Scott Coonrod, Ph.D.
Epigenetic Reprogramming in the Murine Egg and Early Embryo
Once ovulated, the terminally differentiated mammalian oocyte will die if it does not bind and fuse with a sperm. If fertilization occurs, however, maternal gene products orchestrate the transformation of the egg into a totipotent zygote within hours.
The main focus of my laboratory is to identify and study novel, highly-abundant and egg-specific molecules that are involved in this reprogramming process. These aims are being carried out by first, utilizing two-dimensional (2D) electrophoresis (as shown below), tandem mass spectrometry, and bioinformatics to scan the egg proteome for previously uncharacterized molecules which are likely to be functionally-relevant and oocyte-specific.
The tissue-specificity of the identified molecules is then confirmed using northern blot analysis and RT-PCR. The mouse genes encoding ovary-specific proteins are then cloned and expressed in bacterial expression vectors. Recombinant forms of the proteins are then expressed and purified and used to generate mono-specific antibodies. The antibodies, recombinant proteins, and anti-sense oligonucleotides are then tested in assays in vitro to investigate the molecule’s function. The murine egg proteome project has currently identified over 250 proteins in the mature mouse egg. Over twenty of the currently identified proteins are entirely novel and likely oocyte-specific and my lab is in the process investigating the function of a number of these maternal factors that will likely be found to be epigenetic regulators of early development.
Given that cellular differentiation is largely controlled by transcriptional regulation, chromatin remodeling will likely play a central role in this reprogramming process. Covalent histone modifications or "marks" provide an attractive storage mechanism for mitotically and meiotically heritable information. By regulating access to underlying DNA, these modifications can dictate correct spatial and temporal gene expression patterns during cellular differentiation. Given these observations, my laboratory has more recently began to focus on the identification and analysis of maternal epigenetic modifiers and to better understand the role that the resultant modifications play in dictating egg and early embryonic gene expression patterns. Further, we hypothesize that specific histone modifications, involved in dictating gene expression patterns during germ-cell differentiation, will be removed from the chromatin template during reprogramming and that these marks are replaced by new modifications required to direct early embryonic gene expression. To begin to investigate which covalent histone modifications are involved in this epigenomic reprogramming event, changes in global levels of a series of histone tail modifications are being studied during oocyte maturation and throughout pre-implantation mouse development. Results show that, at a global level, histone modifications can be classified into two strikingly different categories: stable ‘epigenetic’ marks and dynamic and reversible marks.
To more rigorously test the hypothesis that "resetting" of specific histone modifications leads to changes in expression patterns of target genes we will soon begin performing chromatin immunoprecipitation using anti-modified histone antibodies on oocytes and embryos as well as generating transgenic “knockdown” mice expressing reduced levels of maternal proteins involved in epigenetic reprogramming.
Wright PW, Bolling LC, Calvert ME, Sarmento OC, Berkeley EV, Shea MC, Hao Z, Jayes FC, Bush LA, Shetty J, Shore AN, Reddi PP, Tung KS, Samy E, Allietta MM, Sherman NE, Herr JC, and Coonrod SA (2003). ePAD, an Oocyte and Early Embryo-Abundant Peptidylarginine Deiminase-Like Protein which Localizes to Egg Cytoplasmic Sheets. Developmental Biology. 256:74-89.
Chen MS*, Tung KS*, Coonrod SA*, Takahashi Y, Bigler D, Chang A, Yamashita Y, Kincade PW, Herr JC, White JM. (1999) Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha6beta1: implications for murine fertilization. Proceedings of the National Academy of Sciences U.S.A, 96:11830-11835. * These authors contributed equally.
Shetty J, Wolkowicz MJ, Digilio LC, Klotz KL, Jayes FL, Diekman AB, Westbrook AV, Farris EM, Hao Z, Coonrod SA, Flickinger CJ, Herr JC. (2003) SAMP14, a novel, acrosome membrane associated, GPI anchored member of the Ly-6/uPAR receptor superfamily with a role in sperm-egg interaction. Journal of Biological Chemistry. In Press.
Coonrod SA, Naaby-HansenS, Shetty J, Shibahara H, Chen M, White JM, Herr JC. (1999). Treatment of mouse oocytes with PI-PLC releases 70 kDa (pI 5) and 35-45 kDa (pI 5.5) protein clusters from the egg surface and inhibits sperm-oolemma binding and fusion. Developmental Biology. 207:334-349.