Erweiterte Suche

The effect of growth factor supplemented ART culture media on whole mouse embryo development

Eizellen, Spermien und Embryonenwerden in der Reproduktionsklinik für einen bestimmten Zeitraum in einer künstlichen Umgebung gehandhabt. Die Umgebung sollte der natürlichen Umgebung so ähnlich wie möglich sein. Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) wurde zu einem attraktiven Kandidaten für eine Supplementierung von Kulturmedien. Eine klinische Studie zeigte, dass mit GM-CSF supplementiertes Embryokulturmedium zu einem erhöhten Überleben der Embryonen führt, insbesondere bei Frauen mit früheren Fehlgeburten. Tier- und Zellkulturstudien belegen außerdem einen Effekt auf die Zellzahl und die Pluripotenzsignalwege. Ziel dieser Studie war es, die Supplementierung von GM-CSF in einem humanen ART-Medium oder in einem mausoptimierten Medium und dessen Effekt auf Zellzahl und der Komposition der Zelllinien im Mausembryo zu untersuchen. Unsere Daten zeigen, dass Maus-GM-CSF einen Einfluss auf die Gesamtzellzahl hatte; Sie nahmen im Kulturmedium zu, das mit zunehmenden Konzentrationen von mGM-CSF ergänzt wurde. Diese Erhöhung der Zellzahl wurde bei Embryonen, die in ART-Medien mit oder ohne GM-CSF oder in Kulturmedium mit unterschiedlichen Konzentrationen von humanen GM-CSF kultiviert wurden, nicht gefunden. Darüber hinaus zeigen die Ergebnisse eine ektopische Expression von NANOG unter CDX2-positiven TE-Zellen sowohl in humanen ART-Medien als auch in zunehmenden Konzentrationen in mit Maus und humanem GM-CSF supplementierten Medien.

Titel: The effect of growth factor supplemented ART culture media on whole mouse embryo development
Verfasser: Pock, Tim GND
Gutachter: Nordhoff, Verena GND
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 2019
Publikation in MIAMI: 18.10.2019
Datum der letzten Änderung: 24.10.2019
Schlagwörter: GM-CSF; Embryo; Embryoentwicklung; NANOG
Fachgebiete: Biowissenschaften; Biologie
Lizenz: CC BY-NC-SA 4.0
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-33149682325
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-33149682325
Onlinezugriff:
Inhalt:
List of content ............................................................................................................................ I
I List of Figures ........................................................................................................................ III
II List of tables ........................................................................................................................ VI
III Abbreviations .................................................................................................................... VIII
1 Introduction ............................................................................................................................ 1
1.1 The need of assisted reproduction ................................................................................. 1
1.2 IVF-register and assisted reproductive technologies ...................................................... 1
1.3 In vitro culture ................................................................................................................. 2
1.4 Granulocyte-macrophage colony-stimulating factor ....................................................... 3
1.5 Mouse model for experimental embryology .................................................................... 4
1.6 Mouse embryo assay ...................................................................................................... 4
1.7 Embryo development in mouse ...................................................................................... 5
1.8 Cell fate decision ............................................................................................................ 7
1.9 Aims and hypothesis of the study ................................................................................. 10
2 Methods ............................................................................................................................... 11
2.1 Mice .............................................................................................................................. 11
2.2 Superovulation & recovery of zygotes .......................................................................... 12
2.3 Embryo culture & media ............................................................................................... 12
2.4 Transfer of embryos to pseudo pregnant recipient mice .............................................. 14
2.5 Immunohistochemical staining ......................................................................................... 15
2.6 Image Analysis ............................................................................................................. 15
2.7 Statistical analysis ........................................................................................................ 15
2.8 Project design ................................................................................................................... 16
3 Results ................................................................................................................................ 17
3.1 Zygote collection ........................................................................................................... 18
3.2 ART experiment ............................................................................................................ 19
3.2.1 Development rates in the ART experiment ............................................................ 19
3.2.2 Cell numbers of different cell lineages in the ART experiment .............................. 20
3.3 mGM-CSF/hGM-CSF experiment ................................................................................. 22
3.3.1 Development rates in the m- and hGM-CSF experiment ....................................... 22
3.3.2 Cell numbers of different cell lineages in the mGM-CSF experiment .................... 23
3.4 Blastocysts with ectopic expression of NANOG ........................................................... 28
3.4.1 Amount of blastocysts with ectopic NANOG expression among different growth
factor concentrations ....................................................................................................... 29
3.4.2 Cell lineage distribution of normal blastocysts in the ART experiment compared to
blastocysts with ectopic expression of NANOG. ............................................................. 31
3.4.3 Cell lineage distribution of normal blastocysts in the m- and hGM-CSF experiment
compared to blastocysts with ectopic expression of NANOG. ........................................ 33
3.5 Repeating experiments in a second laboratory at the MPI Muenster ........................... 36
3.5.1 Cell numbers of different cell lineages in the MPI experiment ............................... 36
3.5.2 Amount of blastocysts with ectopic NANOG expression among different growth
factor concentrations in the MPI experiment. .................................................................. 38
3.6 CD1 strain experiments ................................................................................................ 41
3.6.1 Development rates in the CD1 experiment ............................................................ 41
3.6.2 Cell number of different cell lineages in the CD1 experiment ................................ 42
3.6.3 Amount of blastocysts with ectopic NANOG expression among different growth
factor concentrations in the CD1 experiment. ................................................................. 44
3.7 Embryo transfer experiment ......................................................................................... 46
3.7.1 Development rates in the embryo transfer experiment .......................................... 46
3.7.2 Transferred blastocysts, implantation and fetal rate .............................................. 47
3.7.3 Fetus length ........................................................................................................... 48
3.7.4 Fetus weight ........................................................................................................... 49
3.7.5 Placenta weight ...................................................................................................... 50
4 Discussion ........................................................................................................................... 51
4.1 Influence of GM-CSF on early embryo development .................................................... 51
4.2 GM-CSF alters cell identity of trophectodermal cells .................................................... 54
4.3 Excluding mouse strain and laboratory-specific effects ................................................ 55
5 Conclusion & Outlook .......................................................................................................... 57
6 References .......................................................................................................................... 59
6 Appendix ............................................................................................................................. 64
6.1 Supplementary data & statistics ................................................................................... 64
6.1.1 Supplement Tables ................................................................................................ 64
6.1.2 Supplement figures .................................................................................................... 68
6.2 Materials ....................................................................................................................... 73
6.2.1 Antibodies .............................................................................................................. 73
6.2.2 Growth factors ........................................................................................................ 73
6.2.3 Chemicals and reagents ........................................................................................ 73
6.2.4 Culture media ......................................................................................................... 74
6.2.5 Consumables ......................................................................................................... 74
6.2.6 Equipment .............................................................................................................. 75
6.2.7 Software ................................................................................................................. 75
6.3 Power analysis for the ART experiment ....................................................................... 76
7 CV ....................................................................................................................................... 79
8 Danksagung ........................................................................................................................ 84
9 Versicherungen & Eidesstattliche Erklärungen ................................................................... 86
9.1 Versicherung nach § 6 Abs. 3 (2) ................................................................................. 86
9.2 Versicherungen nach § 6 Abs. 3, Nr. 5, 6, 9 ................................................................. 87