Assisted Reproductive Technologies Disrupt Genomic Imprinting in Human and Mitochondria in Mouse Embryos

Infertile couples worldwide use assisted reproductive technologies (ARTs) to help conceive their own biological child. Due to the rising use of ARTs, there is continual emergence of new techniques implemented in human fertility clinics. When treatment is successful, there is an increased risk even within singletons for perinatal complications including preterm birth, intrauterine growth restriction, low and high birth weight and genomic imprinting disorders Beckwith Wiedemann Syndrome, Angelman Syndrome, and Silver-Russel Syndrome. Consequently, there is a need to investigate the effects of these treatments on the manipulated oocyte and preimplantation embryo. To address this, I first analyzed the combined effects of multiple ARTs on imprinted DNA methylation in human day 3 (6 to 8 cells) and blastocyst-stage embryos. As imprinted DNA methylation is acquired during gametogenesis and maintained throughout preimplantation development, I hypothesized that ARTs disrupt this regulation in donated, good quality, human preimplantation embryos. I observed that seventy-six percent of day 3 embryos and fifty percent of blastocysts exhibited perturbed imprinted methylation at the SNRPN, KCNQ1OT1 and/or H19 domains. This frequency was similar to that previously observed in the mouse, and importantly demonstrated that extended culture did not pose a greater risk for imprinting errors. Overall, human preimplantation embryos generated with ARTs possessed a high frequency of imprinted methylation errors. Next, I hypothesized that a single, indispensible ART treatment, ovarian stimulation, disrupts mitochondria in mouse oocytes and preimplantation embryos. Ovarian stimulation led to a decreased total and active mitochondrial pool in high hormone-treated oocytes, and an increase in the percentage of oocytes displaying mislocalization of active mitochondria. Although the total mitochondrial pool was unchanged in hormone-treated preimplantation embryos compared to controls, the active mitochondrial pool was decreased in hormone-treated 1-cell, 2-cell, morula and blastocysts. Ultimately, the lower active mitochondrial pool in treated embryos was associated with a decreased percentage of outer blastomeres containing high amounts of active mitochondria in morula and blastocysts. In blastocysts, this was associated with increased superoxide levels. Overall, my results provide novel insight onto ARTs-induced disruption of imprinted DNA methylation and mitochondria in human and mouse preimplantation embryos, respectively

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant

Why we should not select the faster embryo: lessons from mice and cattle

Many studies have shown that in vitro culture can negatively impact preimplantation development. This necessitates some selection criteria for identifying the best-suited embryos for transfer. That said, embryo selection after in vitro culture remains a subjective process in most mammalian species, including cows, mice and humans. General consensus in the field is that embryos that develop in a timely manner have the highest developmental competence and viability after transfer. Herein lies the key question: what is a timely manner? With emerging data in bovine and mouse supporting increased developmental competency in embryos with moderate rates of development, it is time to question whether the fastest developing embryos are the best embryos for transfer in the human clinic. This is especially relevant to epigenetic gene regulation, including genomic imprinting, where faster developing embryos exhibit loss of imprinted methylation, as well as to sex selection bias, where faster developmental rates of male embryos may lead to biased embryo transfer and, in turn, biased sex ratios. In this review, we explore evidence surrounding the question of developmental timing as it relates to bovine embryo quality, mouse embryo quality and genomic imprint maintenance, and embryo sex

A role for chromatin topology in imprinted domain regulation

Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically-associated domains, lamin-associated domains, nucleolar-associated domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

EyasSAT: a Revolution in Teaching and Learning Space Systems Engineering

This conference features the work of authors from: Georgia Tech’s Space Systems Design Lab, Aerospace Systems Design Lab, School of Aerospace Engineering, Georgia Tech Research Institute; NASA’s Jet Propulsion Laboratory, Marshall Space Flight Center, Goddard Space Flight Center, Langley Research Center; and other aerospace industry and academic institutionsEyasSAT (patent pending) has transformed the spacecraft systems engineering teaching and learning experience. This new development is a fully functional nanosatellite project that is built up, tested, and “flown” in the classroom. EyasSAT has been used in various space education programs involving high school, undergraduate, and professional students. The overall concept and results from two years of experience are presented.AIAA Space Systems Technical Committee ; AIAA Space Transportation Systems Technical Committee ; Space Technology Advanced Research Cente

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20-80 μm control oocytes at three imprinted genes, Snrpn, Peg3 and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition, individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3 or Peg1 in oocytes from Ers2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4-deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences.

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant

An RB-EZH2 Complex Mediates Silencing of Repetitive DNA Sequences

Repetitive genomic regions include tandem sequence repeats and interspersed repeats, such as endogenous retroviruses and LINE-1 elements. Repressive heterochromatin domains silence expression of these sequences through mechanisms that remain poorly understood. Here, we present evidence that the retinoblastoma protein (pRB) utilizes a cell-cycle-independent interaction with E2F1 to recruit enhancer of zeste homolog 2 (EZH2) to diverse repeat sequences. These include simple repeats, satellites, LINEs, and endogenous retroviruses as well as transposon fragments. We generated a mutant mouse strain carrying an F832A mutation in Rb1 that is defective for recruitment to repetitive sequences. Loss of pRB-EZH2 complexes from repeats disperses H3K27me3 from these genomic locations and permits repeat expression. Consistent with maintenance of H3K27me3 at the Hox clusters, these mice are developmentally normal. However, susceptibility to lymphoma suggests that pRB-EZH2 recruitment to repetitive elements may be cancer relevant