Establishment of a Novel Culture Medium for Growing Cattle Embryos Beyond the Blastocyst Stage

This dissertation is dedicated to developing an extended culture medium that will support the growth of cattle embryos outside the uterine environment beyond the current seven-day limit (hatched blastocyst stage).

Given the increasing global demand for milk and meat production, it becomes critical to establish extended culture conditions that can model normal or in vivo-like embryonic development in cattle in vitro. Despite advancements and optimisation of blastocyst production over the years, the quality of IVP embryos still falls short compared to in vivo-sourced embryos.

An extended culture medium would help the scientific community understand molecular events after blastocyst development that have been difficult to study due to the inaccessibility of the embryo within the uterine environment. Also, their extended pre-implantation period makes cattle embryos an excellent model for studying mammalian embryogenesis, offering a comprehensive analysis of the developmental processes.

Chapter 3 chronicles the year-long journey of troubleshooting IVP and addressing the procedural challenges that arose when the lab relocated to a new facility and how we overcame them. Previously, we achieved the industry standard 30% survival rate for cattle embryos on Day 7 in the old laboratory. This success enabled experimentation with extended culture conditions; however, cattle embryos did not survive in the new lab post-relocation. Through this experience, I gained an in-depth understanding of various techniques and processes commonly followed “blindly” during standard IVP procedures. Our main goal was to re-establish a high survival rate of cattle embryos to continue our research on extended in vitro growth. I discovered that mineral oil, commonly used to overlay culture medium, is highly prone to peroxidation. In addition, bisphenol A, especially when exposed to heat and UV, leaches out of the container and accumulates in the oil, progressively increasing toxicity and ROS, thereby affecting embryo development.

In Chapter 4, in consultation with my supervisor, I developed a non-invasive morphological characterisation system to assess embryo viability, inner cell mass grade, hatchability, embryo and ICM diameter. I designed the basal medium based on published uterine fluid concentrations of amino acids, carbohydrates and electrolytes and tested a seven-growth factor cocktail consisting of ACTIVIN-A, ARTEMIN, BMP4, HB-EGF, FGF2, GM-CSF, and LIF as well as the effect of omitting individual cocktail components. It was found that ARTEMIN and BMP4 were highly beneficial, while excluding FGF2 from the growth factor cocktail positively impacted embryonic development. The addition of foetal bovine serum and the stem-cell culture additive ITSX proved crucial for successful culture. Replacing ITSX with B27 improved embryo cultures the most out of all the tested media combinations. The resultant SUM-T(B27) extended culture medium has potential applications in developmental biology research and commercial applications, as it allows for more time for diagnostic testing and improved evaluation of developmental potential.

Chapter 5 focuses on using our extended embryo culture system, based on uterine composition, growth factors and the cell culture additive B27, for growing cattle embryos in vitro beyond embryonic day 7. Here, extended in vitro cultured embryos are compared with uterine-grown counterparts to establish a developmental staging framework useful for understanding developmental events occurring until Day 10. Analysis of ICM/epiblast markers (OCT4, SOX2, and NANOG), hypoblast markers (GATA6, SOX17, and GATA4) and trophoblast genes (CDX2, GATA3, ASCL2 and IFNT), revealed the presence of four developmental stages during this period that can be molecularly distinguished. These are expanded blastocyst, hatched blastocyst, hypoblast layering and early hypoblast migration. Interestingly, NANOG and SOX17 show reciprocal expression at the expanded blastocyst stage, well before SOX2 and GATA6 expression refines to a “salt ‘n pepper” mutually exclusive expression in the ICM at the hatched blastocyst stage. GATA4 expression is only seen from stages when the hypoblast begins migrating around the blastocyst cavity. Intriguingly, trophoblast still expresses GATA6 and OCT4 in all cells during the expanded blastocyst phase, while SOX2 and SOX17 are seen in only some trophoblast cells. By the hypoblast-epiblast layering stage, these proteins are no longer expressed in the trophoblast apart from OCT4, which starts waning in the trophoblast once the hypoblast begins migrating. It is shown that cultured embryos exhibit increased expression of the stress marker TP53 in the epiblast and hypoblast at late stages compared to embryos grown in the uterine environment. Also presented in this chapter are preliminary observations where we investigated the role of the FGF/MEK signalling pathway and its involvement in the second lineage decision using our extended culture SUM-T(B27) medium, either by adding high levels of exogenous FGF2 ligand or by chemically inhibiting the FGFR or MEK1/2 signalling.