Phosphorylation Status of RNA Polymerase II Carboxyl-terminal Domain in Porcine Oocytes and Early Embryos

Fertilization of the oocyte commences embryogenesis during which maternally inherited mRNAs are degraded and the embryonic genome is activated. Transcription of embryonic mRNA is initiated by embryonic genome activation (EGA). RNA polymerase II (RNA Pol II) is responsible for the synthesis of mRNAs and most small nuclear RNAs, and consists of 12 subunits, the largest of which characteristically harbors a unique C-terminal domain (CTD). Transcriptional activity of RNA Pol II is highly regulated, in particular, by phosphorylation of serine residues in the CTD. Here, we have shown the presence of RNA Pol II CTD phosphoisoforms in porcine oocytes and preimplantation embryos. The distribution pattern as well as phosphorylation dynamics in germinal vesicles and during embryogenesis differed in developmental stages with these isoforms, indicating a role of RNA Pol II CTD phosphorylation at the serine residue in transcriptional activation during both oocyte growth and embryonic genome activation. We additionally examined the effects of the RNA Pol II inhibitor, α-amanitin, on embryo development. Our results show that inhibition of polymerase, even at very early stages and for a short period of time, dramatically impaired blastocyst formation. These findings collectively suggest that the functionality of maternal RNA Pol II, and consequently, expression of early genes regulated by this enzyme are essential for proper embryo development

Artificial Oocyte: Development and Potential Application

Millions of people around the world suffer from infertility, with the number of infertile couples and individuals increasing every year. Assisted reproductive technologies (ART) have been widely developed in recent years; however, some patients are unable to benefit from these technologies due to their lack of functional germ cells. Therefore, the development of alternative methods seems necessary. One of these methods is to create artificial oocytes. Oocytes can be generated in vitro from the ovary, fetal gonad, germline stem cells (GSCs), ovarian stem cells, or pluripotent stem cells (PSCs). This approach has raised new hopes in both basic research and medical applications. In this article, we looked at the principle of oocyte development, the landmark studies that enhanced our understanding of the cellular and molecular mechanisms that govern oogenesis in vivo, as well as the mechanisms underlying in vitro generation of functional oocytes from different sources of mouse and human stem cells. In addition, we introduced next-generation ART using somatic cells with artificial oocytes. Finally, we provided an overview of the reproductive application of in vitro oogenesis and its use in human fertility

Changes in histone H3 lysine 36 methylation in porcine oocytes and preimplantation embryos.

Histone H3 lysine 36 (H3K36) methylation is known to be associated with transcriptionally active genes, and is considered a genomic marker of active loci. To investigate the changes in H3K36 methylation in pig, we determined the mono-, di-, and tri-methylations of H3K36 (H3K36me1, H3K36me2 and H3K36me3, respectively) in porcine fetal fibroblasts, oocytes and preimplantation embryos by immunocytochemistry using specific antibodies and confocal microscopy. These analyses revealed that only H3K36me3 in porcine fetal fibroblasts consistently colocalized with transcription sites identified as actively synthesizing RNA based on fluorouridine (FU) incorporation. Treatment of cells with flavopiridol, which blocks transcription elongation, completely abrogated both H3K36me3 signals and RNA synthesis. All three types of H3K36 methylation were present and did not significantly differ during oocyte maturation. In parthenogenetic embryos, H3K36me1 and -me2 were detected in 1-cell through blastocyst-stage embryos. In contrast, H3K36me3 was not detected in most 1-cell stage embryos. H3K36me3 signals became detectable in 2-cell stage embryos, peaked at the 4-cell stage, decreased at the 8-cell stage, and then became undetectable at blastocyst stages in both parthenogenetic and in vitro-fertilized (IVF) embryos. Unlike the case in IVF embryos, H3K36me3 could not be demethylated completely during the 1-cell stage in somatic cell nuclear transfer (SCNT) embryos. These results collectively indicate that H3K36me3, but not H3K36me1 or -me2, is associated with transcription elongation in porcine fetal fibroblasts. H3K36me3 is developmentally regulated and may be a histone mark of embryonic gene activation in pig. Aberrant H3K36 tri-methylation occurred during the nuclear reprogramming of SCNT embryos

P-TEFb Kinase Activity Is Essential for Global Transcription, Resumption of Meiosis and Embryonic Genome Activation in Pig.

Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig

Engineering of polyketide synthases: How close are we to the reality?

<p>(A) Oocytes and parthenogenetic embryos were immunostained with the anti-H3K36me3 antibody, which was then localized with a FITC-conjugated secondary antibody (green). DNA was stained with DAPI (blue). NSN and SN are two types of GV stage oocytes. MI, metaphase I stage oocytes; MII, metaphase II stage oocytes. Scale bar = 20 µm. (B) Relative intensities of fluorescence signals from H3K36me3. Total 23 oocytes and 47 embryos were analyzed in triplicate in this experiment. Bars represent least-squares showed the standard error in each group. P-values <0.05 were considered statistically significant. ^a,b,c Values represent least-squares with different superscripts are significantly different (P<0.05).</p

P-TEFb association with nucleolar proteins in the germinal vesicle oocytes at different stages of growth.

<p><b>A)</b> CDK9 is colocalized with the DFC component, fibrillarin, only at the periphery of the NLB in NSN oocytes. In pNSN oocytes, nucleolar structure is changed and fibrillarin and other nucleolar components move to the periphery of the NLB. In this stage, CDK9 shows partial colocalization with fibrillarin (inset). In SN oocytes, fibrillarin is mostly gone, but CDK9 remains bound to the nucleolar structure. At the periphery of the NLB, CDK9 shows more colocalization with the FC component, UBF, in pNSN (inset). <b>B)</b> The same pNSN oocytes as in A was analyzed further. Eight optical sections, Z-stacks with 0.5 μm, were taken by confocal microscope confirming the colocalization of CDK9 and fibrillarin at the periphery of NLB. <b>C)</b> X and Y axes of a single focal plane of the same image as B focusing on an area corresponding to the NLB periphery, indicating the spatially colocalization of CDK9 and fibrillarin in that area. <b>D)</b> Cyclin T1 also shows colocalization with nucleolar factor, UBF, in pNSN and pSN oocytes. DNA counterstained with DAPI. <b>E)</b> COCs were collected separately from small or large follicles and then were denuded and subjected to Western blotting. The same anti-CDK9 antibody as used for immunostaining was used for Western blot. The bands correspond to two CDK9 isoforms, 42 kD and 55 kD. Fifty oocytes were collected for each group.</p

Effect of CDK9 inhibition on transcriptional activity of GV oocytes.

<p><b>A)</b> COCs were collected from different size of follicles and immediately cultured in a complete maturation medium in presence of 5 mM FU for 1 hour. Then, COCs were denuded and oocytes were fixed and subjected to immunostaining. In NSN oocytes in which CDK9 is distributed throughout the GV, transcriptional activity is at the highest level. In SN oocytes, CDK9 accumulates around the NLB and nascent RNAs become undetectable. <b>B)</b> COCs were subjected or not to α-amanitin, actinomycin D (ActD), or Flavopiridol for 1 hour. Treatment with α-amanitin started 1 hour ahead of the other compounds. At the same time, nascent transcripts were labeled with FU. Then, COCs were denuded and oocytes were fixed and subjected to immunostaining. In α-amanitin-treated oocytes, nucleoplasmic transcription decreased dramatically, but a rim of transcripts remained around the nucleolus. In oocytes treated with ActD, no transcriptional activity was detected. In Flavopiridol-treated oocytes, similarly, almost all the RNA transcription was abolished. Similar result was obtained when oocytes treated with CDK9 Inhibitor II (CAN508).</p