Painting Qa-2 onto Ped slow preimplantation embryos increases the rate of cleavage.

PROBLEM: Qa-2 protein, the Ped gene product, is linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Some GPI-linked proteins can be spontaneously incorporated into the membranes of cells via a technique called "protein painting."We investigated whether Qa-2 could be painted onto T cells and embryos and whether the painted protein would be functional. METHOD OF STUDY: Incorporation of Qa-2 into the membranes of T cells and embryos was measured by FACScan and Immuno-PCR, respectively. Function of Qa-2 was measured by cell proliferation. RESULTS: Qa-2 was incorporated by T cells and embryos and was functional. CONCLUSION: GPI-linked Qa-2 protein "painted" onto both T cells and preimplantation embryos is functional, as shown by increased proliferation of T cells after cross-linking with anti-Qa-2 antibody, and increased rate of cleavage division of the embryos.

Expression of caspase and BCL-2 apoptotic family members in mouse preimplantation embryos.

Apoptosis, as determined by blastomere and DNA fragmentation, occurs in many preimplantation mouse embryos. To investigate which genes contribute to apoptosis in preimplantation embryos, we used the reverse transcription-polymerase chain reaction to assess mRNA levels for seven genes in the caspase family and seven genes in the BCL-2 family. All caspase mRNAs were detectable in oocytes, while expression in preimplantation embryos varied in a stage-specific manner. An assay for group II caspase enzymatic activity showed that although transcripts for these caspases could not be detected in zygotes, proteolytic activity could be detected in polar bodies, fragmented zygotes, and zygotes treated with staurosporine. This suggests that maternal caspases are inherited during oogenesis. Transcripts for some members of the BCL-2 family could be detected at every stage of preimplantation development. Transcripts for other members were rarely detected. When BCL-2 and BAX protein levels were assessed using immunofluorescence, both proteins were detected in zygotes and in blastocysts. When fragmented blastocysts were compared to normal blastocysts, levels of BCL-2 immunofluorescence tended to be lower in fragmented blastocysts. This result supports a model in which the ratio of BCL-2 to BAX is altered in apoptotic embryos.

Selection in favor of the Ped fast haplotype occurs between mid-gestation and birth.

The preimplantation embryo development (Ped) gene that encodes the class Ib major histocompatibility complex protein Qa-2 influences the rate of embryonic cleavage during the preimplantation stages of development. Embryos from strains of mice that lack the Ped gene cleave slowly, while embryos that have a functional Ped gene cleave more rapidly. This effect is observed both in vivo and in vitro with the Ped fast haplotype showing dominance over the Ped slow haplotype. The Ped gene is associated with pleiotropic effects on reproduction. Certain strains of mice lacking the Ped gene (Ped slow) have smaller litters and the pups weigh less at birth and at weaning. Previously our laboratory reported that in litters derived from Ped fast/slow F1 mice backcrossed to the slow/slow parent, there were significantly more Ped fast pups than the 50% expected, at two months of age. This implies that there is selection in favor of the Ped fast haplotype at some point during development. The present study was designed to determine at what point during development selection occurs. Using a polymerase chain reaction assay, we determined that selection does not occur by days post coitus 14.5. However, our results show that there are significantly more Ped fast pups than Ped slow pups remaining in backcross litters just after birth, indicating that selection in favor of the Ped fast haplotype occurs between day 14.5 and birth.

Genetic regulation of egg and embryo survival.

In both mice and humans, 15-50% of embryos die during the preimplantation period from mechanisms that are largely unknown. Two major criteria predict preimplantation embryo quality, the rate of development and the degree of fragmentation. We review evidence that both of these criteria have a genetic basis. Rate of development and subsequent embryo survival are controlled by a gene, Ped, we discovered in the mouse. Although progress is being made in the search for the human homologue of the mouse Ped gene, it has not yet been identified. Fragmentation, observed in both mouse and human embryos, is probably the result of apoptosis. We analysed transcription of two genes that regulate apoptosis, bcl-2 and bax, and found that both are transcribed in mouse and human preimplantation embryos. Overall, the literature reviewed and new data presented in this paper support the concept that there is a genetic basis for preimplantation egg and embryo survival.

Differential expression of Ped gene candidates in preimplantation mouse embryos.

The Ped (preimplantation embryonic development) gene influences the rate of mouse preimplantation embryonic development and subsequent survival. Four similar tandem genes in the Q region of the major histocompatibility complex-Q6, Q7, Q8, and Q9-were identified as Ped gene candidates. In this study, expression of these genes during preimplantation development was examined and quantitated by reverse transcription-polymerase chain reaction and single nucleotide primer extension assays in order to investigate their contribution to the Ped gene phenotype. The Q7/Q9 gene pair was found to be transcribed in preimplantation mouse embryos, whereas transcription of the Q6/Q8 gene pair was undetectable. Both Q7 and Q9 are expressed in embryos from one Ped fast strain, C57BL/6, while only the Q9 gene is expressed in another Ped fast strain, B6.K2. These results suggest that both the Q7 and Q9 genes can function as the Ped gene in the mouse. Interestingly, the expression pattern of the Q7 and Q9 genes in preimplantation embryos is the same as in splenic lymphocytes. However, the Q6 and Q8 genes are expressed in splenic lymphocytes but not in preimplantation embryos. Treatment of mouse preimplantation embryos with interferon gamma (gamma-IFN) did not induce expression of the Q6/Q8 genes but enhanced expression of the Q7/Q9 genes. The mechanism of this differential transcription pattern is currently under investigation.

Genetic regulation of preimplantation mouse embryo survival.

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.

Role of the Ped gene and apoptosis genes in control of preimplantation development.

PURPOSE: The properties of the mouse Ped gene and the genes that mediate apoptosis in mediating preimplantation embryonic survival were reviewed. METHODS: Preimplantation mouse oocytes and embryos were evaluated microscopically and biochemically for rate of development, degree of fragmentation, and gene expression to correlate these characteristics with embryo mortality, Biochemical assays included PCR for DNA analysis, RT-PCR for mRNA analysis, immuno-PCR for protein analysis, and TUNEL assay for assessment of apoptosis. RESULTS: Using the mouse as a model system we have identified a gene that controls the rate of development, the Ped gene. The Ped gene product is a class Ib major histocompatibility complex protein called the Qa-2 antigen. Research to understand the molecular mechanisms of Ped gene action and to identify the human homologue of the Ped gene is under way. We have also shown using the mouse model, that fragmented embryos show the morphological and biochemical characteristics of apoptosis. Genes in the two major gene families that regulate apoptosis, the caspase and Bcl-2 families, are expressed in mouse oocytes and preimplantation embryos. CONCLUSIONS: Preimplantation embryonic survival depends on two major morphological parameters: rate of development and degree of fragmentation. A fast rate of development and a low degree of fragmentation lead to a better chance of producing live offspring. Both rate of development and degree of fragmentation are genetically controlled, the former by the Ped gene and the latter most likely by genes that mediate apoptosis. It seems probable that regulation of apoptosis will prove to be a major mechanism that mediates oocyte and preimplantation embryonic survival.

Sequence and transcription of Qa-2-encoding genes in mouse lymphocytes and blastocysts.

The protein product of the mouse preimplantation embryo development (Ped) gene, which controls the rate of preimplantation embryonic cleavage division and subsequent embryo survival, is the Qa-2 antigen. This major histocompatibility complex (MHC) class I b protein is encoded by four genes, Q6, Q7, Q8, and Q9. The present study was undertaken to begin to elucidate which of the four Qa-2-encoding genes are responsible for the Ped gene phenotype in the C57BL/6 mouse (H2(b)). First, restriction maps of the four genes, using 25 restriction enzymes, were created. The RE maps confirmed that Q6 is similar to Q8 and Q7 is similar to Q9, but that the Q6/Q8 gene pair differs from the Q7/Q9 gene pair. The genomic DNA sequences of Q6 and Q8 were determined, as well as the DNA sequences of exons 4 - 8 of Q9, and the 5' regulatory regions of Q6, Q8, and Q9. This DNA sequence information, combined with the published DNA sequence information for the entire Q7 gene and exons 1 - 3 of Q9, allowed us to design primers for reverse transcription-polymerase chain reaction that could distinguish which of the four genes were transcribed in mouse lymphocytes and embryos. It was found that all four genes are transcribed in lymphocytes, but only Q7 and Q9 are transcribed in mouse embryos. Thus, both Q7 and Q9 are candidates for the genes responsible for the Ped gene phenotype.

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.