Biology of Reproduction
The adverse effects of maternal diabetes on embryo development and pregnancy outcomes have recently been shown to occur as early as the one cell zygote stage. Recent work from our lab has shown that maternally inherited mitochondria in oocytes from diabetic mice are abnormal and thus may be responsible in part for this latency of developmental compromise. In ovulated oocytes from diabetic mice, transmission electron microscopy revealed an alteration in mitochondrial ultrastructure and the
... tative analysis of mtDNA copy number demonstrated an increase. The levels of ATP and TCA cycle metabolites in diabetic oocytes were markedly reduced compared to controls, suggesting a mitochondrial metabolic dysfunction. Abnormal distribution of mitochondria within maturing oocytes also was seen in diabetic mice. Furthermore, oocytes from diabetic mice displayed a higher frequency of spindle defects and chromosome misalignment in meiosis, resulting in increased aneuploidy rates in ovulated oocytes. Collectively, our results suggest that maternal diabetes results in oocyte defects that are transmitted to the fetus by two routes: first, meiotic spindle and chromatin defects result in non-disjunction leading to embryonic aneuploidy; second, structural and functional abnormalities of oocyte mitochondria, through maternal transmission, provide the embryo with a dysfunctional complement of mitochondria that may be propagated during embryogenesis. The consequences of this abnormal complement of mitochondria is not yet know but may be responsible for the growth abnormalities experienced by offspring of both type 1 and type 2 diabetes. 3 . The concept that certain non-communicable adult diseases, such as hypertension, type 2 diabetes and dyslipidaemia, can originate from events occurring in utero arose from epidemiological studies in humans but has since been supported by numerous animal-based studies, the vast majority of which have been conducted in the sheep and rat where the effects of global nutrient restriction and low protein diets have been investigated. The long-term programming effects of specific dietary nutrients on key epigenetic processes during gametogenesis and pre-implantation development have been the focus of recent investigations at Nottingham. In the sheep we have demonstrated that physiologically relevant reductions in the dietary supply of specific B-vitamiiys (i.e. vitamin B12, folate) and methionine to intending mothers can epigenelically modify DNA in their progeny (determined by Restriction Landmark Genome Scanning) and lead to adult offspring that are obese, have altered immune responses to antigenic challenge, are insulin resistant, and have elevated blood pressure. Intriguingly, these eipigenetic and phenotypic effects are most pronounced in male offspring. Parallel studies in the rat reveal common phenotypic effects which are also male specific. These studies set a precedent for the long-term programming effects of specific micronutrients during gametogenesis and pre-implantation development, emphasizing the need for more basic research into these phenomena so that appropriate nutritional advice can be given to intending mothers and modifications made to existing empirically derived oocyte and embryo culture media. Maternal nutrition around the time of conception in several animal models has been shown to influence the plasticity of the developmental programme leading to altered postnatal phenotype affecting growth, physiological and metabolic parameters, often associated with onset of adult disease. Moreover, culture of preimplantation embryos from different animal species, using procedures practiced in assisted conception treatment and related technologies, can also result in postnatal abnormalities. We have investigated such environmental effects on development in rodent models in particular with a maternal low protein diet administered for the period of preimplantation development after mating, with normal control nutrition provided for the remainder of gestation and postnatally. This protein restriction model has revealed cardiovascular dysfunction, notably relative hypertension and associated disturbance in arterial vessel relaxation potential, behavioural abnormalities, metabolic effects and altered growth in offspring, with evidence of gender-specific sensitivities. Analysis of preimplantation embryos collected from diet-challenged mothers indicate programming of developmental plasticity has occurred by the blastocyst stage. Our results indicate that the extra-embryonic lineages derived from the blastocyst, namely the visceral yolk sac and trophoblast, change their pattern of development in response to maternal diet. These changes in proliferation, behaviour and function induced by maternal low protein diet can be interpreted as compensatory mechanisms to increase the efficiency of subsequent nutrient retrieval and likely to protect foetal growth and stabilise competitive fitness in the next generation. Activation of these compensatory responses and resulting changes in foetal development appear to involve physiological and epigenetic components and are mediated through 'sensing' by the early embryo of the maternal nutrient environment. Collectively, our rodent models provide evidence to support the concept that the developmental origins of adult disorders can be traced back to maternal-embryonic interactions prior to implantation. Small RNAs play central roles in a diversity of cell fate decisions. We have found a key role for small RNAs in embryonic stem (ES) cell self-renewal and differentiation. In the absence of the protein Dgcr8, a RNA binding protein required for the biogenesis of all canonical microRNAs, ES cells show a slowed proliferation rate and an inability to silence self-renewal when induced to differentiate. The former defect is partly due to a family of microRNAs highly enriched in ES cells that maintain the unique cell cycle structure of the self-renewing ES cell. These miRNAs act to suppress the Gl/S restriction point promoting a rapid transition into S phase. These miRNAs are downstream of Myc and they can replace Myc in promoting the de-differentiation of somatic cells to induced pluripotent stem cells. Our findings show a central role for microRNAs in maintaining/promoting key properties of early embryonic sternness. . Small Regulatory RNAs. Eric C. Lai. Memorial Sloan Kettering Cancer Center, New York, NY, USA Recent years have seen phenomenal advances in our understanding of regulatory pathways mediated by small RNA/Argonaute complexes. We seek to define the small regulatory RNAs of Drosophila using high throughput sequencing and bioinformatics, and to probe their biogenesis and function using biochemical and genetic strategies. These efforts yielded not only additional members of the conventional microRNA gene class, but also revealed several novel aspects of microRNA biogenesis and a plethora of endogenous siRNA-generating loci. The existence of these new pathways increases the complexity of the small RNA-mediated regulatory network, raises new questions about the segregation and sorting of RNA substrates through these various pathways, and may potentially lend insight into improving the exploitation of regulatory RNAs for designed purposes. . In eukaryotic cells, RNA-binding proteins play critical roles in transcription and post-transcriptional events. In the testis, the DNA/RNA-binding protein MSY2 is essential for fertility in both male and female mice. MSY2 selectively binds mRNAs that encode stored or translationally-delayed, male gamete-specific transcripts. In the nucleus MSY2 marks these germ cell mRNAs for cytoplasmic storage, thereby linking transcription and mRNA storage in meiotic and post-meiotic germ cells. Among the many classes of small non-coding RNAs are microRNAs and PIWIinteracting RNAs (piRNAs). Applying a cross-linking and immunoprecipitation procedure (the CLIP assay) with an affinity purified antibody to MSY2, we have recently discovered that the MSY2 protein selectively binds a novel population of small testicular RNAs (MSY-RNAs). MSY-RNAs are -26-32 nucleotides, often initiate with a 5' adenine, and are expressed throughout germ cell differentiation and in somatic cells. Although most of the MSY-RNAs are derived from annotated genes, a small number (16 of a total of 230 clones sequenced) are piRNAs. piRNAs are an extremely abundant (>500,000 copies) group of non-coding germ cell RNAs that are evolutionarily conserved, but not sequence conserved. All piRNAs are believed to be processed from long transcripts by a MlWI-dependent mechanism. To our surprise, the piRNAs that selectively bind MSY2 are expressed in mice lacking Miwi and the temporal expression of these piRNAs differs greatly from other known piRNAs. In contrast to piRNAs, most MSY-RNAs are derived from widely distributed (nonclustered) sites. MSY-RNAs are present in both nuclei (enriched in chromatin) and cytoplasm and in both ribonucleoprotein particles and polysomes suggesting multiple cellular functions for this new class of small RNA. In summary, we have a identified a new group of MlWI-independent small RNAs that selectively bind to Y-box proteins such as MSY2 and are expressed in both male germ cell and somatic cells. ABSTRACTS In virtually all metazoan, oocytes become arrested at the prophase of the first meiotic division. This first meiotic arrest may last up to a few years in Xenopus and several decades in humans, and is characterized by synthesis and storage of large quantities of dormant mRNA. When later translated, these maternal mRNAs drive the oocytes' re-entry into meiosis and control the rate of mitosis during the cleavage divisions of the embryo. The resumption of meiosis marks the onset of oocyte maturation. In almost all vertebrates, nuclear and cytoplasmic changes associated with oocyte maturation are completed by the metaphase of the second meiotic division, when oocytes become arrested for a second time and await fertilization. A complex network of translational activation and repression of stored maternal mRNAs accompanies oocyte maturation, while transcription is limited at best. Transcriptional silencing that begins with oocyte maturation persists during the initial mitotic divisions of the embryo. Consequently, gene expression during oocyte maturation, fertilization, and early embryo development until zygotic genome activation (ZGA) is independent of transcription and relies on translational activation of maternally-derived mRNAs. The best-characterized mechanism for translational activation during eary development is cytoplasmic poyadenylation and involves regulatory RNA-binding proteins that interact with 3' untranslated region (3' UTR) of activated mRNAs. Two such proteins, cytoplasmic polyadenylation element binding protein (CPEB) and embryonic poly(A) binding protein (ePAB) play key roles in the regulation of gene expression during early development and are necessary for oogenesis in mammals. . Epigenetic Effects of Manipulating Mouse Oocytes and Preimplantation Embryos. Rocio M. Rivera. University of Missouri, Columbia, MO, USA Genomic imprinting is defined as parent-specific gene expression. Imprinted genes are a small subset of genes found in clusters throughout the mammalian genome. The correct allelic expression of these genes is directed by epigenetic modifications (established during gametogenesis) of the cluster's regulatory region (ICR). These genes are involved in the regulation of growth and development of both fetus and placenta as well as in neurobehavioral processes. Several syndromes exist that are the result of misregulation of imprinted genes. Recent retrospective studies have cautioned of an apparent increase in the incidence of loss-of-imprinting (LOI) syndromes in children conceived through assisted reproductive technologies (ART). In the mouse, we and others have shown that manipulations of gametes (i.e. superovulation) and preimplantation embryos (i.e. culture and/or transfer) can result in LOI. We showed that embryo transfer induced biallelic expression of several imprinted genes in extraembryonic tissues of d9.5 concepti. Furthermore, a superimposed embryo culture treatment caused a more severe misregulation of these genes in extraembryonic tissues. In addition, embryo culture also resulted in LOI in the fetus. Further, expression of the repressed allele of two imprinted genes was associated with loss of methylation at their ICRs. Although a relationship between ART and LOI has been established, the cellular and molecular mechanisms involved in this outcome are not known. Ongoing investigations are aimed at elucidating how each of theses procedures affect global DNA methylation and the expression of proteins involved in regulating the epigenome in oocytes and preimplantation embryos. 0 . Living with the Past: Developmental Origins of Health and Disease. Elena Zambrano. Inst Nac de Ciencias Medicas y Nutricion SZ, Mexico City, Mexico The Developmental Origins of Health and Disease is a concept based on epidemiological, clinical and animal research studies. Organogenesis and life time function depend on gene-environment interactions during critical plastic fetal and early post-natal developmental stages. Previously we have demonstrated that maternal protein restriction (MPR; isocaloric diet with 50% normal protein intake) during fetal development and/or lactation in rats results in delayed sexual maturation and premature aging of reproductive function, delays the natural neonatal leptin peak and adversely affects glucose and insulin metabolism in male and female first and second generation offspring in a gender-specific manner also dependent on the developmental period of exposure. I will present our recent progress on how MPR impacts maternal liver lipid metabolism and its effect on the development of fetal brain. Maternal docosahexanoic acid (DHA) can be obtained directly from diet or by hepatic synthesis from essential fatty acids by desaturases and elongases. Fetal tissues produce only small amounts of long chain polyunsaturated fatty acids (LC-PUFAs) due to a relative lack of desaturases and elongases. Thus the fetal brain depends on the maternal source of LC-PUFAs. Protein restriction significantly reduced maternal liver desaturase and elongase gene expression, and formation of the LC-PUFAs: arachidonic and DHA acids. The fetus from MPR mothers exhibited a low body weight, as well as reduced liver and brain fat, including the content of DHA in the brain. The present study showed that protein restriction during pregnancy negatively impacts normal fetal brain development by changes in maternal lipid metabolism.