- Email: [email protected]
8. Understanding cryoinjuries: Effect of cryopreservation on expression of imprinted genes related to birth defects
Abstracts / Cryobiology 65 (2012) 339–366 183–192). Diffractographic analysis was applied and compared with respective profile curves: mean FAM values vs. mean GAM and CAM values calculating OPC values according with Perez Campos et al. (Transplantation Proceedings 40 (2008) 668–674). According to Bragg’s Law, the comparative distance between the planes of the crystalline design of collagen mesh (d space) was calculated relating to each procedure vs. FAM. Additional Raman scattering comparative considerations were obtained. Results: Both GAM and CAM diffractographic curves had the same design related to FAM. However, respective OPC values (FAM vs. GAM = 14.76; and FAM vs. CAM = 42.02) show a higher relative collagen mesh ordering in GAM respective CAM comparing with control FAM. But when comparing the maximum diffractive peak GAM and CAM vs. FAM there was a mismatch with GAM samples of 0.4° in the incidental X-Ray angle: two theta values (h) of Bragg’s Law. On the contrary no mismatch was observed in CAM samples related with FAM. Comparative Raman spectra profiles between FAM and CAM showed punctual differences at ranges of 1260, 1442, and 1667 cm 1 where increased relative intensity values in FAM related to CAM were observed. Conclusions: X-ray diffraction profiles show higher relative ordering of GAM related to CAM but glycerolization modified the structural crystalline design of collagen mesh. Cryopreservation modifies fibril collagen of human amnion I, III and V in chemical residues measured with Raman spectra technique, according to Frank, C. & McCreey, R. (Anal. Chem. 67 (1995) 777–783), and Frushour & Koenig. (Biopolymers 14 (1975) 363–377). Conflict of interest: None declared. Source of funding: Supported by CSIC and Pedeciba. E-mail address: [email protected] (H.P. Campos) http://dx.doi.org/10.1016/j.cryobiol.2012.07.008
8. Understanding cryoinjuries: Effect of cryopreservation on expression of imprinted genes related to birth defects. A. Baer, E. Szurek, L.C. Layman, A. Eroglu, Institute of Molecular Medicine and Genetics, Department of Medicine, Department of OB/GYN, The Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA Assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and embryo cryopreservation became an indispensible part of infertility treatments. Currently, ARTs account for up to 3% of annual births in industrialized countries and are considered safe. However, recent studies reported an increased incidence of epigenetic birth defects among children conceived by ARTs. Since the timing of ARTs coincides with re-establishment of epigenetic marks (genomic imprinting) during gametogenesis and embryogenesis, it is likely that suboptimal ARTs may alter normal methylation patterns, and thus expression of imprinted genes, leading to epigenetic birth defects. The objective of this study was to investigate the effect of oocyte cryopreservation on expression of the imprinted genes involved in epigenetic birth defects. To this end, mouse metaphase II (M II) oocytes were cryopreserved by using either standard slow freezing (i.e., cooling to 35 °C at 0.3 °C/min in 1.5 M Me2SO + 0.1 M sucrose, and then plunging into liquid nitrogen [LN2]) or vitrification technique (i.e., plunging of oocytes within a 6 1-ml volume of 15% Me2SO + 15% ethylene glycol + 0.5 M Sucrose into LN2). After warming at 37 °C and removal of cryoprotectants by stepwise dilution, cryopreserved oocytes along with untreated controls were inseminated and cultured to the blastocyst stage to determine their gene expression levels. To do so, blastocysts derived from cryopreserved and control oocytes were subjected first to reverse transcription and subsequently to real-time PCR. Gene expression levels in three replicates of experiments were analyzed by the comparative CT method using Rest Software. So far, our results suggest that both cryopreservation techniques downregulate the expression level of two imprinted genes (i.e., H19, and Ube3a). These findings warrant further studies to address the effect of oocyte cryopreservation on imprinted genes. Conflict of interest: The authors declare no conflict of interest related to this work. Source of funding: This study was supported by an extramural grant from the National Institutes of Health (Grant No. R01HD049537 from the NICHD) to A.E. and an intramural award from Georgia Health Sciences University (IRP00003) to A.E. and L.L. E-mail address: [email protected] (A. Eroglu) http://dx.doi.org/10.1016/j.cryobiol.2012.07.009
How to do it? Workshops
9. Proteomics analysis. Germán L. Rosano, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET Facultad de Ciencias Bioquı´micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina
341
The proteome is the full set of proteins expressed in a cell or organism at a given time and condition. Proteomics analysis provides a snapshot of the proteome and allows the identification and quantification of virtually any protein. Also, proteome variation under different conditions can be detected (comparative proteomics). Proteomics techniques can be divided between gel-based methods (2D electrophoresis, 2DE) and mass spectrometry (MS)-based methods. 2D electrophoresis is the workhorse of proteomics, with an unparalleled resolution and ease of use. The workflow of a 2DE protocol is simple regardless of the origin of the sample. Although technically straightforward and with superior resolution, 2DE is not without pitfalls and limitations. A 2DE analysis may not detect low-abundant proteins and hydrophobic membrane proteins. Moreover, reproducibility among laboratories is still an issue. On the other hand, there have been major advances in MS-based techniques in the last decade. Combined with the resolving power of liquid chromatography, it is possible to detect scarce and abundant proteins in complex mixtures simultaneously. If cells are subjected to different treatments, then the proteomes can be compared by the newly developed techniques of ICAT, SILAC and iTRAQ. When absolute quantification is needed, an AQUA protocol should be considered. However, the expensive equipment required is a high-entry barrier for most laboratories. Also, MS-based technologies produce great loads of raw data. Its processing and analysis is still a major bottleneck. Correct sample preparation is essential to both groups of techniques. Protein extraction, avoidance of proteolysis, sample buffer formulation and elimination of impurities are key steps that need to be perfected before running a 2DE or an MS protocol. Only by standardizing and refining these procedures, then the results can be considered reliable. Despite some issues that need to be overcome, proteomics has reached a level of maturity comparable to other ‘‘omics”, such as genomics and transcriptomics. Advances in instrumentation have allowed high throughput analysis with high resolution and sensitivity in shorter times. Thanks to this, proteomics analysis is living in a golden era, making a deep impact in biological research, especially in systems biology. Conflict of interest: None declared. Source of funding: None declared. E-mail address: [email protected] (G.L. Rosano) http://dx.doi.org/10.1016/j.cryobiol.2012.07.010
10. Stem cells for clinical use. Patricia Pranke, Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul and Stem Cell Research Institute. Porto Alegre, RS, Brazil Mesenchymal stem cells (MSCs) are cells derived from adult tissue found in various locations, such as bone marrow, adipose tissue, dental pulp tissue and umbilical cord. The MSCs are pluripotent cells and are therefore capable of differentiation into all types of cells such as osteoblasts, chondrocytes, adipocytes and cells in the nervous system. Electrospinning (ES) is an attractive method used in bioengineering to produce nanofibrous scaffolds that mimick the native extracellular matrix, serving as supports for cell growth. The use of polymeric matrices or scaffolds composed of nanofibers produced by electrospinning (ES) can serve as templates in which the cells can adhere, proliferate and differentiate, contributing to the development of a new tissue. The cell growth in the scaffolds can be stimulated through the controlled release of bioactive molecules, such as growth factors, which thereby enhance tissue regeneration. Conflict of interest: None declared. Source of funding: None declared. E-mail address: [email protected] (P. Pranke) http://dx.doi.org/10.1016/j.cryobiol.2012.07.011
11. Vitrification of bovine oocytes and embryos. G. Dalvit, C. Gutnisky, G. Alvarez, P. Cetica, School of Veterinary Sciences, University of Buenos Aires, Argentina Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology. Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this work is to challenge vitrification
8. Understanding cryoinjuries: Effect of cryopreservation on expression of imprinted genes related to birth defects. A. Baer, E. Szurek, L.C. Layman, A. Eroglu, Institute of Molecular Medicine and Genetics, Department of Medicine, Department of OB/GYN, The Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA Assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and embryo cryopreservation became an indispensible part of infertility treatments. Currently, ARTs account for up to 3% of annual births in industrialized countries and are considered safe. However, recent studies reported an increased incidence of epigenetic birth defects among children conceived by ARTs. Since the timing of ARTs coincides with re-establishment of epigenetic marks (genomic imprinting) during gametogenesis and embryogenesis, it is likely that suboptimal ARTs may alter normal methylation patterns, and thus expression of imprinted genes, leading to epigenetic birth defects. The objective of this study was to investigate the effect of oocyte cryopreservation on expression of the imprinted genes involved in epigenetic birth defects. To this end, mouse metaphase II (M II) oocytes were cryopreserved by using either standard slow freezing (i.e., cooling to 35 °C at 0.3 °C/min in 1.5 M Me2SO + 0.1 M sucrose, and then plunging into liquid nitrogen [LN2]) or vitrification technique (i.e., plunging of oocytes within a 6 1-ml volume of 15% Me2SO + 15% ethylene glycol + 0.5 M Sucrose into LN2). After warming at 37 °C and removal of cryoprotectants by stepwise dilution, cryopreserved oocytes along with untreated controls were inseminated and cultured to the blastocyst stage to determine their gene expression levels. To do so, blastocysts derived from cryopreserved and control oocytes were subjected first to reverse transcription and subsequently to real-time PCR. Gene expression levels in three replicates of experiments were analyzed by the comparative CT method using Rest Software. So far, our results suggest that both cryopreservation techniques downregulate the expression level of two imprinted genes (i.e., H19, and Ube3a). These findings warrant further studies to address the effect of oocyte cryopreservation on imprinted genes. Conflict of interest: The authors declare no conflict of interest related to this work. Source of funding: This study was supported by an extramural grant from the National Institutes of Health (Grant No. R01HD049537 from the NICHD) to A.E. and an intramural award from Georgia Health Sciences University (IRP00003) to A.E. and L.L. E-mail address: [email protected] (A. Eroglu) http://dx.doi.org/10.1016/j.cryobiol.2012.07.009
How to do it? Workshops
9. Proteomics analysis. Germán L. Rosano, Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET Facultad de Ciencias Bioquı´micas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina
341
The proteome is the full set of proteins expressed in a cell or organism at a given time and condition. Proteomics analysis provides a snapshot of the proteome and allows the identification and quantification of virtually any protein. Also, proteome variation under different conditions can be detected (comparative proteomics). Proteomics techniques can be divided between gel-based methods (2D electrophoresis, 2DE) and mass spectrometry (MS)-based methods. 2D electrophoresis is the workhorse of proteomics, with an unparalleled resolution and ease of use. The workflow of a 2DE protocol is simple regardless of the origin of the sample. Although technically straightforward and with superior resolution, 2DE is not without pitfalls and limitations. A 2DE analysis may not detect low-abundant proteins and hydrophobic membrane proteins. Moreover, reproducibility among laboratories is still an issue. On the other hand, there have been major advances in MS-based techniques in the last decade. Combined with the resolving power of liquid chromatography, it is possible to detect scarce and abundant proteins in complex mixtures simultaneously. If cells are subjected to different treatments, then the proteomes can be compared by the newly developed techniques of ICAT, SILAC and iTRAQ. When absolute quantification is needed, an AQUA protocol should be considered. However, the expensive equipment required is a high-entry barrier for most laboratories. Also, MS-based technologies produce great loads of raw data. Its processing and analysis is still a major bottleneck. Correct sample preparation is essential to both groups of techniques. Protein extraction, avoidance of proteolysis, sample buffer formulation and elimination of impurities are key steps that need to be perfected before running a 2DE or an MS protocol. Only by standardizing and refining these procedures, then the results can be considered reliable. Despite some issues that need to be overcome, proteomics has reached a level of maturity comparable to other ‘‘omics”, such as genomics and transcriptomics. Advances in instrumentation have allowed high throughput analysis with high resolution and sensitivity in shorter times. Thanks to this, proteomics analysis is living in a golden era, making a deep impact in biological research, especially in systems biology. Conflict of interest: None declared. Source of funding: None declared. E-mail address: [email protected] (G.L. Rosano) http://dx.doi.org/10.1016/j.cryobiol.2012.07.010
10. Stem cells for clinical use. Patricia Pranke, Hematology and Stem Cell Laboratory, Faculty of Pharmacy, Federal University of Rio Grande do Sul and Stem Cell Research Institute. Porto Alegre, RS, Brazil Mesenchymal stem cells (MSCs) are cells derived from adult tissue found in various locations, such as bone marrow, adipose tissue, dental pulp tissue and umbilical cord. The MSCs are pluripotent cells and are therefore capable of differentiation into all types of cells such as osteoblasts, chondrocytes, adipocytes and cells in the nervous system. Electrospinning (ES) is an attractive method used in bioengineering to produce nanofibrous scaffolds that mimick the native extracellular matrix, serving as supports for cell growth. The use of polymeric matrices or scaffolds composed of nanofibers produced by electrospinning (ES) can serve as templates in which the cells can adhere, proliferate and differentiate, contributing to the development of a new tissue. The cell growth in the scaffolds can be stimulated through the controlled release of bioactive molecules, such as growth factors, which thereby enhance tissue regeneration. Conflict of interest: None declared. Source of funding: None declared. E-mail address: [email protected] (P. Pranke) http://dx.doi.org/10.1016/j.cryobiol.2012.07.011
11. Vitrification of bovine oocytes and embryos. G. Dalvit, C. Gutnisky, G. Alvarez, P. Cetica, School of Veterinary Sciences, University of Buenos Aires, Argentina Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology. Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this work is to challenge vitrification