Bela Patel

The Primate Embryo Gene Expression Resource: A Novel Resource to Facilitate Rapid Analysis of Gene Expression Patterns in Non-Human Primate Oocytes and Preimplantation Stage Embryos

Abstract Detailed molecular studies of preimplantation stage development in a suitable nonhuman primate model organism have been inhibited due to the cost and scarcity of embryos. To circumvent these limitations, we have created a new resource for the research community, designated as the Primate Embryo Gene Expression Resource (PREGER). The PREGER sample collection currently contains over 160 informative samples of oocytes, obtained from various sized antral follicles, and embryos obtained through a variety of different protocols. The PREGER makes it possible to undertake quantitative gene-expression studies in rhesus monkey oocytes and embryos through simple and cost-effective hybridization-based methods. The PREGER also makes available other molecular tools to facilitate nonhuman primate embryology. We used PREGER here to compare the temporal expression patterns of five housekeeping mRNAs and three transcription factor mRNAs between mouse and rhesus monkey. We observed noticeable differences in temporal expression patterns between species for some mRNAs, but clear similarities for others. Our results also provide new information related to genome activation and the effects of embryo culture conditions on gene expression in primate embryos. These results provide one illustration of how the PREGER can be employed to obtain novel insight into primate embryogenesis.

Comparison of Gene Expression During Preimplantation Development Between Diploid and Haploid Mouse Embryos

Abstract Haploid development is a normal part of the life cycle for some animals, but it has not been observed in mammals. Studies in mice have revealed that the preimplantation developmental potential of haploid embryos is significantly impaired relative to diploid embryos. The reasons for the severely limited developmental potential of haploid embryos in mammals have not been discerned. To examine the effects of haploid development on gene expression, and in particular on X-linked gene expression, and to evaluate to what degree newer techniques of producing and culturing such embryos might affect developmental potential, haploid and diploid parthenogenetic and androgenetic embryos were produced and reevaluated for developmental potential, genomic integrity, and relative expression levels of specific autosomal and X-linked gene transcripts. Our data confirm the previously observed restriction in haploid developmental potential, eliminate chromosomal abnormalities as a major factor in this restriction, and reveal subtle alterations in gene expression. Haploid parthenogenones display only very subtle alterations in the expression of most mRNAs but a consistent elevation in X-linked Bex1 mRNA expression. Haploid androgenones seem to lack repression of the Pgk1 gene that is seen in diploid androgenones, but this may reflect ongoing loss of those haploid androgenones that experience X chromosome inactivation. The significance and possible explanations for these differences are discussed.

Effects of Follicle Size and Oocyte Maturation Conditions on Maternal Messenger RNA Regulation and Gene Expression in Rhesus Monkey Oocytes and Embryos

Abstract The relationship between alterations in gene expression and differences in developmental potential in primate oocytes and embryos was examined. Oocytes from 3 sources were used for these studies: 1) in vivo-matured oocytes from monkeys stimulated with FSH and hCG, 2) in vitro-matured oocytes from large follicles of monkeys primed with FSH, and 3) in vitro-matured oocytes from small follicles from nonstimulated (NS) monkeys. Following in vitro fertilization, embryos from these oocytes displayed high, moderate, and low developmental competence, respectively. Oocytes from NS females displayed aberrant accumulation of a number of maternal mRNAs, followed by precocious loss of many maternal mRNAs by the 2-cell stage. Embryos from NS oocytes displayed alterations in expression of key transcription factors after the 8-cell stage. Oocytes and embryos from FSH-stimulated females also displayed alterations in gene expression relative to hCG-stimulated females, but these alterations were much less severe than those observed for NS oocytes and embryos. Our data are consistent with the hypothesis that continued development and maturation of the oocyte within the ovarian follicle in vivo facilitates the production of oocytes of the highest developmental potential, and that in vitro conditions may not support this process as effectively due to differences in the extracellular milieu. These observations are relevant to understanding the role of the in vivo environment on oocyte maturation, and the potential effects of in vitro maturation on human assisted reproduction methods.

Expression of Genes Encoding Chromatin Regulatory Factors in Developing Rhesus Monkey Oocytes and Preimplantation Stage Embryos: Possible Roles in Genome Activation

Abstract One of the most critical events of preimplantation development is the successful activation of gene transcription. Both the timing and the array of genes activated must be controlled. The ability to regulate gene transcription appears to be reduced just prior to the time of the major genome activation event, and changes in chromatin structure appear essential for establishing this ability. Major molecules that modulate chromatin structure are the linker and core histones, enzymes that modify histones, and a wide variety of other factors that associate with DNA and mediate either repressive or activating changes. Among the latter are chromatin accessibility complexes, SWI/SNF complexes, and the YY1 protein and its associated factors. Detailed information about the expression and regulation of these factors in preimplantation stage embryos has not been published for any species. In order to ascertain which of these factors may participate in chromatin remodeling, genome activation, and DNA replication during early primate embryogenesis, we determined the temporal expression patterns of mRNA encoding these factors. Our data identify the predominant members of these different functional classes of factors expressed in oocytes and embryos, and reveal patterns of expression distinct from those patterns seen in somatic cells. Among each of four classes of mRNAs examined, some mRNAs were expressed predominantly in the oocyte, with these largely giving way to others expressed stage specifically in the embryo. This transition may be part of a global mechanism underlying the transition from maternal to embryonic control of development, wherein the oocyte program is silenced and an embryonic pattern of gene expression becomes established. Possible roles for these mRNAs in chromatin remodeling, genome activation, DNA replication, cell lineage determination, and nuclear reprogramming are discussed.

Effects of X Chromosome Number and Parental Origin on X-Linked Gene Expression in Preimplantation Mouse Embryos

Abstract Diploid androgenetic mouse embryos, possessing two sets of paternally inherited chromosomes, and control fertilized embryos were used to examine the relative effects of X chromosome number and parental chromosome origin on androgenone viability and X-linked gene expression. A significant difference in efficiency of blastocyst formation was observed between XX and XY androgenones in some experiments, but this difference was not uniformly observed. Significant effects of both X chromosome number and parental origin on X-linked gene expression were observed. Male and female control embryos expressed the Xist RNA initially. This expression was followed by a preferential reduction in Xist RNA abundance in male embryos, indicating that dosage compensation for the X chromosome may normally require the downregulation of Xist RNA expression in male embryos, in conjunction with the production of stable Xist transcripts in female embryos. By the late blastocyst stage, XX control embryos expressed significantly more Xist RNA than did XY embryos. Unlike their normal counterparts, XX androgenones did not express significantly more Xist RNA than did XY androgenones at the late blastocyst stage. Androgenones exhibited severe repression of the Pgk1 gene, but during development to the late blastocyst stage Pgk1 mRNA expression increased in XX androgenones and decreased in XY androgenones. Thus, the initial repression of the Pgk1 gene in XX androgenones was lost as the Xist RNA declined in abundance, and this loss was correlated with a failure of XX androgenones to express significantly more Xist RNA than did XY androgenones. These results indicate that androgenones may lack a factor that is expressed from the maternal genome and required for dosage compensation in preimplantation embryos. The results also indicate that early dosage compensation in preimplantation embryos may normally be reversible, thus providing flexibility to meet different developmental requirements of the embryonic and extraembryonic lineages.

Hybrid Vigor and Transgenerational Epigenetic Effects on Early Mouse Embryo Phenotype

Abstract Mouse embryos display a strain-dependent propensity for blastomere cytofragmentation at the two-cell stage. The maternal pronucleus exerts a predominant, transcription-dependent effect on this phenotype, with lesser effects of the ooplasm and the paternal pronucleus. A parental origin effect has been observed as an inequality in the cytofragmentation rate of embryos produced through genetic crosses of reciprocal F1 hybrid females. To understand the basis for this, we conducted an extensive series of pronuclear transfer studies employing different combinations of inbred and F1 hybrid maternal and paternal genotypes. We find that the parental origin effect is the result of a transgenerational epigenetic modification, whereby the inherited maternal grandpaternal contribution interacts with the fertilizing paternal genome and the ooplasm. This result indicates that some epigenetic information related to grandparental origins of chromosomes (i.e., imprinting of chromosomes in the mother) is retained through oogenesis and transmitted to progeny, where it affects gene expression from the maternal pronucleus and subsequent embryo phenotype. These results reveal for the first time that mammalian embryonic development can be affected by the epigenotype of at least three individuals. Additionally, we observe a significant suppression of fragmentation by F1 hybrid ooplasm when it is separated from the F1 hybrid maternal pronucleus. This latter effect is a striking example of heterosis in the early mammalian embryo, and it provides a new opportunity for examining the molecular mechanisms of heterosis. These results are relevant to our understanding of the mechanisms of epigenetic effects on development and the possible fertility effects of genetic and epigenetic interactions in reproductive medicine.

Growth Hormone and Gene Expression of In Vitro-Matured Rhesus Macaque Oocytes

Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r‐hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r‐hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo‐matured metaphase II (MII) oocytes and germinal vesicle (GV)‐stage oocytes. Only 2 of 17 genes, insulin‐like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r‐hGH treatment group compared with other IVM treatment groups, implicating insulin‐like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV‐stage or MII oocytes or cumulus cells. Mol. Reprod. Dev. 77: 353–362, 2010.

Systems Genetics Implicates Cytoskeletal Genes in Oocyte Control of Cloned Embryo Quality

Cloning by somatic cell nuclear transfer is an important technology, but remains limited due to poor rates of success. Identifying genes supporting clone development would enhance our understanding of basic embryology, improve applications of the technology, support greater understanding of establishing pluripotent stem cells, and provide new insight into clinically important determinants of oocyte quality. For the first time, a systems genetics approach was taken to discover genes contributing to the ability of an oocyte to support early cloned embryo development. This identified a primary locus on mouse chromosome 17 and potential loci on chromosomes 1 and 4. A combination of oocyte transcriptome profiling data, expression correlation analysis, and functional and network analyses yielded a short list of likely candidate genes in two categories. The major category—including two genes with the strongest genetic associations with the traits (Epb4.1l3 and Dlgap1)—encodes proteins associated with the subcortical cytoskeleton and other cytoskeletal elements such as the spindle. The second category encodes chromatin and transcription regulators (Runx1t1, Smchd1, and Chd7). Smchd1 promotes X chromosome inactivation, whereas Chd7 regulates expression of pluripotency genes. Runx1t1 has not been associated with these processes, but acts as a transcriptional repressor. The finding that cytoskeleton-associated proteins may be key determinants of early clone development highlights potential roles for cytoplasmic components of the oocyte in supporting nuclear reprogramming. The transcriptional regulators identified may contribute to the overall process as downstream effectors.

Rhesus Monkey Cumulus Cells Revert to a Mural Granulosa Cell State After an Ovulatory Stimulus

Follicular somatic cells (mural granulosa cells and cumulus cells) and the oocyte communicate through paracrine interactions and through direct gap junctions between oocyte and cumulus cells. Considering that mural and cumulus cells arise through a common developmental pathway and that their differentiation is essential to reproductive success, understanding how these cells differ is a key aspect to understanding their critical functions. Changes in global gene expression before and after an ovulatory stimulus were compared between cumulus and mural granulosa cells to test the hypothesis that mural and cumulus cells are highly differentiated at the time of an ovulatory stimulus and further differentiate during the periovulatory interval. The transcriptomes of the two cell types were markedly different (>1500 genes) before an ovulatory hCG bolus but converged after ovulation to become completely overlapping. The predominant transition was for the cumulus cells to become more like mural cells after hCG. This indicates that the differentiated phenotype of the cumulus cell is not stable and irreversibly established but may rather be an ongoing physiological response to the oocyte.

A novel isoform of TET1 that lacks a CXXC domain is overexpressed in cancer

Abstract TET1 oxidizes methylated cytosine into 5-hydroxymethylcytosine (5hmC), resulting in regulation of DNA methylation and gene expression. Full length TET1 (TET1FL) has a CXXC domain that binds to unmethylated CpG islands (CGIs). This CXXC domain allows TET1 to protect CGIs from aberrant methylation, but it also limits its ability to regulate genes outside of CGIs. Here, we report a novel isoform of TET1 (TET1ALT) that has a unique transcription start site from an alternate promoter in intron 2, yielding a protein with a unique translation start site. Importantly, TET1ALT lacks the CXXC domain but retains the catalytic domain. TET1ALT is repressed in embryonic stem cells (ESCs) but becomes activated in embryonic and adult tissues while TET1FL is expressed in ESCs, but repressed in adult tissues. Overexpression of TET1ALT shows production of 5hmC with distinct (and weaker) effects on DNA methylation or gene expression when compared to TET1FL. TET1ALT is aberrantly activated in multiple cancer types including breast, uterine and glioblastoma, and TET1 activation is associated with a worse overall survival in breast, uterine and ovarian cancers. Our data suggest that the predominantly activated isoform of TET1 in cancer cells does not protect from CGI methylation and likely mediates dynamic site-specific demethylation outside of CGIs.