Genetics and imaging to assess oocyte and preimplantation embryo health

Two major criteria are currently used in human assisted reproductive technologies (ART) to evaluate oocyte and preimplantation embryo health: (1) rate of preimplantation embryonic development; and (2) overall morphology. A major gene that regulates the rate of preimplantation development is the preimplantation embryo development (Ped) gene, discovered in our laboratory. In mice, presence of the Ped gene product, Qa-2 protein, results in a fast rate of preimplantation embryonic development, compared with a slow rate of preimplantation embryonic development for embryos that are lacking Qa-2 protein. Moreover, mice that express Qa-2 protein have an overall reproductive advantage that extends beyond the preimplantation period, including higher survival to birth, higher birthweight, and higher survival to weaning. Data are presented that suggest that Qa-2 increases the rate of development of early embryos by acting as a cell-signalling molecule and that phosphatidylinositol-3′ kinase is involved in the cell-signalling pathway. The most likely human homologue of Qa-2 has recently been identified as human leukocyte antigen (HLA)-G. Data are presented which show that HLA-G, like Qa-2, is located in lipid rafts, implying that HLA-G also acts as a signalling molecule. In order to better evaluate the second criterion used in ART (i.e. overall morphology), a unique and innovative imaging microscope has been constructed, the Keck 3-D fusion microscope (Keck 3DFM). The Keck 3DFM combines five different microscopic modes into a single platform, allowing multi-modal imaging of the specimen. One of the modes, the quadrature tomographic microscope (QTM), creates digital images of non-stained transparent cells by measuring changes in the index of refraction. Quadrature tomographic microscope images of oocytes and preimplantation mouse embryos are presented for the first time. The digital information from the QTM images should allow the number of cells in a preimplantation embryo to be counted non-invasively. The Keck 3DFM is also being used to assess mitochondrial distribution in mouse oocytes and embryos by using the k-means clustering algorithm. Both the number of cells in preimplantation embryos and mitochondrial distribution are related to oocyte and embryo health. New imaging data obtained from the Keck 3DFM, combined with genetic and biochemical approaches, have the promise of being able to distinguish healthy from unhealthy oocytes and embryos in a non-invasive manner. The goal is to apply the information from our mouse model system to the clinic in order to identify one and only one healthy embryo for transfer back to the mother undergoing an ART procedure. This approach has the potential to increase the success rate of ART and to decrease the high, and undesirable, multiple birth rate presently associated with ART.

232SEARCH FOR THE BOVINE HOMOLOG OF THE MURINE PED GENE

The aim of the current study was to identify the bovine homolog of the murine Ped (preimplantation embryo development) gene, which regulates mouse preimplantation embryonic growth, including cleavage rate and embryo survivability, and to characterize the expression pattern of this gene during bovine preimplantation embryo development. Experiment (I): The National Center for Biotechnology Information (NCBI) GenBank/EBI EST databases were searched for bovine-expressed sequence tags (EST) that were homologous with the murine Ped gene (Accession number: NM_010394). The resulting ESTs were aligned and assembled in to one complete sequence (841bp), which was shown to be homologous with the Murine Ped gene and the Bovine Major Histocompatibility Complex class I 4221.1 gene (Accession No.: AJ010865, length 1090bp). The expression of the protein product of the Ped gene by bovine tissue was confirmed using Western Blot analysis. Experiment (II): The expression pattern of the Ped gene homolog during in vivo and in vitro bovine preimplantation embryo development was characterized using real time PCR. Embryos at the same stage for age were compared (Day 1: 2-cell; Day 2: 4-cell; Day 3: 8-cell; Day 4: 16-cell; Day 5: early morula;; Day 6, compact/late morula;; Day 7: blastocyst). The relative transcript abundance was consistently lower in the in vitro-cultured embryos at all stages of preimplantation development the differences were significant on Days 2, 4, 5, 6, and 7. The relative transcript abundance was significantly lower on Days 1, 2, and 3 of in vivo culture than on Days 4, 5, 6, and 7 and was significantly higher in Day 7 blastocysts than in Day 5 early morula. In in vitro-cultured embryos the relative transcript abundance was significantly higher in Day 7 blastocysts compared to all other stages of the preimplantation period. Experiment (III): A quantitative analysis of the Ped gene homolog was carried out on replicates of pools of ten 2-cell embryos collected at 25, 28, 32, and >36hpi from three different fertilizations. Transcript relative abundance was highest in those embryos that had cleaved by 25hpi. By 28hpi abundance had decreased slightly;; as time to cleavage increased further to 32 and >36hpi, the relative abundance decreased significantly. In conclusion, we have successfully identified a potential bovine homolog of the murine Ped gene. Furthermore, we have characterized the expression pattern of this gene during preimplantation embryo development in cattle and we have shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality and greater developmental potential.