Naomi Dicks

Endoplasmic Reticulum Stress, Genome Damage, and Cancer

Endoplasmic reticulum (ER) stress has been linked to many diseases, including cancer. A large body of work has focused on the activation of the ER stress response in cancer cells to facilitate their survival and tumor growth; however, there are some studies suggesting that the ER stress response can also mitigate cancer progression. Despite these contradictions, it is clear that the ER stress response is closely associated with cancer biology. The ER stress response classically encompasses activation of three separate pathways, which are collectively categorized the unfolded protein response (UPR). The UPR has been extensively studied in various cancers and appears to confer a selective advantage to tumor cells to facilitate their enhanced growth and resistance to anti-cancer agents. It has also been shown that ER stress induces chromatin changes, which can also facilitate cell survival. Chromatin remodeling has been linked with many cancers through repression of tumor suppressor and apoptosis genes. Interplay between the classic UPR and genome damage repair mechanisms may have important implications in the transformation process of normal cells into cancer cells.

Efficacy of the porcine species in biomedical research

Since domestication, pigs have been used extensively in agriculture and kept as companion animals. More recently they have been used in biomedical research, given they share many physiological and anatomical similarities with humans. Recent technological advances in assisted reproduction, somatic cell cloning, stem cell culture, genome editing, and transgenesis now enable the creation of unique porcine models of human diseases. Here, we highlight the potential applications and advantages of using pigs, particularly minipigs, as indispensable large animal models in fundamental and clinical research, including the development of therapeutics for inherited and chronic disorders, and cancers.

Relief of endoplasmic reticulum stress enhances DNA damage repair and improves development of pre-implantation embryos

Early-cleaving embryos are known to have better capacity to reach the blastocyst stage and produce better quality embryos compared to late-cleaving embryos. To investigate the significance of endoplasmic reticulum (ER) stress on early embryo cleavage kinetics and development, porcine embryos produced in vitro were separated into early- and late-cleaving groups and then cultured in the absence or presence of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Developing embryos were collected at days 3 to 7 of culture for assessment of ER stress status, incidence of DNA double-strand breaks (DSBs), development and total cell number. In the absence of TUDCA treatment, late-cleaving embryos exhibited ER stress, higher incidence of DNA DSBs, as well as reductions in development to the blastocyst stage and total embryo cell numbers. Treatment of late-cleaving embryos with TUDCA mitigated these effects and markedly improved embryo quality and development. These results demonstrate the importance of stress coping responses in early developing embryos, and that reduction of ER stress is a potential means to improve embryo quality and developmental competence.

Somatic Cell Nuclear Transfer and the Creation of Transgenic Large Animal Models

Transgenic animals have been instrumental in biomedical and genetic research, particularly through the use of genetically modified mice. To facilitate translational research to humans, the development of larger species of transgenic animals is necessary. Furthermore, these transgenic large animal models have numerous other applications in biomedicine and agriculture. Various techniques to produce genetically altered animals have been developed throughout the years including pronuclear microinjection, sperm-mediated gene transfer (SMGT), electroporation, oocyte/embryo transduction and somatic cell nuclear transfer (SCNT). A major advantage of the SCNT technique is the ability to genetically modify donor cells and select for these transgenic cells prior to the cloning procedure, resulting in 100 % transgenic efficiency. This has particularly facilitated the production of transgenic large animals, for which embryonic stem cells that can be cultured and reliably modified in the laboratory are not fully established. In addition, new gene-editing technologies, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have further assisted the creation of transgenic animals, notably gene-disrupted animals. Demand for these large transgenic animal models is increasing as a result of their numerous possible applications including the development of higher quality production animals, creation of stem cells for tissue repair (therapeutic cloning), production of protein-based pharmaceuticals (animal pharming), creation of organ donors for xenotransplantation and the creation of large animal models for biomedical research.

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis: MICHALOVIC et al.

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus–oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle‐stimulating hormone [FSH] treated and three untreated) and eight FSH‐treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real‐time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

Double-strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos

DNA double‐strand breaks (DSBs) are less frequent than single‐strand breaks but have more harmful consequences on cell survival and physiology. Homologous recombination (HR) and nonhomologous end‐joining (NHEJ) are the two main pathways that are responsible for DSB repair in eukaryotic cells, but their importance for the preservation of genome stability in totipotent blastomeres of early developing embryos has not been determined. In this study, we observed that the chemical inhibition of HR or both pathways, but not NHEJ alone, increased the number of DSBs, reduced embryo development to the blastocyst stage, and resulted in embryos with higher proportions of apoptotic cells. Targeted knockdown of ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related; HR regulators) and DNA‐dependent protein kinase (NHEJ regulator) mRNAs revealed that the attenuation of HR or both HR and NHEJ regulators severely impaired blastocyst formation and quality. Attenuation of ATM alone resulted in a higher incidence of DSBs, lower development and embryo quality, and increased mRNA abundance of genes that are involved in either repair pathway. These findings indicate that HR is the main pathway responsible for the promotion of DSB repair in early developing embryos, and that ATM seems to be more important than ATR in the regulationofthe HR pathwayinmammalianembryos.— Bohrer, R. C., Dicks, N., Gutierrez, K., Duggavathi, R., Bordignon, V. Double‐strand DNA breaks are mainly repaired by the homologous recombination pathway in early developing swine embryos. FASEB J. 32, 1818–1829 (2018). www.fasebj.org

70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9)+1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9+1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9+sgRNA-1+sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1+sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Students t-test or Tukey-Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a=59.8±4.9%, b=58.8±5.3%, c=68.86±2.2%, d=66.4±5.9%, and e=70.10±1.9%), but development to the blastocyst stage was substantially reduced (P<0.05) in the groups injected with Cas9+sgRNAs (a=18±4.5%, b=16±1.5%, and c=5.3±2.8%) compared with controls (d=33.7±6.2% and e=31.4±1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9+sgRNA-1+sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.