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
page: 3

RESULTS

The visible quality of chromatin varied widely between analyzed cells. In unfertilized oocytes with good metaphase spread, chromosomes had a normal appearance in some cells but were more condensed or fragmented in others. When PCC was present, paternal chromosomes were slightly condensed (with prometaphase appearance), while maternal ones were highly condensed and degenerative/fragmented. This latter picture was also observed in the chromosomes of PBI. Chromatin quality varied in the pronuclei of arrested zygotes: apparently normal interphase or slightlyto highly condensed as if undergoing apoptosis. The chromatin of PBII tended to resemble the chromatin of the respective arrested zygote.

The success of FISH analyses in unfertilized oocytes correlated with chromosomal morphology. Successful hybridization was detected in 24 of 40 analyzed metaphases (60.0%). Both centromere- and locus-specific signals were detected in nine of these 24 cells (37.50%) (see Figure 1). All these oocytes had been shown to have good metaphase plates by Giemsa staining. The other FISH positive metaphases (15 of 24 cells, 62.50%) displayed only the centromere signals. These oocytes displayed two types of progressive chromatin degeneration as extremely condensed (Figure 2) or “fuzzy” chromosomes (Figures 3 and 4). The 16 oocytes with no FISH signals had more pronounced chromatin degeneration.

Fourteen unfertilized oocytes contained prematurely condensed sperm chromosomes (PCC) with prophase or prometaphase appearance beside the oocyte plate (Figure 3). Maternal chromosomes were visibly degenerated and displayed no signals. However, in nine of these cells (64.29%), the FISH reaction was positive in the group of sperm chromosomes (Figures 3 and 4).

The 34 analyzed zygotes displayed two pronuclei on the day of embryo transfer. Their chromatin condensation varied between the cells and between the pronuclei in the same cell. In 13 zygotes (38.24%) no fluorescent signals were detected. The negative FISH reaction was associated with highly condensed chromatin. In the other 21 zygotes (61.76%), the centromere probes labeled at least one of the pronuclei (Figure 5).

Of the 15 polar bodies, seven were obtained from unfertilized oocytes (PBI) and eight from zygotes, and were regarded to be PBII because of their interphase chromatin appearance. The PBIf had highly condensed, visibly degenerated chromosomes. Hybridization success was low: only three of seven PBI (42.86%) showed centromere signals (Figure 6) and none reacted with the locus-specific probe. In six of eight PBII (75.0%, Figure 7), the chromatin was better preserved and positive FISH signals for centromere- and locus-specific probes were obtained.




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