIOSIS by Mehmet Erman Karasu A Dissertation Presented to the Faculty of the Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY November, 2018 Scott Keeney, PhD Date Dissertation Mentor Copyright © Mehmet Erman Karasu 2018 DEDICATION I would like to dedicate this thesis to my parents, Mukaddes and Mustafa Karasu. I have been so lucky to have their support and unconditional love in this life. ii ABSTRACT Cyclins and cyclin dependent kinases (CDKs) lie at the center of the regulation of the cell cycle. Cyclins as regulatory partners of CDKs control the switch-like cell cycle transitions that orchestrate orderly duplication and segregation of genomes. Similar to somatic cell division, temporal regulation of cyclin-CDK activity is also important in meiosis, which is the specialized cell division that generates gametes for sexual production by halving the genome. Meiosis does so by carrying out one round of DNA replication followed by two successive divisions without another intervening phase of DNA replication. In budding yeast, cyclin-CDK activity has been shown to have a crucial role in meiotic events such as formation of meiotic double-strand breaks that initiate homologous recombination. Mammalian cells express numerous cyclins and CDKs, but how these proteins control meiosis remains poorly understood. Cyclin B3 was previously identified as germ cell specific, and its restricted expression pattern at the beginning of meiosis made it an interesting candidate to regulate meiotic events.
    [Show full text]
  • Aspects of Sexual Dimorphism in Mammalian Meiosis
    REPRODUCTIONREVIEW Focus on Meiosis Not all germ cells are created equal: Aspects of sexual dimorphism in mammalian meiosis Meisha A Morelli and Paula E Cohen Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA Correspondence should be addressed to P E Cohen; Email: [email protected] Abstract The study of mammalian meiosis is complicated by the timing of meiotic events in females and by the intermingling of meiotic sub-stages with somatic cells in the gonad of both sexes. In addition, studies of mouse mutants for different meiotic regulators have revealed significant differences in the stringency of meiotic events in males versus females. This sexual dimorphism implies that the processes of recombination and homologous chromosome pairing, while being controlled by similar genetic pathways, are subject to different levels of checkpoint control in males and females. This review is focused on the emerging picture of sexual dimorphism exhibited by mammalian germ cells using evidence from the broad range of meiotic mutants now available in the mouse. Many of these mouse mutants display distinct differences in meiotic progression and/or dysfunc- tion in males versus females, and their continued study will allow us to understand the molecular basis for the sex-specific differences observed during prophase I progression. Reproduction (2005) 130 761–781 Introduction most of the defining events that differentiate meiosis from mitosis. This includes homolog pairing as well as double Meiosis is a specialized cell division process that is essen- stranded break (DSB) formation and resolution leading to tial for the propagation of all sexually reproducing organ- crossover/recombination between homologus chromo- isms.
    [Show full text]
  • Direct Effects of Heat Stress During Meiotic Maturation on Bovine Oocyte and Cumulus RNA
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2009 Direct Effects of Heat Stress During Meiotic Maturation on Bovine Oocyte and Cumulus RNA Rebecca R. Payton University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Animal Sciences Commons Recommended Citation Payton, Rebecca R., "Direct Effects of Heat Stress During Meiotic Maturation on Bovine Oocyte and Cumulus RNA. " PhD diss., University of Tennessee, 2009. https://trace.tennessee.edu/utk_graddiss/628 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Rebecca R. Payton entitled "Direct Effects of Heat Stress During Meiotic Maturation on Bovine Oocyte and Cumulus RNA." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Animal Science. J. Lannett Edwards, Major Professor We have read this dissertation and recommend its acceptance: Cheryl Kojima, Arnold Saxton, F. Neal Schrick, Neal Stewart Accepted for the Council: Carolyn
    [Show full text]
  • TBP2 Is Essential for Germ Cell Development by Regulating Transcription and Chromatin Condensation in the Oocyte
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press TBP2 is essential for germ cell development by regulating transcription and chromatin condensation in the oocyte Emese Gazdag,1,4 Ange`le Santenard,1,2,4 Ce´line Ziegler-Birling,1,2 Gioia Altobelli,1,3 Olivier Poch,3 La`szlo` Tora,1,5 and Maria-Elena Torres-Padilla1,2,6 1Department of Functional Genomics, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), UMR 7104 CNRS, UdS, INSERM U964, BP 10142, F-67404 Illkirch Cedex, CU de Strasbourg, France; 2Department of Developmental and Cell Biology, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), UMR 7104 CNRS, UdS, INSERM U964, BP 10142, F-67404 Illkirch Cedex, CU de Strasbourg, France; 3Bioinformatics and Integrative Biology Laboratory, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), UMR 7104 CNRS, UdS, INSERM U964, BP 10142, F-67404 Illkirch Cedex, CU de Strasbourg, France Development of the germline requires consecutive differentiation events. Regulation of these has been associated with germ cell-specific and pluripotency-associated transcription factors, but the role of general transcription factors (GTFs) remains elusive. TATA-binding protein (TBP) is a GTF involved in transcription by all RNA polymerases. During ovarian folliculogenesis in mice the vertebrate-specific member of the TBP family, TBP2/ TRF3, is expressed exclusively in oocytes. To determine TBP2 function in vivo, we generated TBP2-deficient mice. We found that Tbp2À/À mice are viable with no apparent phenotype. However, females lacking TBP2 are sterile due to defective folliculogenesis, altered chromatin organization, and transcriptional misregulation of key oocyte-specific genes.
    [Show full text]
  • I N P R E S E N T I N G T H E D I S S E R T a T I O N a S a P a R T I a L F U L F I L L M E N T O F the Requirements for an Adva
    In presenting the dissertation as a partial fulfillment of the requirements for an advanced degree from the Georgia Institute of Technology, I agree that the Library of the Institute shall make it available for inspection and circulation in accordance with its regulations governing materials of this type. I agree that permission to copy from, or to publish from, this dissertation may be granted by the professor under whose direction it was written, or, in his absence, by the Dean of the Graduate Division when such copying or publication is solely for scholarly purposes and does not involve potential financial gain. It is under­ stood that any copying from, or publication of, this dis­ sertation which involves potential financial gain will not be allowed without written permission. s7 MEIOTIC STAGES IN THE FEMALE CHINESE HAMSTER (CRICETULUS GRISEUS MILNE-EDW.) A THESIS Presented to The Faculty of the Graduate Division by Brenda Maxine Waldon In Partial Fulfillment of the Requirements for the Degree ster of Science in The School of Applied Biology Georgia Institute of Technology March, 1969 MEIOTIC STAGES IN THE: FEMALE CHINESE HAMSTER (CRICETULUS GRISEUS MILNE-EDW.) APPROVED: CHAIRMAN DATE APPROVED BY CHAIRMAN: 3 -// ~69 ii ACKNOWLEDGMENTS My warm and sincere gratitude is expressed to Dr. R. H. Fetner for his guidance and assistance during my entire graduate program. I also extend my appreciation to Dr. A. B. Eschenbrenner and to Dr. H. S. Min for their assistance. I am indeed grateful to my friends Corinne Morgan and Ginny Zehr for their encouragement and assistance. This thesis is dedicated to Dr.
    [Show full text]
  • Two Distinct Pathways of Pregranulosa Cell Differentiation Support Follicle Formation in the Mouse Ovary
    Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary Wanbao Niua,b and Allan C. Spradlinga,b,1 aHoward Hughes Medical Institute Research Laboratories, Carnegie Institution for Science, Baltimore, MD 21218; and bDepartment of Embryology, Carnegie Institution for Science, Baltimore, MD 21218 Contributed by Allan C. Spradling, July 2, 2020 (sent for review March 25, 2020; reviewed by Brigid L. M. Hogan and Melissa Pepling) We sequenced more than 52,500 single cells from embryonic day (EPG) cell population begins production at least by E14.5, also 11.5 (E11.5) postembryonic day 5 (P5) gonads and performed from progenitors in the ovarian surface epithelium (22, 23). These lineage tracing to analyze primordial follicles and wave 1 medullar cells express Lgr5 and eventually differentiate as granulosa cells follicles during mouse fetal and perinatal oogenesis. Germ cells on second wave follicles (8, 21–23). The cellular origins, division clustered into six meiotic substages, as well as dying/nurse cells. timing, and gene expression programs underlying both groups of Wnt-expressing bipotential precursors already present at E11.5 are PG cells need to be more precisely defined. Ultimately, the extent followed at each developmental stage by two groups of ovarian to which these cells control the different properties of the two pregranulosa (PG) cells. One PG group, bipotential pregranulosa follicular waves remains of interest. (BPG) cells, derives directly from bipotential precursors, expresses Evolutionary conservation provides another potentially valu- Foxl2 early, and associates with cysts throughout the ovary by able source of insight into ovarian follicle development. In both E12.5.
    [Show full text]
  • Radiation Damage in the Mouse Female Germ Cell: a Two-Part Study
    V UCRL-52530 ) RADIATION DAMAGE IN THE MOUSE FEMALE GERM CELL: A TWO-PART STUDY Barbara Wuebbles (M.A. Thesis) %/# July 1978 Biomedical Sciences Division This paper was submitted as a Thesis to California State University, Hayward Work performed under the auipices of the U.S Department of Energy by the UCLLL under contract number W-7405-ENG-48. 111 • LAWRENCE IU3 UVERMORE •^LABORATORY MsnummcM at TSIS DOCIT ONT it unumtm m LAWRFNCE LIVERMORE LABORATORY University ot CaHomia/Livermore, Ca/«fomia/94550 UCRL-52S30 RADIATION DAMAGE IN THE MOUSE FEMALE GERM CELL: A TWO-PART STUDY (M.A. Thesis) July 1978 Biomedical Sciences Division This paper was submitted as a Thesis to California State University, Hayward I Thii "NOTICE r Willy « »„»»„, r„ J,, ^ ™™ JJJW ^ DISTKI?!TTTrc:; X" TITTS noCTTItnSNTIE TW>J.MITEI> Abstract This research consists of two parts. In one part irradiation was used to determine the radiosensitivity of prenatal female germ cells. Mice were given 18 rads of 60Co r-radiation (1 rad/min) at various times in utero. The effect of this treatment was measured by light microscopic enumeration of unilaminar follicles remaining in the ovary at U9 days after birth. Greatest sensitivity was seen for irradiation on day 11 post conception. However the germ cells at this period were not as sensitive as are dictyate oocytes of the juvenile mouse. The other part of this study involved y-irradiation of lit-day-old female mice. Electron microscopy was used to examine the ultra- structural changes taking place in the dictyate oocyte as a response to the radiation.
    [Show full text]
  • Heterochromatin Morphodynamics in Late Oogenesis and Early Embryogenesis of Mammals
    cells Review Heterochromatin Morphodynamics in Late Oogenesis and Early Embryogenesis of Mammals Irina Bogolyubova * and Dmitry Bogolyubov Laboratory of Cell Morphology, Institute of Cytology of the Russian Academy of Sciences, 4 Tikhoretsky ave., 194064 St. Petersburg, Russia; [email protected] * Correspondence: [email protected] Received: 13 May 2020; Accepted: 16 June 2020; Published: 19 June 2020 Abstract: During the period of oocyte growth, chromatin undergoes global rearrangements at both morphological and molecular levels. An intriguing feature of oogenesis in some mammalian species is the formation of a heterochromatin ring-shaped structure, called the karyosphere or surrounded “nucleolus”, which is associated with the periphery of the nucleolus-like bodies (NLBs). Morphologically similar heterochromatin structures also form around the nucleolus-precursor bodies (NPBs) in zygotes and persist for several first cleavage divisions in blastomeres. Despite recent progress in our understanding the regulation of gene silencing/expression during early mammalian development, as well as the molecular mechanisms that underlie chromatin condensation and heterochromatin structure, the biological significance of the karyosphere and its counterparts in early embryos is still elusive. We pay attention to both the changes of heterochromatin morphology and to the molecular mechanisms that can affect the configuration and functional activity of chromatin. We briefly discuss how DNA methylation, post-translational histone modifications, alternative histone variants, and some chromatin-associated non-histone proteins may be involved in the formation of peculiar heterochromatin structures intimately associated with NLBs and NPBs, the unique nuclear bodies of oocytes and early embryos. Keywords: mammalian early development; heterochromatin configuration; karyosphere; oocytes; pre-implantation embryos 1. Introduction The development of germ cells, the formation of a zygote and the subsequent cleavage of the embryo are amazing phenomena in nature.
    [Show full text]
  • Two Distinct Pathways of Pregranulosa Cell Differentiation Support Follicle Formation in the Mouse Ovary
    Two distinct pathways of pregranulosa cell differentiation support follicle formation in the mouse ovary Wanbao Niua,b and Allan C. Spradlinga,b,1 aHoward Hughes Medical Institute Research Laboratories, Carnegie Institution for Science, Baltimore, MD 21218; and bDepartment of Embryology, Carnegie Institution for Science, Baltimore, MD 21218 Contributed by Allan C. Spradling, July 2, 2020 (sent for review March 25, 2020; reviewed by Brigid L. M. Hogan and Melissa Pepling) We sequenced more than 52,500 single cells from embryonic day (EPG) cell population begins production at least by E14.5, also 11.5 (E11.5) postembryonic day 5 (P5) gonads and performed from progenitors in the ovarian surface epithelium (22, 23). These lineage tracing to analyze primordial follicles and wave 1 medullar cells express Lgr5 and eventually differentiate as granulosa cells follicles during mouse fetal and perinatal oogenesis. Germ cells on second wave follicles (8, 21–23). The cellular origins, division clustered into six meiotic substages, as well as dying/nurse cells. timing, and gene expression programs underlying both groups of Wnt-expressing bipotential precursors already present at E11.5 are PG cells need to be more precisely defined. Ultimately, the extent followed at each developmental stage by two groups of ovarian to which these cells control the different properties of the two pregranulosa (PG) cells. One PG group, bipotential pregranulosa follicular waves remains of interest. (BPG) cells, derives directly from bipotential precursors, expresses Evolutionary conservation provides another potentially valu- Foxl2 early, and associates with cysts throughout the ovary by able source of insight into ovarian follicle development. In both E12.5.
    [Show full text]
  • REP-19-0077.Pdf
    158 5 REPRODUCTIONANNIVERSARY REVIEW FERTILITY PRESERVATION Progress and prospects for developing human immature oocytes in vitro Evelyn E Telfer Institute of Cell Biology and Genes and Development Group CDBS, The University of Edinburgh, Edinburgh, UK Correspondence should be addressed to E E Telfer; Email: [email protected] This paper forms part of an anniversary issue on Fertility Preservation. The Guest Editor for this section was Professor Roger Gosden, College of William and Mary, Williamsburg, Virginia, USA Abstract Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.
    [Show full text]
  • Occur in Simple Chemically Defined Media Used for the Culture of the Cleavage Stages
    THE PATTERN OF ENERGY METABOLISM IN THE MOUSE OOCYTE AND ZYGOTE* BY J. D. BIGGERS, D. G. WHITTINGHAM, AND R. P. DONAHUE DIVISION OF POPULATION DYNAMICS, JOHNS HOPKINS SCHOOL OF HYGIENE AND PUBLIC HEALTH, AND DIVISION OF MEDICAL GENETICS, DEPARTMENT OF MEDICINE, JOHNS HOPKINS SCHOOL OF MEDICINE, BALTIMORE Communicated by W. D. McElroy, May 29, 1967 In vitro methods for the quantitative culture of cleaving mouse embryos on chemically defined medial' 2 have demonstrated that their basic patterns of energy metabolism differ from those found in later embryonic stages and the adult. The late 2-cell stage can only use lactate, pyruvate, phosphoenolpyruvate (PEP), and oxaloacetate to support cleavage.3 As development proceeds, more compounds can serve as energy sources, and at the 8-cell stage such compounds as malate, a- ketoglutarate, and glucose can be utilized.4 These observations pose a funda- mental question: Are the unique patterns of energy metabolism in the early mammalian embryo established prior to fertilization? To answer this question we have compared the energy requirements of the maturing oocyte and the zygote. This comparison has become possible because of recent advances in the hitherto independently studied fields of o6cyte and zygote culture. First, Edwards5 showed that mouse o6cytes, liberated from their ovarian follicles, will mature in medium 1996 supplemented with bovine serum. The maturation process involves breakdown of the arrested dictyate (germinal vesicle) stage of meiosis, and passage through metaphase I, anaphase I, and telophase I with first polar body extrusion, ending in an arrest of the chromosomes in a metaphase II configuration. We have now shown that maturation of the mouse o6cyte will occur in simple chemically defined media used for the culture of the cleavage stages.
    [Show full text]