| Summary: | During preimplantation embryo development the zygote is transformed into the blastocyst. Metabolism during preimplantation development has been well studied but a major challenge is to understand the underlying control mechanisms. THe hypothesis tested in this thesis is that control mechanisms may be revealed by examining regulatory metabolic enzymes at the level of gene and protein expression alongside measurements of their biochemical activity. The gene expression, protein localisation and biochemical activity of three key enzymes of metabolism: Hexokinase (HK), Creatine Kinase (CK) and the Na+/K+ ATPase, were therefore studied during pig and mouse preimplantation embryo development. HK is the first enzyme of glycolysis, CK functions to buffer cellular ATP/ADP ratios and the Na+/K+ ATPase is a major consumer of ATP at cavitation. Pig zygotes were produced by in vitro fertilisation of in vitro-matured abattoir-derived immature oocytes; mouse zygotes were derived in vivo. Both pig and mouse zygotes were cultured to t~e blastocyst stage in vitro. Some in vivo-derived pig embryos at various stages of preimplantation development were also studied. The gene expression profiles of multiple isoforms ofHK and CK and the Na+/K+ ATPase al subunit were determined using quantitative RT-PCR. The biochemical activity of HK and CK were profiled in both species, using ultramicrofluorescence, with CK activity also measured during human preimplantation embryo development. There was little relationship between gene expression and biochemical activity of HK and CK throughout preimplantation -development. Protein expressio~ patterns of CKB and Na+/K+ ATPase al were determined at all stages of mouse preimplantation development and revealed ubiquitous expression of CK and a possible interaction with the mitotic spindle. The work suggests that the previously little studiedCK plays an important role in the metabolism of the developing preimplantation embryo ~d, consistent with the original hypothesis, that biochemical activity needs to be studied alongside gene and protein expression to understand fully the factors involved in the control of energy metabolism.
|
|---|
