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

Abstract

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.

MicroRNA Expression during Bovine Oocyte Maturation and Fertilization

“…It appears likely that many of the molecular processes driving oocyte maturation have the potential to be regulated by the dynamic pattern of miRNA expression. Upon germinal vesicle breakdown, it is it widely recognized that there is a widespread decrease in the level of oocyte transcriptional activity that lasts until the onset of zygotic transcription which is generally thought to happen at the 8–16 cell stage [ 32 , 33 ]. MicroRNA-155 is a major miRNA induced at this time point and one of the major functional clusters identified in miR-155 targets is the regulation of transcription from RNA polymerase II transcription factor activity, suggesting associations between the two processes.…”

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes

“…Porcine ovaries were obtained at a local slaughterhouse and transported to the laboratory in a Dulbecco's phosphate-buffered saline supplemented with 0.01% polyvinyl-alcohol (PBS-PVA) at 30–35°C. After three times washing with PBS-PVA, the GV stage oocytes were aspirated from large antral follicles (3–5 mm, in diameter) as previously reported (Barnetova et al , 2012). The oocytes surrounded by several layers of compact cumulus cells (cumulus–oocyte complexes – COCs) were selected and washed three times in M2 medium.…”

Reconstitution of chromatin reorganization during mammalian oocyte development

“…Next, we asked whether RNAPII degradation is physiologically relevant to NSN-to-SN transition in vivo. Previous work reported that the levels of phosphorylated RNAPII are lower in SN oocytes as compared to NSN oocytes [38][39][40][41][42][43][44][45][46][47] , but whether this observation reflects simply dephosphorylation or depletion at protein level is not clear. To this end, we compared the abundance of phosphorylated as well as of total RNAPII in NSN and SN oocytes by immunofluorescence staining and immunoblotting.…”