Corticotrophin releasing hormone (CRH) and CRH receptor (CRH-R) appear to play a number of important roles in human pregnancy. The purpose of the first part of my project was to clone and sequence CRH-R subtypes from human myometrial biopsies. In order to understand the molecular mechanisms that direct the expression of the CRH-R gene, the objective of the second part of my research project was to clone and characterise the promoter region of the human CRH-R2 gene. Our results demonstrated the presence of multiple CRH-R mRNAs in the human myometrium. Six subtypes of the CRH receptor, 1a, 1b, 1c, 2a, 2b, and 2g, were found in the pregnant human myometrium, whereas only three subtypes, 1a, 1b, and 2b were found in the nonpregnant myometrium. Multiple CRH-R mRNAs have been identified in human myometrium with differential expression pattern during pregnancy, which argues for multiple roles for CRH and/or related peptides in myometrial function and suggests distinct functional roles for each receptor during pregnancy. These findings suggest that CRH and its receptor play an important modulatory role in myometrial function. The genomic organisation of human CRH-R2 alternative exons has been determined in the project. The coding region of the gene spans over 18kb. The genomic orders of the alternative exons are CRH-R2b-2g-2a. The 5'-flanking region of the human CRH-R2 gene was cloned by the genomic walking method. Using 5'RACE, the transcription start sites were mapped. The results suggest that there may be a single promoter regulating the expression of all (2a, 2b and 2g) subtypes. The CRH-R2 5'-flanking sequence has many characteristics of the housekeeping gene promoter. Therefore, we speculated that the strong and housekeeping gene-like activity of the CRH-2R gene promoter may contribute to the ubiquitous expression of the CRH-R2 gene. For functional analysis, 5'-flanking sequences up to-1393 bp were fused to the Chloramphenicol acetyltransferase (CAT) gene and tested using a HEK-293 cell line by transfection CAT assays. The results confirmed that the DNA region indicated demonstrated strong basal promoter activity.

The Xenopus pronephros is a simple paired organ; each nephron consists of a single large glomus, one set of tubules and a single duct. Previous work done in our laboratory demonstrates that the tubules and duct are specified at stages 12.5 and 14 respectively and the glomus is specified at stage 12.5. The kidney unit functions to control water balance and dispose of wastes. The simple organisation of the pronephros and the amenability of Xenopus laevis embryos to manipulation make the Xenopus pronephros an attractive system in which to study organogenesis. It has been shown that pronephric tubules can be induced to form in presumptive ectodermal tissue by treatment with RA and activin. Members of this laboratory have used this system in a subtractive hybridisation screen that resulted in the cloning of Xenopus laevis annexin IV (Xanx-4). In this thesis it has been demonstrated that Xanx-4 transcripts are specifically located to the developing pronephric tubules, and the protein to the luminal surface of these tubules. Temporal expression shows zygotic transcription is upregulated at the time of pronephric tubule specification. The temporal and spatial expression pattern of Xanx-4 suggests it may have a role in pronephric tubule development. Overexpression of Xanx-4 yields no apparent phenotype. Depletion of Xanx-4 dsRNA was tested and was shown to specifically reduce levels of Xanx-4 protein in oocytes. Xanx-4 siRNA was also tested for efficacy and was shown to cause a reduced pronephric tubule phenotype. However analysis of the effects of dsRNA and siRNA on mRNA levels have proved inconclusive and cast doubt on the usefulness of dsRNAs in Xenopus laevis. Further studies on RNAi in Xenopus will be required before it can be judged a reliable method for interfering with gene expression. Xanx-4 depletion, using morpholinos produces a shortened, enlarged tubule phenotype. The phenotype observed can be rescued by coinjection of Xanx-4 mRNA. Thus, Xanx-4 can be successfully depleted in embryos using morpholino oligos.

All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.