Study of gene expression with molecular biological methods during mammalian embryo preimplantation development. Árpád Baji Gál

Transcription

1 Study of gene expression with molecular biological methods during mammalian embryo preimplantation development PhD theses Árpád Baji Gál Doctoral School in Biology, Head of the School: Prof. Anna Erdei Structural Biochemistry Doctoral Program, Head of the Program: DSc. Prof. László Gráf Faculty of Science, Eötvös Loránd University Supervisor: DSc. András Dinnyés Scientific Advisor, Group Leader Genetic Reprogramming Group Agricultural Biotechnology Center, Gödöllő May 2008

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3 Introduction Understanding the molecular mechanisms that drive cellular processes during animal development has been the aim of developmental biology research for many years; yet the field has only recently whole heartedly embraced molecular and cell-biological approaches that complement its traditional repertoire of genetic and embryology techniques. As a result, modern developmental biology now finds itself at the interface between many other disciplines, as new biochemical, genomic and systems approaches are developed and implemented. There is now a reasonable amount of evidence from studies that information in addition to the primary DNA sequence is inherited across generations and can influence the phenotype of the offspring. Researchers refer to this information as epigenetic, and there is much interest in discovering the molecular basis for this epigenetic information. From the various types of epigenetic marks that regulate the expression of the genome within the life of an organism, are specific modifications of the proteins that package the DNA into chromosomes, termed chromatin. The basic repeating unit of chromatin, nucleosome, is formed by wrapping roughly two superhelical turns of DNA (146 base pair) around a core histone octamer composed of two copies of each histone H2A, H2B, H3 and H4. The organization of the higher-order chromatin structure has been linked to post-translational covalent modifications of the protruding N-terminal and C-terminal tails of the core histones. Dynamic transitions in chromatin structure are controlled by non-histone chromosomal proteins. As part of the hypothesized histone code, overall levels of histone acetylation of the genome are determined by the combined activities of histone acetyltransferases (HATs) and deacetylases (HDACs), acting as a central switch between transcriptionally active and silent states. During mammalian embryo preimplantation development, a number of key molecular events take place, including the embryonic genome activation (EGA) and the first cell differentiation among others. The genetic information of the two gametes and their epigenetic regulation like DNA methylation, genomic imprinting, chromatin remodeling and histone modifications stand behind them, which together control embryonic developmental program through gene expression. Currently, modern embryo technologies can substitute natural fertilization and these unnatural (in vitro) interventions can induce embryo development and epigenetic reprogramming of the genome. Therefore, molecular studies of the gene expression on different types of embryos (in vitro produced, vitrified, parthenogenetically activated, cloned) will help to improve their viability and the efficiency of the techniques, to solve problems inherent in nuclear transfer. Moreover, they will bring new knowledge underlying causes of developmental disorders and help in practical applications (reproductive biology, cell therapy, regenerative curing, drug research and development), which all play an important role in modern biotechnological researches. Among the different molecular biology tools, the quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr) addresses the evident requirement for quantitative data analysis of gene expression in molecular medicine, diagnostics, microbiology and biotechnology and has become the method of choice for the quantification of mrna. 2

4 Aims of the study The overall aim of the Genetic Reprogramming Group of Agricultural Biotechnology Center in Gödöllő, Hungary is to establish, develop and optimize different embryological techniques, such as in vitro embryo production, parthenogenetic activation, vitrification, embryonic stem sell culture and somatic cell nuclear transfer (cloning) in mammals. For successful implementation of its goals, monitoring expression of genes during preimplantation development, embryonic genom activaton, epigenetic reprogramming and pluripotency maintenance is vital. These aims were promoted by my experiments described in the thesis work from molecular biological point of view. Aims of my studies are listed below. (1) Development of a new sesitive protocol which is able to detect gene expression from individual cells and embryos, as opposed to the usual analyses, from mean values of the pooled samples. I undertook the comparison of end-point RT-PCR and real-time RT-PCR methods, development and characterization of a full gene expression protocol and measuring gene expression differences of individual blastomer cells in 4- cell and 8-cell stage mouse embryos. (2) Comparison of solid surface vitrification (SSV) and in-straw vitrification (ISV) methods and their effects on gene expression before and after EGA in mouse. (3) Study of housekeeping gene expression in in vivo, in vitro and parthenogenetically activated mouse embryos and selection of reference genes suitable for normalization and further data analyses. (4) Selection of the best reference housekeeping genes in rabbit in vivo and in vitro embryos. Identification and characterization of two pluripotency marker genes, Pou5f1 (Oct-4) and Nanog. (5) Comparative analysis of gene expression in mouse and bovine preimplantation embryos in relation to minor genome activation and EGA. (6) Detailed examination of the expression patterns of the different HATs and HDACs genes in mouse oocytes and embryos. It would be a big implementation to select that genes, which are involved actively in molecular processes of reprogramming and EGA during preimplantation development. 3

5 Methods The embryo techniques used in the experiments are listed below: in vivo oocyte collection, in vivo embryo collection, in vitro embryo culture, embryo vitrification, separation of blastomer cells, parthenogenetic activation. I applied the following molecular methods on the oocyte and embryo samples: total RNA isolation, mrna isolation, reverse transcription, polymerase chain reaction, agarose gel electrophoresis, real-time polymerase chain reaction, primer design, quantitative PCR data analysis, gene expression stability analysis, other standard molecular methods. Generally, real-time polymerase chain reaction, primer design and quantitative PCR data analysis have vital importance from all methods described here for the creation of the sensitive gene expression protocol and generating results for different applications. 4

6 Theses 1(a) The results of comparison of the two methods indicate that the real-time RT-PCR is two orders of magnitude more sensitive than end-point RT-PCR which can be compensated by pooling embryos or using a larger sample of cdna template for weakly expressed genes. However, for individual early stage embryos or blastomer cells, only real-time RT-PCR would give quantitative results. There can be no doubt that the real-time RT-PCR assay is significantly less variable and even more precise (CV = 19 %) than endpoint RT-PCR procedure (CV = 38 %). 1(b) Based on this real-time RT-PCR, a full gene expression protocol was developed and standardized, including oocyte and embryo collection, mrna isolation, reverse transcription and quantitative data analysis. Moreover, the sensitivity and suitability of the system was also characterized. Real-time PCR results have shown that the variability of three processes together has two main components: PCR alone gives a big part of the whole assay variance and mrna preparation contributes an equivalent amount of additional variability, while RT step makes no significant contribution to variability. I compared for the first time quantitative real-time RT-PCR assay variability with the biological variation of mouse embryos. In comparing various individual embryos of the same developmental stage, the mrna levels were much different between them. Therefore, I could assume that biological differences were much greater than the methodological variance in my experiments, which confirms that the measured variances originated from real biological differences, not from technical errors. I would like to emphasize, however, that qrt-pcr data constitute only a snapshot of information regarding the quantity of a given transcript in a cell, embryo or tissue. 1(c) In verifying the protocol, it was found that Pou5f1 and Nanog mrna levels have remarkable discrete differences within the blastomers of a single 4-cell stage mouse embryo. Similarly, in blastomers of 8-cell stage embryos expression of Nanog gene showed notable differences between them. These differences might be a very early sign of cell fate determination. 2(a) There is a lack of information on vitrification induced subtle changes at molecular level. In 1-cell stage mouse embryos, they showed higher activity of stress related genes in in-straw treated embryos at 3 hr postwarming than SSV-treated and control ones, while almost the same level as SSV-treated embryos at 10 hr post-warming and in blastocyst stage. It is interesting that expression of the stress related genes in this study were induced by vitrification procedures before the EGA. This might be an early signal of reduced viability following in-straw vitrification indicated by the developmental data. 2(b) In contrast to 1-cell stage embryos, there was no clear pattern of gene expression changes after vitrification of 8-cell stage embryos. This might result from compensation of gene expression among blastomers and from the fully active embryonic genome, making gene expression changes in this stage much more complex. It is possible that patterns can be found as well by detecting more genes or in individual blastomer cells at this stage. Although in vitro development of SSV-treated embryos did not differ from 5

7 control embryos, subtle change was observed in gene expression in this case. The developmental data showed that SSV was more efficient than in-straw technique. 2(c) The result of Actb gene expression in 8-cell stage showed that Actb is not ideal to be a reference gene for cryopreservation experiments, as in-straw vitrification induced gene expression changes. Higher concentration of cryoprotectant that was used in in-straw method could have affected microfilament structures of the blastomers. I have showed that Actb is not the ideal housekeeping gene for further studies in gene expression after cryopreservation and the need to search for stable ones. 3(a) One of the interesting observations was the transcript variation of housekeeping genes between the early and late 2-cell stage mouse embryos during EGA. In most studies so far, 2-cell stage was taken in a holistic analysis without due regard for the different time courses. Thus, compared to other stages, the fractional analysis of transcripts at this stage is important, as it enables to identify the most stable genes for normalization even under major transcript shifts during development. 3(b) The in vitro cultured embryos showed higher expression levels until the 8-cell stage, and lower expression levels at the blastocyst stages compared to in vivo control. However, parthenogenetic activation has little effect on expression of different housekeeping genes, and this has been described in mouse embryos for the first time. As activation of the oocytes is part of the nuclear transfer and cloning, the parthenogenetic embryos would serve as controls in future experiments. 3(c) The results have finally enabled us to select the most stable reference genes to be considered for normalization of gene expression in mouse preimplantation stage embryos. The relatively stable expression of the three genes (H2afz, Hprt1 and Ppia) throughout the different preimplantation stages was further verified at the late 2-cell stage, when the transcripts of most other genes were repressed, yet these genes showed a stable expression in all three sample groups. This approach had big advantage to select the appropriate housekeeping genes focused on the use of multiple individual embryos as compared to pooled samples. I calculated the geometric averages of the above three genes for mouse preimplantation stage gene expression result normalizations. It is important to note that these normalization factors are suitable only in case of using the same methods, the same gene expression protocol and the same embryo stages and culture conditions. The frequently used Actb gene was the worst performing gene and my earlier vitrification study revealed this fact as well. 4(a) Similar to the mouse case, I selected 10 reference genes and compared their expression in the rabbit oocytes and different preimplantation stage embryos that were derived in vivo or produced in vitro. This study, for the first time, revealed a detailed reference gene validation and selection for future rabbit embryo studies. In general, most of the genes had the respective lowest levels at the 8-cell stage. This transcript depression at the 8-cell stage coincides with the lag in development occurring during the EGA in rabbit embryos. 6

8 4(b) Variations of gene stability values between the in vivo and in vitro comparisons were narrower than in the earlier mouse study. This may indicate better in vitro culture conditions of rabbit embryos than the in vitro mouse embryo culture. Interestingly, the gene Actb showed wider stability range between the embryo types with better stability in the in vivo samples compared to the in vitro ones, which clearly indicated the inappropriateness of this genes for normalization in rabbit. After thorough analyses, the three most stable reference genes (H2afz, Hprt1 and Ywhaz) were selected and normalization factors were calculated (from the geometric averages of the three selected genes) for further gene expression studies in preimplantation stage rabbit embryos. 4(c) In rabbit experiments I could identify for the first time the Pou5f1 and Nanog genes and characterize the expression pattern of the Pou5f1 in rabbit tissues and different preimplantation stage embryos. Based on analyses, Pou5f1 was expressed in all developmental stages with various signal intensities. Comparatively higher expression levels were observed at the oocyte and zygote stages, and declined gradually to the lowest levels at the 8-cell stage during the EGA. However, starting from the morula stage, the levels increased continuously. In the meantime, the complete Pou5f1 sequence has been described in rabbit following the human genom project. Comparison of my identified Pou5f1 fragment showed perfect alignment to that sequence, which also confirmed my results. In the case of Nanog, I found several pseudogenes published in mouse and human and because of the sequence variances I stayed further analysis in rabbit. 5 The expression of selected genes described for bovine embryos showed the expected pattern in relation to minor genome activation and EGA. However, this profile has less decided difference in mouse. The observed differences might be species-specific alterations. On the other hand, it was not so evident to separate the minor genome activation from EGA in gene expression, as the EGA comes early, at the 2-cell stage in mouse. For further refinement of the results, I took into the experiment more 1-cell stage mouse embryos collected at different time points, the analyses of which is currently in progress. 6 I determined for the first time the overall patterns of all known HDACs and some selected HATs gene expression during mouse preimplantation development and EGA in details. One of the most interesting observations was that almost all genes were presented actively in the oocytes and they showed specific expression pattern. Some of the HDACs were expressed during minor genome activation, while others only during the EGA. This might reveal, that the different HDAC genes are strictly regulated spatially and temporally, and change during preimplantation development. However, there might be functional gene redundancy among them and one HDAC gene can attend to the specific function of the missing one. Most of the HAT genes were expressed in oocytes and they silenced in later stages, except two of them, which were actively presented during the EGA. 7

9 Conclusions (1) I successfully worked out a full sensitive real-time RT-PCR protocol for studying gene expression quantitatively which makes it suitable to determine mrna level of genes simultaneously even from single cells and embryos. With its application I measured effects of the embryo technologies and selected genes expressed during preimplantation development. I could presumably measure a very early sign of cell fate determination in blastomers of 4-cell and 8-cell stage mouse embryos. (2) Comparison of solid surface vitrification and in-straw vitrification revealed direct correlation between expression of the studied genes, and morphological alterations and survival rates of the mouse embryos. (3) I determined the most stable housekeeping genes in in vivo, in vitro cultured and parthenogenetically activated mouse embryos that are suitable as reference genes for normalization in future studies: H2afz, Hprt1 and Ppia. I could show that parthenogenetic activation has little effect on housekeeping gene expression, which makes these embryos suitable control for somatic cell nuclear transfer. (4) For rabbit, I selected H2afz, Hprt1 and Ywhaz as reference genes for the in vivo and in vitro samples. Moreover, I also identified and detected successfully the transcription factor Pou5f1 (Oct-4) during rabbit preimplantation development. (5) From comparison of bovine and mouse gene expression changes during preimplantation development and EGA, I identified species-specific differences which point out limitations of the mouse as general animal model. Results from different species may reveal the variances of molecular processes in reprogramming, EGA and evolutionary alterations. (6) I studied extensively the regulation of histone acetylation mechanism and detected several HDACs and HATs simultaneously in mouse preimplantation stage embryos, which demonstrates that not only individual genes, but well-regulated and orchestrated expressions are responsible for molecular reprogramming and EGA. However, understanding their particular function requires further extensive studies. In summary, I could establish successfully the molecular background for single cell analyses and could get information about several embryo techniques by the developed gene expression protocol. Results of my experiments in this thesis work validates the basic principles of the method for multiparameter analysis and provides proof of its broad spectrum of applications. However, it is evident that recognition and operation of epigenetic processes during mammalian preimplantation development and failures during the desease processes require notable efforts in the future. 8

10 Publications Publications concerning this thesis work Gal AB, Carnwath JW, Dinnyes A, Herrmann D, Niemann H, Wrenzycki C. (2006) Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos. Reprod Fertil Dev.; 18(3): IF: 1,086 (2003); 2,541 (2006) Boonkusol D, Gal AB, Bodo S, Gorhony B, Kitiyanant Y, Dinnyes A. (2006) Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos. Mol Reprod Dev.; 73(6): IF: 2,543 (2003); 2,379 (2006) Mamo S, Baji Gal A, Bodo S, Dinnyes A. (2007) Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro. BMC Dev Biol.; 7: 14. IF: - (2003); IF: 3,5 (2006) Other publications Á. Baji Gál, Sz. Bodó, D. Boonkusol, B. Görhöny, E. Balogh, A. Dinnyés (2005) Different gene expression of individual blastomeres in early mouse embryo detected by real time PCR. (Hungarian Journal of) Animal Production.; 54(3): Á. Baji Gál, Sz. Bodo, A. Dinnyes (2005) Epigenetic code changes during early mouse embryo development - focus on histone deacetylation. (30th FEBS Congress - 9th IUBMB Conference, July , Budapest) FEBS J.; 272(s1): O1-052P. Z. Tancos, J. Kobolak, A. Baji Gal, A. Dinnyes (2006) Identification of Oct-4 and Nanog, the two pluripotency marker genes in rabbit preimplantation stage embryos. (Hungarian Journal of) Animal Production.; 55(6): A. Baji Gal, S. Mamo, S. Bodo, A. Dinnyes (2007) Comparison of housekeeping gene expression in individual 8- cell stage mouse embryos produced under different conditions. (43rd Annual Conference of the International Embryo Transfer Society, Jan , Caribe Royale, Orlando, Japan) Reprod Fertil Dev.; 19(1): S. Mamo, A. Baji Gal, Z. Polgar, A. Dinnyes (2008) Expression profiles of the pluripotency marker POU5F1 and validation of reference genes in rabbit oocytes and preimplantation stage embryos. BMC Mol Biol.; submitted. K. Kiss, J. Kobolak, Z. Tancos, S. Mamo, Z. Polgar, C. Rogel-Gaillard, A. Baji Gal, A. Dinnyes (2008) Promoter analysis of the rabbit pou5f1 gene and it s expression in preimplantation stage embryos. Mol Reprod Dev.; submitted. 9

11 Acknowledgements I would like to thank to everybody for the help provided in bringing to fruition my thesis work, namely: Lídia Lennert (my wife) Lídia, Valéria and Árpád Baji Gál (my children) Dr. András Dinnyés (my supervisor) Prof. László Gráf and the Department of Biochemistry, Eötvös Loránd University Dr. Márta Adorján Dr. Emese Balogh Sándorné Bara Vladimir Benes Dr. Szilárd Bodó Duangjai Boonkusol Stephen A. Bustin Joseph W. Carnwath Edit Deák Dr. Botond Görhöny Jiri Kanka Mikael Kubista Györgyi Kungl Dr. Solomon Mamo Lucie Nemcova Tania Nolan Zsuzsanna Polgár Zsuzsanna Táncos as well as the B.E.Sz.T. Istvánné Klotz, Zsuzsanna Wittmayer, Miklós Bálint (my ex-teachers) Ferenc Olasz, Katalin Csomor, Dr. György Thaler (my ex-leaders). 10