Qa-2, a murine class Ib major histocompatibility complex (MHC) molecule, is a possible functional homolog of human leukocyte antigen G (HLA-G). Both molecules have been implicated in immunoregulation and embryonic development and both occur in membrane-bound and soluble isoforms that arise by alternative splicing. Soluble splice variants have been implicated in the reproductive functions of HLA-G. While soluble variants of Qa-2 have been previously detected in T lymphocytes, we now demonstrate the presence of mRNA for one of the two known soluble forms of Qa-2 in eight-cell embryos and in blastocysts. Qa-2 is glycosylphosphatidy-linositol (GPI) linked in the outer leaflet of the cell membrane and is found in lipid raft microdomains where other raftassociated proteins transduce signals into the cell. In contrast, HLA-G has a truncated six amino acid cytoplasmic tail. By fluorescence co-localization in JEG-3 cells, using fluorescent cholera toxin β subunit (a lipid raft marker) and anti-HLA-G antibody, we have demonstrated that membrane-bound HLA-G also localizes to lipid rafts, consistent with functional homology between the two molecules. Finally, our experiments in which we have purified Qa-2 and transferred it via a process known as protein painting to Qa-2 negative cells represent a model for potential therapy involving HLA-G.

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-32 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.

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Fyn, PI-3 kinase, and Akt are required for the activation of T cells by cross-linking Qa-2.

The tolerogenicity of neonatal skin grafts derives in part from natural expression of IL-10 by the graft. Another possible contribution to tolerogenicity may be the inability of neonatal antigen presenting cells to up-regulate class II fully. Low expression of class II by neonatal cells is not attributable to epidermal IL-10 secretion.

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