Final Report Summary - OOAGEMEIOSIS (Age-related Changes in Chromosome Segregation in Female Meiosis)
Project objectives
It has been shown that mammalian meiosis is associated with high incidence of errors, leading into numerical chromosomal aberrations, which are more prevalent in female gametes and which seem to increase with maternal age. If transferred to newly formed embryo, oocyte aneuploidy has severe consequences, including pregnancy loss and birth defects. According to the prevailing hypothesis, aneuploidy in oocytes requires coincidence of two errors; first is an error in early meiotic program, for example during recombination, the second is a failure of mechanisms controlling chromosome segregation after resumption of meiosis. In our project we focused our effort first on establishing an appropriate animal model, which could be used to study the origins of oocyte and embryo aneuploidy. Long time established and most widely used mammalian model systems, which are based on laboratory rodents, have multiple advantages, including reasonable body size, easy handling and short generation time just to name a few. However, the close comparison between human and mouse oocytes and oocytes from large mammals, such as cattle or pig, showed that the latter are significantly more similar to human oocytes, particularly the duration of meiotic events and cell size is quite similar.
This is the reason we initially studied the effect of aging on oocyte aneuploidy using porcine oocytes. Cells were obtained from minipigs breed established in our institute and also from large farm animals. To obtain a complete picture about segregation pattern of each particular chromosome during meiosis, we have adapted method known from human reproduction medicine and cancer research, which could be used to identify each individual chromosome in a single cell. The method, called Comparative Genomic Hybridisation (CGH), is the most accurate technique for scoring aneuploidy in a single cell, which is available at the moment, and in our experiments was used for the first time to study chromosomes in animal oocyte, although there were some challenges, such as getting reasonable signal from a reduced DNA content of a single haploid cell. This method lacks the usual problems of chromosome spreads and fluorescent in situ hybridisation (FISH), allowing reduction of the number of cells to obtain statistically significant results. This was important for our study since the number of oocytes, which could be obtained from ovaries of aged animals, is reduced and also the availability of aged animals is limited; therefore the cells are extremely valuable. To confirm results obtained from oocytes we used a corresponding polar body as a control. Initial optimisation of the method was necessary for detection of both major sources of oocyte aneuploidy non-disjunction of the whole bivalent and premature segregation of sister chromatids.
Our results showed, that the overall incidence of aneuploidy in porcine oocytes is remarkably lower than it was found previously using other methods. Surprisingly, we were not able to detect any increase of the frequency of aneuploidy in oocytes from aged animals. Although we have analysed the segregation pattern of more than 2600 chromosomes, the frequency of aneuploidy was very similar in groups of animals separated by more than 6.5 years, with the oldest animals reaching the lifespan of its species. Considering the high frequency of aneuploidy seen in aged human and mice eggs, the finding that porcine oocytes are not affected by this defect was unexpected. Our recent experiments are focused on analysing of major differences in chromosome segregation between human, mouse and porcine oocytes. We believe that this might help our understanding of the aetiology of maternal age-related aneuploidy.
It has been shown that mammalian meiosis is associated with high incidence of errors, leading into numerical chromosomal aberrations, which are more prevalent in female gametes and which seem to increase with maternal age. If transferred to newly formed embryo, oocyte aneuploidy has severe consequences, including pregnancy loss and birth defects. According to the prevailing hypothesis, aneuploidy in oocytes requires coincidence of two errors; first is an error in early meiotic program, for example during recombination, the second is a failure of mechanisms controlling chromosome segregation after resumption of meiosis. In our project we focused our effort first on establishing an appropriate animal model, which could be used to study the origins of oocyte and embryo aneuploidy. Long time established and most widely used mammalian model systems, which are based on laboratory rodents, have multiple advantages, including reasonable body size, easy handling and short generation time just to name a few. However, the close comparison between human and mouse oocytes and oocytes from large mammals, such as cattle or pig, showed that the latter are significantly more similar to human oocytes, particularly the duration of meiotic events and cell size is quite similar.
This is the reason we initially studied the effect of aging on oocyte aneuploidy using porcine oocytes. Cells were obtained from minipigs breed established in our institute and also from large farm animals. To obtain a complete picture about segregation pattern of each particular chromosome during meiosis, we have adapted method known from human reproduction medicine and cancer research, which could be used to identify each individual chromosome in a single cell. The method, called Comparative Genomic Hybridisation (CGH), is the most accurate technique for scoring aneuploidy in a single cell, which is available at the moment, and in our experiments was used for the first time to study chromosomes in animal oocyte, although there were some challenges, such as getting reasonable signal from a reduced DNA content of a single haploid cell. This method lacks the usual problems of chromosome spreads and fluorescent in situ hybridisation (FISH), allowing reduction of the number of cells to obtain statistically significant results. This was important for our study since the number of oocytes, which could be obtained from ovaries of aged animals, is reduced and also the availability of aged animals is limited; therefore the cells are extremely valuable. To confirm results obtained from oocytes we used a corresponding polar body as a control. Initial optimisation of the method was necessary for detection of both major sources of oocyte aneuploidy non-disjunction of the whole bivalent and premature segregation of sister chromatids.
Our results showed, that the overall incidence of aneuploidy in porcine oocytes is remarkably lower than it was found previously using other methods. Surprisingly, we were not able to detect any increase of the frequency of aneuploidy in oocytes from aged animals. Although we have analysed the segregation pattern of more than 2600 chromosomes, the frequency of aneuploidy was very similar in groups of animals separated by more than 6.5 years, with the oldest animals reaching the lifespan of its species. Considering the high frequency of aneuploidy seen in aged human and mice eggs, the finding that porcine oocytes are not affected by this defect was unexpected. Our recent experiments are focused on analysing of major differences in chromosome segregation between human, mouse and porcine oocytes. We believe that this might help our understanding of the aetiology of maternal age-related aneuploidy.