P-531 Influence of tandemly repetitive pericentromeric DNA transcripts inactivation on the formation of membraneless RNP structures at the end of human oocytes maturation

Abstract

Abstract Study question Does inactivation of tandemly repetitive (TR) pericentromeric DNA transcription affect the size and the number of DDX4-containing RNPs at the end of human oocytes maturation? Summary answer Antisense oligonucleotides (AO) can inactivate the transcription of HS2/HS3. Inactivation leads to disaggregation of the DDX4-based membraneless RNP structures. What is known already Earlier we had described for the first time the transcription of the pericentromeric TR DNA of HS2/HS3 families in late human oogenesis, during the transition of oocytes from the GV stage to the MI stage. Both HS2/HS3 strands were transcribed. The presence of HS2/HS3 transcripts was demonstrated by bioinformatic analysis, qPCR and RNA-DNA-FISH. HS2/HS3 RNAs were found in RNPs which contained DDX5 and DDX4 RNA helicases, located near mitochondria. We suggested that the RNPs could be the site of spatial sequestration of RNA and proteins at the end of human oocytes maturation. Study design, size, duration For studying the inactivation of HS2/HS3 transcription, oocytes were subjected to cytoplasmic microinjection using a micromanipulator. Before the experiment, oocytes were divided into 3 groups (n = 11). Group 1 (control, n = 4) got a dose of saline, group 2 (n = 3) – 8.5 fmol AO, group 3 (n = 4) – 12.75 fmol AO. AO or saline was injected into oocytes, the cells were then placed in a CO2 incubator, and after 48 hours, they were fixed and examined. Participants/materials, setting, methods GV and MI human oocytes are not used for the bank of donor oocytes and were transferred for research. The informed consent of the donors, as well as the permission of the ethical committee of the clinic, was obtained. The distribution of HS2/HS3 transcripts and DDX4 helicase was studied using the immuno-FISH. Fluorescent signals were analyzed using the ImageJ program. Statistical processing of the count data was carried out using GraphPad Prism 9 software. Main results and the role of chance Statistically significant inactivation of TR RNA HS2/HS3 (p = 0.01) in group 3 (AO, 12.75 fmol) was observed. The fluorescence of the HS2/HS3 RNA hybridization signal dropped to 25.68±1.777 as compared to the control group 1 (35.50±2.938). In group 2, this value (34.47 ± 1.984) did not change significantly as compared to the control group. Also, in the 3rd group, there was a tendency (p = 0.16) of an increase in the fluorescence of the antibodies signal against DDX4 up to 26.23 ± 2.223 in comparison with group 1 (19.57 ± 2.496). In group 2, this indicator (21.63 ± 3.095) changed to a lesser extent as compared to the control group. At the same time, in the oocytes of the 3rd group, there was a tendency (p = 0.19) to an increase in the number of individual cytoplasmic foci stained with the antibody against DDX4 up to 76±18.56 as compared with group 1 (44±8.994 signals). In group 2 this value (26±5.508 signals) changed less compared to control group 1. We assume, after the inactivation of HS2/HS3 transcription, DDX4-containing RNPs dissociate since an increase in the number of individual signals of the corresponding DDX4 was revealed. Limitations, reasons for caution The small sample size did not allow us to obtain statistically reliable data on the fluorescence of DDX 4. Data accumulation will be continued in future studies. Wider implications of the findings The data presented confirm the existence of DDX4-based RNPs in human preovulatory oocytes. These membraneless structures are involved in the processes of oocyte maturation. The suggested approach of transcripts inactivation can be used for oocytes without removing zona pellucidae increasing their survival after inactivation. Trial registration number not applicable