Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1738-2432(Print)
ISSN : 2288-0151(Online)
Reproductive & developmental Biology Vol.36 No.4 pp.237-241
DOI : https://doi.org/10.12749/RDB.2012.36.4.237

Temporal Expression of RNA Polymerase II in Porcine Oocytes and Embryos

Reza Oqani, Min Gu Lee, Lin Tao, Dong Il Jin
Department of Animal Science & Biotechnology, Research Center for Transgenic Cloned Pigs, Chungnam National University
(Received: 26 September 2012/ Accepted: 12 December 2012)

Abstract

Embryonic genome activation (EGA) is the first major transition that occurs after fertilization, and entails a dramaticreprogramming of gene expression that is essential for continued development. Although it has been suggested thatEGA in porcine embryos starts at the four-cell stage, recent evidence indicates that EGA may commence even earlier;however, the molecular details of EGA remain incompletely understood. The RNA polymerase II of eukaryotes transcribesmRNAs and most small nuclear RNAs. The largest subunit of RNA polymerase II can become phosphorylatedin the C-terminal domain. The unphosphorylated form of the RNA polymerase II largest subunit C-terminaldomain (IIa) plays a role in initiation of transcription, and the phosphorylated form (IIo) is required for transcriptionalelongation and mRNA splicing. In the present study, we explored the nuclear translocation, nuclear localization, andphosphorylation dynamics of the RNA polymerase II C-terminal domain in immature pig oocytes, mature oocytes,two-, four-, and eight-cell embryos, and the morula and blastocyst. To this end, we used antibodies specific for theIIa and IIo forms of RNA polymerase II to stain the proteins. Unphosphorylated RNA polymerase II stained stronglyin the nuclei of germinal vesicle oocytes, whereas the phosphorylated form of the enzyme was confined to the chromatinof prophase I oocytes. After fertilization, both unphosphorylated and phosphorylated RNA polymerase II beganto accumulate in the nuclei of early stage one-cell embryos, and this pattern was maintained through to theblastocyst stage. The results suggest that both porcine oocytes and early embryos are transcriptionally competent, andthat transcription of embryonic genes during the first three cell cycles parallels expression of phosphorylated RNApolymerase II.

INTRODUCTION

F1.jpg119.4KB

Embryogenesis commences at fertilization and is characterized by gradual degradation of maternally inherited messages and activation of the embryonic genome; thus, by initiation of embryonic transcription and translation of embryonic proteins. If the embryonic genome is not activated, development is irreversibly arrested because basic metabolic processes cannot be further supported. It has been suggested that embryonic genome activation (EGA) of porcine embryos occurs at the four-cell stage, but the molecular details of EGA in pigs are not understood in detail. The maternal-zygotic transition is a critical event of early mouse embryogenesis; the highly differentiated oocyte is transformed into a totipotent blastomere as early as the two-cell stage. At this time, maternal mRNAs are degraded and the embryonic genome is activated (Schultz, 2002; Walser and Lipshitz, 2011). Genome activation triggers replacement of transcripts common to both the oocyte and embryo and also, the generation of new transcripts necessary for further development (Kanka, 2003; Bogolyubova, 2011). Mouse embryos that cannot accomplish genome activation cease to develop at the two-cell stage. In the mouse, two transcriptional stages have been identified. A minor transcriptional wave is evident at the one-cell stage, and a second major transcriptional wave occurs at the two-cell stage (Forlani et al., 1998).

In eukaryotes, RNA polymerase II is transcribes m-RNAs and most small nuclear RNAs. Transcription of class II genes requires coordinated assembly of a protein complex containing RNA polymerase II and six accessory factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. The latter six proteins have been termed “general transcription factors” (Sikorski and Buratowski, 2009). Transcription begins upon formation of the first phosphodiester bond and phosphorylation of the serine-5 residue, catalyzed by TFIIH, in the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD of RNA polymerase II, which is a highly conserved tandem repeat of a heptapeptide motif (YSPTSPS), is subjected to extensive phosphorylation and  dephosphorylation during the transcription cycle. This oscillation of CTD phosphorylation regulates recruitment of various factors that are required at different stages of transcription (Sims et al., 2004). P-TEFb (CDK9/Cyclin- T1), the metazoan RNA polymerase II CTD kinase, regulates transcription elongation via phosphorylation of the serine-2 residue of the CTD (Price, 2000; Xie et al., 2006; Bres et al., 2008). RNA polymerase II can assume three different forms varying in electrophoretic mobility. These forms are the unphosphorylated (IIa) enzyme, a hyperphosphorylated form of the enzyme (IIo), and the IIb form. Synthesis of mRNA in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II, and alkaline phosphatase can convert the IIo form of the enzyme to the hypophosphorylated IIa form by removing phosphates from the CTD of RNA polymerase II. If the enzyme is to catalyze transcription, the enzyme must be present in an active form prior to commencement of transcription. Phosphorylation of the CTD of RNA polymerase II is also required when co-transcriptional mRNA processing is underway in vivo; the enzyme serves to bind factors required for 5' end-capping of mRNA, splicing, 3' end-processing, and chromatin modification (Pirngruber et al., 2009). Previously we showed that phosphorylation of RNA polymerase II by CDK9 is essential for embryonic genome activation in the mouse (Oqani et al., 2011).

Pig embryos develop to the four-cell stage in vitro even if neither the maternal nor paternal contribution to the genome is transcribed. At the four-cell stage, the embryonic genome becomes activated. Both nuclear translocation of RNA polymerase II and the phosphorylation of the CTD of RNA polymerase II have been suggested to be major triggers of EGA in mammals. Although we reported earlier that the phosphorylation of the serine-2 residue of RNA polymerase II CTD in porcine embryos (Oqani et al., 2012), the expression level of the phosphorylated or unphosphyrylated RNA polymerase II has not been explored in detail in either porcine oocytes or embryos. In the present study, we explored changes in RNA polymerase II phosphorylation status in such oocytes and embryos. We found that signals triggered by both unphosphorylated and phosphorylated RNA polymerase II are in play even in the preovulatory porcine oocyte, and this state of affairs continues through to the blastocyst stage.

MATERIALS AND METHODS

Oocyte and Embryo Production

Pig ovaries were collected at a local slaughterhouse. Antral follicles were manually aspirated. Cumulus-oocyte complexes (COCs) were resuspended in TL-HEPES containing 0.01% (v/v) polyvinyl alcohol (PVA). COCs were suspended in 500 ml amounts of TCM-199 medium containing EGF, L-cysteine, FSH, and LH. COCs were allowed to mature for 44 h and were next denuded and prepared for in vitro fertilization or immunostaining. After in vitro maturation as described above, oocytes were washed with  mTBM containing 0.1% (w/v) BSA (“IVF medium”). After washing, oocytes were placed in drops of IVF medium and the drops covered with mineral oil. Pig semen was next added. After several hours, the oocytes were removed from these fertilization drops and cultured in PZM3 medium.

Immunostaining and Confocal Microscopy

Oocytes and embryos were washed twice in PBS containing 0.1% (v/v) PVA (PBS-PVA) and next fixed in 2.5% (v/v) paraformaldehyde in PBS with 0.2% (v/v) Triton X-100 for 25 min at room temperature (RT). The oocytes and embryos were next permeabilized by incubation in 0.5% (v/v) Triton X-100 in PBS for 20 min at RT. The tissues were next washed three times in PBS-PVA (10 min for each wash) and blocked in a 2.5% (w/v) solution of BSA in PBS for 1 h at RT. Oocytes and embryos were next incubated, overnight at 4 ℃, with primary antibodies dissolved in blocking solution. After three washes with PBS-PVA, oocytes and embryos were stained with secondary antibodies for 1 h at RT and subsequently extensively washed in PBSPVA. Chromatin was counterstained with DAPI for 10 min at RT.

Immunocytochemistry and Confocal Microscopy

Oocytes and embryos collected after culture were washed and fixed in PBS containing 4% (w/v) paraformaldehyde and 0.2% (v/v) Triton X-100 for 40 min. Fixed tissues were next blocked with goat serum and BSA for 45 min and subsequently incubated with primary antibodies. These antibodies were a mouse monoclonal anti-phosphorylated CTD (Clone H5; Abcam) and a mouse monoclonal anti-nonphosphorylated CTD (Clone 8WG16; Upstate,). After washing, oocytes and embryos were incubated with secondary antibodies; these were FITC-labeled goat anti-mouse IgG (Santa Cruz), and Texas Red-labeled goat anti-mouse IgG (Santa Cruz), for 40 min at RT. Chromosomes were stained with DAPI or propidium iodide and the oocytes and embryos were mounted on slides. Immunocytochemical images were taken with the aid of a confocal scanning laser microscope (Zeiss).

RESULTS

Accumulation of RNA Polymerase II during Oocyte Maturation

To localize RNA polymerase II in porcine oocytes, the oocytes were immunocytochemically stained using the 8WG16 and H5 antibodies (both of which carry immunofluorescent tags). These antibodies recognize the unphosphorylated (IIa) and phosphorylated CTD (IIo) of RNA polymerase II, respectively. Nuclei were stained with propidium iodide. No unphosphorylated RNA polymerase II was evident in prophase I or metaphase II oocytes. However, strong nuclear staining commenced at the germinal vesicle stage (Fig. 1). Phosphorylated RNA polymerase II was located in chromatin of prophase I oocytes; no such staining was observed in oocytes at the germinal vesicle or metaphase II stages.

Figure 1. Expression and subcellular localization of RNA polymerase II in porcine oocytes. The time course of RNA polymerase II expression at defined oocyte stages was explored using an immunocytochemical technique. Polyclonal antibodies detecting the phosphorylated CTD (antibody H5) and the nonphosphorylated CTD (antibody 8WG16) served to immunolocalize the CTD regions of RNA polymerase II. Chromatin was counterstained with propidium iodide.

Nuclear Accumulation of RNA Polymerase II in Zygotes at the One-Cell Stage

To locate RNA polymerase II in fertilized porcine zygotes, the cells were immunostained as above; nuclei were stained with DAPI. Pronuclear zygotes expressed both unphosphorylated and phosphorylated RNA polymerase II but the unphosphorylated form of the enzyme was expressed predominantly in the pronucleus (Fig. 2). Both forms of RNA polymerase II began to accumulate in nuclei after the late one-cell stage.

Figure 2. Nuclear translocation of RNA polymerase II after fertilization. Porcine single-cell embryos were fixed and immunostained with polyclonal antibodies detecting the phosphorylated CTD (antibody H5) and the nonphosphorylated CTD (antibody 8WG-16). The test was performed just after fertilization. Chromatin was counterstained with DAPI.

RNA Polymerase in the Nucleus at Later Embryonic Stages

Further staining studies were performed to define the expression pattern of RNA polymerase II in the later stages of porcine embryos. Both unphosphorylated and phosphorylated enzyme became localized in the nucleus at the two-cell stage and remained there through to the late blastocyst stage (Fig. 3). In general, the level of phosphorylated RNA polymerase II was greater than was that of the unphosphorylated RNA polymerase II all embryonic stages studied; this was particularly true of embryos at the four-cell stage or later.

Figure 3. Expression and subcellular localization of RNA polymerase II in preimplantation porcine embryos. The time course of RNA polymerase II expression at defined preimplantation stages was determined using an immunocytochemical technique. Polyclonal antibodies detecting the phosphorylated CTD (antibody H5) and the nonphosphorylated CTD (antibody 8WG16) served to immunolocalize the CTD regions of RNA polymerase II. Chromatin was counterstained with DAPI.

DISCUSSION

In the present study, we found that unphosphorylated and phosphorylated RNA polymerase II were expressed in both immature and mature porcine oocytes and at all preimplantation stages of embryonic development. The levels of the proteins decreased only when oocyte development matured. As both proteins are expressed at a stage as early as the oocyte, it appears that RNA polymerase II present at the preimplantation stage synthesizes maternal messages and transcribes the embryonic genome.

Phosphorylated RNA polymerase II was predominantly nuclear in immature oocytes (Fig. 1). This observation is in the line with the finding that the oocyte is transcriptionally active (Wrenzycki et al., 2007; Tan et al., 2009). During oogenesis, an important transition takes place at the level of gene expression. Transcriptionally active chromatin becomes silenced during meiosis. This suggests that the chromatin becomes modified and that transcription factors are generally excluded from compacted chromosomes. Although the kinase active to phosphorylate the CTD of RNA polymerase II (P-TEFb) is recruited to mitotic chromosomes of somatic cells (Yang et al., 2008; Dey et al., 2009), recruitment to meiotic chromosomes has not been recorded.

After fertilization, both unphosphorylated and phosphorylated RNA polymerase II were clearly present in both the pronucleus and nucleus (Fig. 2). At the pronuclear stage, unphosphorylated RNA polymerase II predominated in both female and male pronuclei. At the middle or late one-cell stage, the level of nuclear unphosphorylated RNA polymerase II increased, in line with the observation that transcription is activated earlier in the male (compared to the female) pronucleus (Bouniol et al., 1995). RNA polymerase II accumulation in pronuclei is contemporaneous with detection of the hyperphosphorylated form of the RNA polymerase II (Bellier et al., 1997); this is also the time at which embryonic genome transcription is activated (Oqani et al., 2012).

RNA polymerase II subunits, and other components of the basal transcription machinery, are maternally contributed to the cytoplasm of the early embryo and are translocated from the cytoplasm to the nucleus just before EGA is triggered. For example, mouse oocytes contain large amounts of RNA polymerase II; the level of the enzyme decreases during oocyte maturation and increases after fertilization, at the end of the one-cell stage; the enzyme level is linked to development of EGA. Translocation of RNA polymerase II from the cytoplasm to the nucleus occurs the late one-cell stage, and preferentially to the male pronucleus (Bellier et al., 1997). It is unclear whether translocation of the basal transcription machinery triggers transcription or whether basal transcription components accumulate in nuclei of the early embryo because of transcriptional activation. We hypothesize that nuclear translocation of factors (including CDK9) required for the transition from transcription initiation to elongation regulate the onset of productive embryonic transcription (Oqani et al., 2011). In the present work, we observed a strong phosphorylated RNA polymerase II signal at the four-cell stage. At this time, major EGA is evident in the pig. Two transcriptional stages have been defined in the mouse embryo; these are a minor transcriptional wave at the one-cell stage and a major transcriptional wave at the two-cell stage (Schultz, 2002). However, analogous transcriptional stages have not been identified in pig embryos. Our results suggest that prominent nuclear accumulation of both unphosphorylated and phosphorylated RNA polymerase II, commencing at the one-cell stage, may indicate existence of minor EGA in the pig embryo, thus similar from what has been observed in the mouse with regard to the major embryonic genome activation.

In conclusion, the results of the present study suggest that porcine oocytes and early embryos are transcriptionally competent; RNA polymerase II is both expressed and functional. Further, optimization of the transcriptional apparatus during early embryogenesis is essential if the embryo is to develop beyond the stage at which EGA is triggered.

Reference

1.Bellier S, Chastant S, Adenot P, Vincent M, Renard JP, Bensaude O (1997): Nuclear translocation and carboxyl- terminal domain phosphorylation of RNA polymerase II delineate the two phases of zygotic gene activation in mammalian embryos. EMBO J 16:6250-6262.
2.Bogolyubova IO (2011): Transcriptional activity of nuclei in 2-cell blocked mouse embryos. Tissue Cell 43: 262–265.
3.Bouniol C, Nguyen E, Debey P (1995): Endogenous transcription occurs at the 1-cell stage in the mouse embryo. Exp Cell Res 218:57-62.
4.Bres V, Yoh SM, Jones KA (2008): The multi-tasking P-TEFb complex. Curr Opin Cell Biol 20:334-340.
5.Dey A, Nishiyama A, Karpova T, McNally J, Ozato K (2009): Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription. Mol Biol Cell 20:4899-4909.
6.Forlani S, Bonnerot C, Capgras S, Nicolas JF (19998): Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication. Development125:3153-3166.
7.Kanka J (2003): Gene expression and chromatin structure in the pre-implantation embryo. Theriogenology 59:3-19.
8.Oqani RK, Kim HR, Zhang JY, Diao YF, Jin DI (2011): The CDK9/Cyclin T1 subunits of P-TEFb in mouse oocytes and preimplantation embryos: a possible ro le in embryonic genome activation. BMC Developmental Biology 11:33
9.Oqani RK, Zhang JY, Lee MG, Diao YF, Ijn DI (2012): Phosphorylation status of RNA polymerase II carboxyl- terminal domain in porcine oocytes and early embryos. Asian-Australasian J Anim Sci 25:789-793.
10.Pirngruber J, Shchebet A, Johnsen SA (2009): Insights into the function of the human P-TEFb component CDK9 in the regulation of chromatin modifications and co-transcriptional mRNA processing. Cell Cycle 8:3636-3642.
11.Price DH (2000): P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol 20:2629-2634.
12.Reese JC (2003): Basal transcription factors. Curr Opin Genet Dev 13:114-118.
13.Schultz RM (2002): The molecular foundations of the maternal to zygotic transition in the preimplantation embryo. Hum Reprod Update 8:323-331.
14.Sikorski TW, Buratowski S (2009): The basal initiation machinery: beyond the general transcription factors. Curr Opin Cell Biol 21:344-351.
15.Sims RJ 3rd, Mandal SS, Reinberg D (2004): Recent highlights of RNA-polymerase-II-mediated transcription.Curr Opin Cell Biol 16:263-271.
16.Tan JH, Wang HL, Sun XS, Liu Y, Sui HS, Zhang J(2009): Chromatin configurations in the germinalvesicle of mammalian oocytes. Mol Hum Reprod 15:1-9.
17.Walser CD, Lipshitz HD (2011): Transcript clearance during the maternal-to-zygotic transition. Curr Opin Genet Develop 21:431-443.
18.Wrenzycki C, Herrmann D, Niemann H (2007): Messenger RNA in oocytes and embryos in relation to embryo viability. Theriogenology 68: 77-83
19.Xie SQ, Martin S, Guillot PV, Bentley DL, Pombo A (2006): Splicing speckles are not reservoirs of RNApolymerase II, but contain an inactive form, phosphorylated on serine2 residues of the C-terminal domain. Mol Biol Cell 17:1723-1733.
20.Yang Z, He N, Zhou Q (2008): Brd4 recruits PTEFb chromosomes at late mitosis to promote G1 gene expression and cell cycle progression. Mol Cell Biol 28:967-976.