CHROMATIN QUALITY AS A CRUCIAL FACTOR FOR THE SUCCESS OF FLUORESCENT IN SITU HYBRIDIZATION ANALYSES OF UNFERTILIZED OOCYTES, POLAR BODIES AND ARRESTED ZYGOTES
Zhivkova RS1,*, Delimitreva SM1, Toncheva DI2, Vatev IT1
*Corresponding Author: Ralitsa S. Zhivkova, 1, St. Georgi Sofiiski Str., Laboratory of IVF and Preimplantation Embryology, Department of Biology, Medical Faculty, Medical University of Sofia, 1431, Sofia, Bulgaria; Tel.: +3592-91-72-678; Fax: +3592-91-72-660; E-mail: zhivkova@medfac.acad.bg
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DISCUSSION

The FISH analyses of human unfertilized oocytes and arrested zygotes revealed some disadvantages related to chromatin changes before fixation. Hybridization efficiency varied between the cells and between different chromatin groups in the same cell. The results were dependent on the type of fluorescent probe (centromere- or locus-specific) in the same chromatin. The observed differences correlated with chromatin condensation and fragmentation: results of FISH were informative when the chromatin was well preserved (Figures 1 and 7 for both types of probes and in PCC in Figures 3 and 4). Fluorescent in situ hybridization was less informative when chromatin quality was poor (Figures 2 and 6).

In arrested 2PN zygotes, FISH efficiency was different and correlated with chromatin morphology. In pronuclei with visibly normal interphase or prometaphase chromatin, hybridization was successful: in slightly condensed pronuclei, centromere signals were registered (shown in Figure 5); in pronuclei with highly condensed and/or fragmented chromatin, FISH was unsuccessful. In other words, pronuclear chromatin displayed different levels of condensation and fragmentation and FISH results were dependent on these changes. These chromatin changes during the prolonged culture period could be attributed to cell ageing and death. In the present study, ageing chromatin of 2PN zygotes followed the typical pattern of apoptotic changes previously observed by us in blastomeres of preimplantation embryos with poor quality and low developmental potential [35].

In highly condensed oocyte and PBI metaphase chromosomes, FISH success was low, especially with locus-specific probes. This could be explained by at least two factors, both associated with prolonged culture time. First, the high degree of chromosome condensation, which is a well known characteristic even of normal oocyte metaphase plates [36,37], could advance so much that target sequences would become inaccessible for probes. Second, in some cases, a high degree of chromatin condensation could be a sign of degeneration and be accompanied with actual destruction of target sequences. It should be mentioned that unlike some similar studies using FISH for identification of up to 12 or all chromosomes, where interpretation of hybridization results was not reliable because of signal overlapping/merging [32,38], we applied a small number of probes. This allowed us to conclude that the low success rate of FISH was due to degradation or inaccessibility of target regions in unfertilized human oocytes and PBIs. Although relatively few reports have addressed these processes in gametes, recent in vivo and in vitro studies indicate that not only polar bodies but also oocytes, similar to somatic cells, can undergo apoptosis [39,40].

The chromatin of PBII and prematurely condensed sperm chromatin (PCC groups in oocytes) was more suitable for FISH analysis with both centromere- and locus-specific probes. This could be explained with their later formation as chromatin groups in cells and, hence, shorter culture time.

The condensation and fragmentation of the chromatin are the first signs of cell ageing processes ahead of fragmentation of the nuclei and cytoplasm during apoptotic cell death [41,42]. Chromatin changes were related to the period of culture before cell fixation and FISH results were dependent on these changes. This was shown in cases of different hybridization success in differently condensed/ fragmented chromatin groups in the same analyzed cell, positive FISH reaction in PCC combined by negative FISH reaction in the oocyte metaphase plate. Unlike the oocyte chromosomes, in prematurely condensed sperm chromatin (PCC) the visible signs of ageing were not advanced. We could suppose that DNA fragmentation was also more pronounced in the oocyte plate than in the later formed PCC chromosomes. Another factor could be the different level of chromatin condensation of these two groups influencing the accessibility of the target regions to the probes. The difference in FISH success between PBI and PBII could be explained in the same way: At the moment of cell fixation, chromosome condensation and fragmentation were more advanced in PBI than in the later formed PBII.

The observed difference in hybridization success between centromere- and locus-specific reaction suggests that the target sequences of locus-specific probes are more sensitive to degeneration changes. This could be due to the fact that locus-specific probes detect unique DNA sequences, while centromere probes bind to DNA repeats.

Our results concerning polar bodies are of interest with regard to preimplantation genetic screening. Polar bodies are regarded as a source of information for maternal meiotic errors but these cells are normally destined for destruction. Since chromatin degradation begins early through programed cell death, FISH failure due to chromosome fragmentation could be misinterpreted as aneuploidy. This is especially true for the PBI, which is formed at an early stage.

We found that FISH success correlated with the presence of chromatin changes during the culture of human oocytes, polar bodies and bipronuclear zygotes. This emphasizes the significance of early signs of in vitro cell ageing for successful FISH analysis and for the interpretation of results in analysis of unfertilized human ova, polar bodies and arrested zygotes.




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