Research interests

Oocytes and Embryos; Stem Cells and Cancer

Two of the most compelling questions in biology today are: how are the nuclei from differentiated cells (gametes on fertilization, or somatic cell nuclei when transferred) reprogrammed into totipotent stem cells in the ooplasm, and how are stem cells induced into normal differentiation or into genome instability and tumor formation? We investigate the molecules and molecular processes occurring in the egg cytoplasm during reprogramming of egg and sperm nuclei to form totipotent stem cells of the preimplantation embryo, as well as early markers at the outset of mammary tumorigenesis, a process that may be considered inappropriate stem cell/progenitor cell activation. This laboratory group is composed of young investigators who are transitioning to independent careers. This report highlights their individual work as well as the work we have all done together.

Oocyte to Embryo Transition

The oocyte is hormonally activated into growth and differentiation within the ovary. Once it has reached its full growth potential, transcription ceases. Oocyte maturation, ovulation, fertilization, completion of meiosis and of the first mitotic cell cycle are all accomplished in the absence of new transcription. Indeed, the embryonic genome is not considered activated until the end of the 2-cell stage.

To gain insight into the nature of the stable maternal transcripts still present at the end of the 2-cell stage, Dr. Evsikov analyzed cDNA libraries prepared from 2-cell embryos, documenting the 5,000 most abundant genes expressed in the 2-cell embryo. Delayed translation is an important factor during the transcriptionally silent oocyte-to-embryo transition. RNA-binding proteins, known to stabilize transcribed RNAs and prevent translation, and members of the protein complex that controls delayed translation, are among these stable transcripts. Gene ontology classification of these maternal transcripts revealed that transcripts for numerous kinases, phosphatases, and factors controlling protein degradation are abundant. These are the types of gene products likely to drive metamorphosis of a differentiated gamete to a pluripotent cell type.

An outstanding characteristic of the 2-cell-stage embryo is the prominence of transcribed retrotransposons in the transcriptome. To explore the significance of this finding, Dr. Peaston documented a burst of reverse transcriptase beginning early in the 2-cell stage lasting throughout the cleavage stage. This suggests that one of the classes of retrotransposons expressed in the 2-cell stage embryo may replicate, and also serve to catalyze the replication of other such elements that do not encode their own reverse transcriptase.

In the first of a series of works analyzing the 6,000 most abundant genes expressed in the full grown oocyte, Drs. Evsikov and Peaston noted that over 10% of these transcripts are retrotransposon-derived; furthermore, they found that the most abundant classes of retrotransposons differ between the full-grown oocyte and the 2-cell-stage embryo. Remarkably, there are many novel chimeric transcripts, composed of a viral Long Terminal Repeat (LTR) virus spliced into a host gene, which supply new transcription start sites. In some cases the chimeric gene product is foreshortened, possibly changing the function of the normal gene’s cognate protein. In other cases the addition of the retrotransposon-derived 5’UTR merely ensures that the host gene is expressed, ensuring the presence of a group of proteins during oocyte growth. Decades ago, the late Barbara McClintock suggested retrotransposons could be considered master switches controlling expression of many genes simultaneously. Our results can be regarded as proof of her postulate. Both sense and antisense transcripts of one of the retroviral genes expressed in the oocyte and 2-cell embryo suggest that these gene products might be under the control of an RNAi-like mechanism. The genome of most species is littered by multiple, integrated transposons, many of which no longer have functional gene products. Because of the abundant transcription we describe in gamete formation, we suggest they are hallmarks of, or contribute to, a mechanism for reprogramming the embryonic genome. Dr. Keith Hutchison, a professor from the University of Maine who is spending a sabbatical year in our laboratory, is identifying the primary sites of transcription of these retrotransposons in the mouse genome.

In a collaborative effort, Dr. Evsikov searched the full-grown oocyte library for a G-linked receptor that Dr. Laurinda Jaffee, University of Connecticut, hypothesized to be present. Once identified, she found this gene product to be responsible for maintaining meiotic arrest. Dr. Evsikov has found a diversity of G-linked receptors and ligands characterize the full-grown oocyte.

Dr. de Vries studies the role of specific genes in the oocyte-embryo transition and in documenting the onset of the paternal genome expression. She previously made and characterized a mouse transgenic for a zona pellucida glycoprotein 3-promoted cre transgene (Zp3-cre), which conditionally deletes genes in the full-grown oocyte. Using this mouse line and various floxed alleles of β-catenin, and cadherin 1 (or E-cadherin), she has approached the function of these gene products in the oocyte and pre- and peri-implantation embryo. Deletion of the floxed E-cadherin allele results in complete absence of E-cadherin in the oocyte. Deleting the N-terminus of β-catenin removes a large portion of the N-terminus synthesis, but an in frame transcript and truncated protein are made. E-cadherin and β-catenin mediate blastomere adhesion. In their absence, blastomeres do not adhere until sufficient protein is synthesized from the paternal allele of β-catenin in the 6- to 8-cell stage, or from the E-cadherin paternal allele at the 16-cell stage. Females producing E-cadherin-null oocytes are fully fertile, while those producing the truncated β-catenin are subfertile. In the absence of E-cadherin, the truncated and normal forms of β-catenin are found in the zygotic pronuclei and 2-cell-stage embryo nuclei. To determine when the conventional Wnt pathway, mediated by β-catenin, is activated, mice carrying a different mutant conditional β-catenin allele were crossed with the Zp3-cre transgenic line. These embryos have a stabilized, toxic form of β-catenin. These embryos develop to blastocysts, suggesting that the Wnt pathway is not necessary until that point in embryogenesis. However, endoderm formation is compromised. These studies suggest that (1) full-length β-catenin is not a requirement for preimplantation development to blastocyst in vitro; (2) that β-catenin has an alternative nuclear role, uncoupled from the Wnt/β-catenin signaling pathway, at the onset of preimplantation embryo development; and (3) that alternative Wnt signaling pathways are involved in controlling preimplantation development.

Tumor Cell Transition

Karen Fancher is a graduate student in Functional Genomics at the University of Maine, with two Jackson-Laboratory-based mentors, Dr. Churchill in biostatistics and Dr. Knowles in tumor genetics. She has used mice transgenic for a whey acidic protein-promoted viral oncogene to determine changes in gene expression that occur during the earliest stages of tumorigenesis in the mammary gland. These female mice typically develop frank tumors at a year of age, but the gene expression profile in the mammary gland is altered up to 6 months earlier, at a time when atypical cells and ductal carcinoma in situ are detected histologically.

Publications

• Solter D, deVries WN, Peaston A, Evsikov A, Knowles BB. 2002. Fertilization and

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