The inability of fully grown germinal vesicle stage oocyte cytoplasm to transcriptionally silence transferred transcribing nuclei

Abstract

For somatic cell nuclear transfer cytoplasts from metaphase II, oocytes are exclusively used. However, it is evident that certain reprogramming activities are present in oocytes even at earlier stages of maturation. These activities are, however, only poorly characterised. The main reason for this is that even the intrinsic oocyte processes are insufficiently understood. The mammalian oocyte is a highly specialised cell that harbours many specific characteristics. One of these is its particularly large size when compared to somatic cells. As the oocyte enters the growth phase its volume, as well as the amount of material, increases considerably. Thus, it is clear that the oocyte must possess the machinery to accomplish this incredible material accumulation. When the growth phase is completed, the transcription ceases and the oocyte becomes transcriptionally inactive. In our study, we have used the model system of oocyte fusion (transcribing x non-transcribing germinal vesicle (GV) stage oocytes) as a substitute for a somatic cell nuclear transfer schemes where the somatic cell nucleus would be introduced into a cytoplast obtained from a GV stage oocyte. We wanted to determine if the fully grown GV stage oocyte could induce reprogramming of transcriptionally active transferred nucleus by suppressing this activity. In order to evaluate possible changes in transcriptional properties after nuclear transfer, we also investigated the mechanism of transcriptional silencing taking place when the oocyte reaches its full size as well as the fate of the components namely of the RNA polymerase II (Pol II) transcriptional and splicing machinery. Here, we show that while the Pol II is degraded in fully grown GV stage oocytes and the splicing proteins undergo significant rearrangement, these oocytes are unable to induce similar changes in transcriptionally active nuclei even after a prolonged culture interval.

Global Gene Silencing is Caused by the Dissociation of RNA Polymerase II from DNA in Mouse Oocytes

“…2B), which was consistent with a previous report [23]. Although Fulka et al [24] recently reported that they were unable to detect unphosphorylated RPB1 in full-grown oocytes, this apparent discrepancy may have resulted from the anti-RPB1 CTD antibody they used for immunoblotting. The antibody they used reacts with both the phosphorylated and unphosphorylated forms of RPB1 but appears to have a much lower affinity for the unphosphorylated form.…”

RNA polymerase II transcriptional silencing in growing and fully grown germinal vesicle oocytes isolated from gonadotropin‐stimulated and non‐stimulated gilts

“…Similar studies were previously performed in the mouse (Fulka et al, 2009; Abe et al, 2010), but the authors interpreted their results differently. Fulka et al (2009) concluded that RNA polymerase II is degraded during oocyte growth. The authors also used the CTD4H8 and 8WG16 clones for immunofluorescence and Western blots.…”

“…Moreover, the dephosphorylation of POLR2A was followed by the release of RNA polymerase II from DNA (Abe et al, 2010). The findings of Abe et al (2010) are in contrast to the study by Fulka et al (2009), who did not observe any form of RNA polymerase II in fully grown, mouse GV oocytes, and therefore concluded that the RNA polymerase II is degraded in these cells (Fulka et al, 2009).…”

“…This observation is in contrast to the observation of Abe et al (2010), who detected similar amounts of hypophosphorylated POLR2A in growing and fully grown oocytes. Abe et al (2010) suggest that the CTD4H8 clone used by Fulka et al (2009) had different affinities for the two forms of POLR2A. Yet, the antibody used by Fulka et al (2009) had been verified on several cell lysates, and the conclusion was not based solely on evaluation with the CTD4H8 clone.…”

Extending the knowledge in histochemistry and cell biology

“…Upon follicular recruitment, the oocyte enters the growth phase and after completing the growth phase the oocyte becomes transcriptionally inactive (Mehlmann 2005). Fulka et al (2009) used an oocyte fusion model to determine if the fully grown germinal vesicle stage oocyte could induce suppression of transcription activity in transcribing germinal vesicle stage oocytes. Although the authors identiWed mechanisms for silencing transcription, such as RNA polymerase II degradation and rearrangement of splicing proteins in mature oocytes, these oocytes were insuYcient to induce these changes in transcriptionally active nuclei, which led the authors to discuss the selection of cytoplasts in nuclear transfer experiments.…”