Serum supplementation during bovine embryo culture affects their development and proliferation through macroautophagy and endoplasmic reticulum stress regulation

Serum supplementation during bovine embryo culture has been demonstrated to promote cell proliferation and preimplantation embryo development. However, these desirable outcomes, have been associated with gene expression alterations of pathways involved in macroautophagy, growth, and development at the blastocyst stage, as well as with developmental anomalies such as fetal overgrowth and placental malformations. In order to start dissecting the molecular pathways by which serum supplementation of the culture medium during the preimplantation stage promotes developmental abnormalities, we examined blastocyst morphometry, inner cell mass and trophectoderm cell allocations, macroautophagy, and endoplasmic reticulum stress. On day 5 post-insemination, > 16 cells embryos were selected and cultured in medium containing 10% serum or left as controls. Embryo diameter, inner cell mass and trophectoderm cell number, and macroautophagy were measured on day 8 blastocysts (BL) and expanded blastocysts (XBL). On day 5 and day 8, we assessed transcript level of the ER stress markers HSPA5, ATF4, MTHFD2, and SHMT2 as well as XBP1 splicing (a marker of the unfolded protein response). Serum increased diameter and proliferation of embryos when compared to the no-serum group. In addition, serum increased macroautophagy of BL when compared to controls, while the opposite was true for XBL. None of the genes analyzed was differentially expressed at any stage, except that serum decreased HSPA5 in day 5 > 16 cells stage embryos. XBP1 splicing was decreased in BL when compared to XBL, but only in the serum group. Our data suggest that serum rescues delayed embryos by alleviating endoplasmic reticulum stress and promotes development of advanced embryos by decreasing macroautophagy.

Adapting CRISPR/Cas9 Lentivirus Technology for Functional Studies of GATA6 & GATA4 during Bovine Preimplantation Embryogenesis

<p>In recent times, cattle embryology has been under the spotlight of investigation due to its apparent economic values. This is especially relevant in the case of New Zealand, owing to its high percentage of livestock export. Specifically, the period of peri-implantation development has been of particular relevance. During this stage, the developing zygote will establish 3 key lineages – epiblast, hypoblast and trophoblast. Previous studies have elucidated that a significant number of embryos die prior to implantation, therefore highlighting the importance of correctly establishing these 3 lineages to overall embryonic survival. However, while embryological stages of the preimplantation embryo have been extensively studied in their eutherian cousin, mice, the molecular regulation of that of cattle remains much less addressed. Whereas the regulation of bovine embryo development is orchestrated by many transcriptional regulators, or genetic regulatory networks (GNP), we aimed to focus our studies on 2 key transcriptional regulators, GATA4 and GATA6. During early embryogenesis, both these transcriptional factors are known molecular regulators that drive the establishment of the hypoblast lineage in mice. By and large, while their respective expression has been documented in cattle embryos, functional studies towards these markers have not yet been performed. Latest advances in molecular biology have given us novel methods to study the mechanism of bovine embryogenesis. To this end, the continuing perfection of CRISPR technologies in the last decade - in particular its delivery through lentiviral vectors, has established an ability to generate stable, targeted knock-out mutants. Therefore, it is aimed in this thesis to design and test lentiviral particles that induce knock-out mutants of GATA4 and GATAT6, to test their efficacy in primary cell cultures (bovine cumulus cells) and to functionally analyse the effect of GATA4 and GATA6 knockdowns in early bovine embryos.</p>

Adapting CRISPR/Cas9 Lentivirus Technology for Functional Studies of GATA6 & GATA4 during Bovine Preimplantation Embryogenesis

<p>In recent times, cattle embryology has been under the spotlight of investigation due to its apparent economic values. This is especially relevant in the case of New Zealand, owing to its high percentage of livestock export. Specifically, the period of peri-implantation development has been of particular relevance. During this stage, the developing zygote will establish 3 key lineages – epiblast, hypoblast and trophoblast. Previous studies have elucidated that a significant number of embryos die prior to implantation, therefore highlighting the importance of correctly establishing these 3 lineages to overall embryonic survival. However, while embryological stages of the preimplantation embryo have been extensively studied in their eutherian cousin, mice, the molecular regulation of that of cattle remains much less addressed. Whereas the regulation of bovine embryo development is orchestrated by many transcriptional regulators, or genetic regulatory networks (GNP), we aimed to focus our studies on 2 key transcriptional regulators, GATA4 and GATA6. During early embryogenesis, both these transcriptional factors are known molecular regulators that drive the establishment of the hypoblast lineage in mice. By and large, while their respective expression has been documented in cattle embryos, functional studies towards these markers have not yet been performed. Latest advances in molecular biology have given us novel methods to study the mechanism of bovine embryogenesis. To this end, the continuing perfection of CRISPR technologies in the last decade - in particular its delivery through lentiviral vectors, has established an ability to generate stable, targeted knock-out mutants. Therefore, it is aimed in this thesis to design and test lentiviral particles that induce knock-out mutants of GATA4 and GATAT6, to test their efficacy in primary cell cultures (bovine cumulus cells) and to functionally analyse the effect of GATA4 and GATA6 knockdowns in early bovine embryos.</p>