Only about 5 percent of DNA is made up of protein-coding genes; the other 95 percent is noncoding and referred as junk DNA. Most of the non-coding DNA is repeats either scattered through genome (dispersed repeats) or tandemly organized (tandem repeats, TR or satellite DNA). These repeats are transcribed and the transcripts are involved in different processes including early preimplantation embryogenesis. However it is still unknown are the tandem repeats transcripts accumulated during oogenesis or the repeats are transcribed de novo in early embryo prior after the genome activation. The aim of the study was to check the presence of pericentromeric TR transcripts in preovulatory oocytes and to reveal its intracellular distribution. Methods. The TR of pericentromeric satellite 3 (HS3) as well as its transcripts distribution in GV, MI and MII oocytes was studied with DNA-DNA and RNA-DNA FISH, respectively. The colocalisation of HS3 DNA and RNA with RNA-helicase p68 (DDX5) involved in the HS3 transcription regulation was estimated by immunoFISH. The GV and MI preovulatory oocytes were obtained from donors after the informed consent had been signed. GV and MI donor oocytes are not used for gametes banking and are usually discarded. They were donated for scientific purposes after the ethic committee permission was granted. The presence of HS3 transcripts in transcriptome was verified with bioinformatics’ methods of analysis of published preovulatory oocytes transcriptome (Zhang et al., 2018). Results. The probe used was hybridized to pericentromeric regions of all chromosomes excluding 2,6,8,11,12,18,19 on lymphocytes chromosome spreads in DNA-DNA FISH experiments. In GV oocytes, the probe was revealed as the part of condensed heterochromatic ring included in the inverted karyosphere . In M1 and M2, the probe was localized to pericentromeric chromosome regions. In DNA-RNA FISH, the probe revealed 1 mkm granules in ooplasm in M1, M2 but not in GV oocytes. Pretreatment with RNAse eliminated the hybridization signal. The HS3 RNA granules were associated with the granules of RNA helicase p68 (DDX5). The analysis of transcriptome revealed a polyadenilated HS3 transcript, that contained a sequence homological to the probe used in the experiment. The transcript contained also the sequence we describe previously as actively transcribed in senescent and malignant cells (Enukashvily et al., 2007). Conclusion. HS3 transcripts are accumulated in human oocyte after the transition from GV to M1. The transcripts are not associated with chromosomes but are adjacent to granules of RNA helicase p68.
Предметные области Scopus
- Биохимия, генетика и молекулярная биология (все)