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This Article Abstract Full Text (PDF) All Versions of this Article: 69/6/1890    most recent biolreprod.103.018945v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tani, T. Articles by Tsunoda, Y. Search for Related Content PubMed PubMed Citation Articles by Tani, T. Articles by Tsunoda, Y. Agricola Articles by Tani, T. Articles by Tsunoda, Y.

Reprogramming of Bovine Somatic Cell Nuclei Is Not Directly Regulated by Maturation Promoting Factor or Mitogen-Activated Protein Kinase Activity¹

Laboratory of Animal Reproduction, College of Agriculture, Kinki University, Nara, 631-8505, Japan

	ABSTRACT

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Cloned mammals with normal fertility have been produced by nuclear transfer. Thus, oocyte cytoplasm has the ability to convert differentiated somatic cell nuclei into a state that resembles the conditions that occur at fertilization (nuclear reprogramming). Despite the long-held assumption that reprogramming factors are present in mammalian oocytes, the molecular nature of these factors is not known. The present study demonstrates that the process of nuclear reprogramming is not directly regulated by maturation promoting factor or mitogen-activated protein kinase activity. The potential for nuclear-transferred oocytes to develop to the blastocyst stage was not different when somatic cells at the M phase were fused with oocytes activated with ionomycin and cycloheximide 1–5 h before (12%–22%) but was significantly decreased when oocytes were activated 6 h before (1%). Further molecular studies on the differences between oocytes with and without reprogramming potential are required and will be useful for the identification of reprogramming factors.

developmental biology, early development, embryo, gamete biology, gene regulation

	INTRODUCTION

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The nuclei of bovine preimplantation embryos can be reprogrammed in either oocytes at the second metaphase (MII, nonactivated) with high maturation-promoting factor (MPF) activity or in oocytes at the S phase (activated) with low MPF activity [1]. When these nuclei are introduced into nonactivated oocytes, the nuclear membrane breaks down rapidly, and the chromosomes are exposed to oocyte cytoplasm and reprogrammed. When the nuclei of bovine preimplantation embryos are transferred into oocytes at the S phase, the nuclear membrane does not break down, but the chromosomes of donor cells are reprogrammed during nuclear enlargement. Reprogramming of somatic cell nuclei, however, only occurs in nonactivated oocytes [2]. When bovine somatic cells are fused with S-phase oocytes that are parthenogenetically activated 6 h before fusion, the nuclear-transferred oocytes stop developing at the eight-cell stage, which is when bovine embryonic genome activation occurs [3], irrespective of the cell cycle phase of the donor cells. This indicates that bovine MII oocytes have "reprogramming factors" in their cytoplasm, but the activity disappears after parthenogenetic activation. The point at which the ability of oocyte cytoplasm to reprogram somatic cell nuclei is not known. Mammalian oocytes are arrested in the MII phase of meiosis by a cytostatic factor that stabilizes MPF activity [4, 5]. MPF is not inactivated until fertilization, but fertilization can be mimicked by treatment with chemicals and by electric stimulation [6, 7]. Treatment of bovine oocytes with ionomycin induces meiotic release but does not induce pronuclear formation [8]. Combined treatment of bovine oocytes with ionomycin and cycloheximide (CHY) or 6-dimethyl aminopurine (6-DMAP) is effective for pronuclear formation and further preimplantation development [7].

The nature of the reprogramming factors in mammals, however, is not clear. The fact that cultured bovine cumulus cells fused with enucleated MII oocytes are reprogrammed, but not when they are fused with oocytes activated 6 h previously [2], indicates that MPF is a candidate reprogramming factor. Although Gonda et al. [9] recently demonstrated that a single protein (FRGY2) from Xenopus oocytes triggers reversible disassembly of somatic cell nuclei, it is not clear whether the FRGY2 protein can convert somatic cell nuclei to totipotent cells. The mitogen-activated protein kinase (MAPK) cascade, i.e., Mos-MEK (MAPK kinase)-ERK1/2 (extra cellular signal-regulated protein kinase)-p90RSK1, is another principal regulator of transition from the MII phase to pronuclear formation after fertilization or parthenogenetic activation [10]. The present study examined whether the process of nuclear reprogramming is regulated by MPF and MAPK activity.

	MATERIALS AND METHODS

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Nuclear Transfer

Nuclear transfer was performed as described previously [11]. Briefly, immature oocytes were isolated from slaughtered bovine ovaries and cultured in TCM199 (Gibco, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) for 22–24 h. Cumulus cells were passaged seven to 10 times and induced into the G0/G1 phase by serum starvation [11] or into the M phase by nocodazole treatment [2]. Donor cells were electrically fused with nonactivated and activated oocytes whose maternal chromosomes were previously removed by two direct-current (150-V/mm) pulses for 25 µsec with a 0.1-sec interval in Zimmerman fusion medium. When nonactivated oocytes were used, fused oocytes were activated following treatment with 10 µg/ml of CHY (Sigma Chemical Co., St. Louis, MO) with or without 10 µM U0126 (Promega, Madison, WI) or with 2 mM 6-DMAP (Sigma) for 6 or 4 h, respectively. Oocytes whose chromosomes were previously removed were also used 1–6 h after activation by exposure to 5 µM ionomycin for 5 min following treatment with CHY with or without U0126 or 6-DMAP. In this case, CHY, with or without U0126, or 6-DMAP was added to the micromanipulation medium (TCM199 supplemented with 10% FBS), and nuclear-transferred oocytes were further cultured in each medium for activation so that the total treatment time was 6 h (CHY with or without U0126) or 4 h (6-DMAP). Fused and activated oocytes were cultured in vitro for 9 days to examine the developmental potential as reported previously [2]. Each experiment was repeated more than three times, and the development to two-cell and eight-cell, morula, and blastocyst stages was evaluated on Days 3, 6–7, and 8–9, respectively. (Day 1 was the day of nuclear transfer.) To examine the morphologic changes of donor nuclei, nuclear-transferred oocytes were mounted on slides, fixed, and stained with 1% aceto orcein 3, 6, and 15 h after fusion. The cross-sectional area of nuclei in 30 oocytes was measured in each group using Quantity One software (Bio-Rad, Tokyo, Japan).

Western Blotting

Ten oocytes frozen on dry ice with cell lysis buffer (Cell Signaling Technology, Beverly, MA) were added to Laemmli buffer and boiled at 100°C for 4 min. SDS-PAGE was conducted using 10% polyacrylamide gels (Bio-Rad) and analyzed by Western blotting with antibodies specific for phospho-p90RSK1, phospho-MEK1/2, phospho-ERK1/2, MEK1/2 (Cell Signaling Technology), p90RSK1, ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-rabbit horseradish peroxidase-conjugated secondary antibody (Bio-Rad) as described previously [12].

Cdc2 Kinase Assay

MPF activity was determined by cdc2 kinase assay using a cdc2 kinase ELISA kit (MBL, Nagoya, Japan) as described previously [13]. This assay uses a synthetic peptide as a substrate for cdc2 kinase and a monoclonal antibody that recognizes the phosphorylated form of the peptide substrate. Ten oocytes were used for each group, and each assay was repeated five times.

Data Analysis

The developmental rates were analyzed by ² analysis, and the sizes of the nuclei were compared using the Student t-test.

	RESULTS

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We examined the point at which the ability of oocytes to reprogram somatic cell nuclei disappears after parthenogenetic activation by ionomycin exposure following CHY treatment (Table 1). The developmental potential of nuclear-transferred oocytes is clearly different between donor cells at the G0/G1 phase and those at the M phase. The potential of oocytes receiving donor cells from the G0/G1 phase, which have nuclear membranes, to develop to the blastocyst stage decreased significantly when nuclei were fused with enucleated oocytes activated 1 h before. The developmental potential of oocytes receiving somatic cells at the M phase was not different when somatic cells were fused with enucleated oocytes activated 1–5 h before but significantly decreased when oocytes were activated 6 h before.

Inactivation of MPF and the MAPK Cascade After Parthenogenetic Activation

MPF activity, determined by cdc2 kinase in oocytes whose maternal chromosomes were present (Fig. 1A) or previously removed (Fig. 1B), decreased 1 h after activation with ionomycin and CHY, and pronuclear formation occurred in all parthenogenetic oocytes 5 h after treatment. Therefore, MPF is not a candidate reprogramming factor in somatic cells.

View larger version (64K): [in this window] [in a new window]   FIG. 1. MAP kinase cascade and inactivation of cdc2 kinase after activation stimuli in MII oocytes with (A) and without (B) the maternal chromosomes by Western blotting. Oocytes were activated by ionomycin and CHY without (left) or with (middle) U0126 and ionomycin and 6-DMAP (right) for 0–6 h. X-axis in A and B show hours after activation. The bar graph in the lower figure of A shows the percentage of oocytes with pronucleus(ei). Ten oocytes were used in each treatment for Western blotting. Each treatment was repeated at least three times and produced consistent results. For the cdc2 kinase assay, 10 oocytes were used and each treatment was repeated three times

We examined the effect of U0126, a specific MEK inhibitor, and 6-DMAP, a common protein kinase inhibitor, on the developmental potential of nuclear-transferred oocytes and p90RSK1, MEK 1/2, ERK 1/2, and MPF activity. MPF activity decreased 1 h after activation in both groups, but formation of the pronucleus started 1 (CHY plus U0126) or 2 (6-DMAP) h earlier than in the CHY treatment group. Dephosphorylation of p90RSK1, MEK 1/2, and ERK 1/2 was not observed in enucleated oocytes 5 h after treatment with CHY plus U0126 (Fig. 1B). The number of nuclear-transferred oocytes that developed into blastocysts was significantly lower than that of nuclear-transferred oocytes that were treated for 4 h (Table 2). When enucleated oocytes were treated with 6-DMAP, dephosphorylation of p90RSK1, MEK 1/2, and ERK 1/2 was not observed 4 h after treatment. None of the oocytes treated with 6-DMAP for 4 h that received cumulus cells at the M phase developed into blastocysts (Table 2). The discrepancy between the dephosphorylation of p90RSK1, MEK 1/2, and ERK 1/2 and the development of nuclear-transferred oocytes to the blastocyst stage suggests that reprogramming of the somatic cells is not controlled directly by the MAPK cascade.

Although the pattern of decreasing cdc2 kinase activity in oocytes was the same whether or not maternal chromosomes were removed and regardless of the type of activation procedure used, MAPK activity was different (Fig 1A and B). Dephosphorylation of MAPK in oocytes without maternal chromosomes was delayed at least 1 h irrespective of the activation procedure used.

Nuclear Enlargement Is Necessary for Nuclear Reprogramming

Swelling of the donor nucleus is necessary for successful nuclear reprogramming [14, 15]. When nuclei at the G0/G1 phase were fused with enucleated oocytes at the M phase (0 h after activation, G0/G1-M), the nuclear membranes of the donor cells were broken down but reformed following parthenogenetic activation. The sizes of the reformed nuclei increased up to 1300 µm2 15 h after fusion (Fig. 2A and B). When nuclei were fused with enucleated oocytes activated 3 (G0/G1-S3) and 6 (G0/G1-S6) h before, the sizes of donor cell nuclei increased slightly. When cumulus cells at the M phase were fused with either oocytes at the M phase (M-M) or oocytes activated 3 h (M-S3) before, the nuclear size increased up to 3000 µm2 15 h after fusion. When donor cells were fused with oocytes activated 6 h (M-S6) before, however, the nuclear size was approximately 1000 µm2 and significantly smaller than that in M-M and M-S3. This finding suggests that the factor(s) involved in nuclear swelling remained at a high level in oocytes even 3 h after activation but decreased 6 h after activation.

View larger version (35K): [in this window] [in a new window]   FIG. 2. Donor nuclei at the G0/G1 or M phase transferred into oocytes, nonactivated (M) or activated 3 or 6 h previously (S3, S6). Oocytes were stained at 3, 6, or 15 h after fusion. A) Morphological change of the nucleus. When a nucleus at the G0/G1 phase was fused with a nonactivated oocyte, the Y axis indicates G0/G1-M. B) Average size of the nucleus of 30 nuclear-transferred oocytes with SD. The group with a different superscript was significantly different (P < 0.005)

	DISCUSSION

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The term nuclear reprogramming is ambiguous. We define the term as the acquisition by nuclei of the ability to produce adult nuclear transplant animals with normal fertility. Under our experimental conditions, enucleated bovine oocytes receiving somatic cells at the G0/G1 phase develop into blastocysts (23%–53%) [11, 16] and also calve (14%–80%) [11, 16] at a relatively high rate compared with the low potential of nuclear-transferred mouse oocytes to develop into young [17] (1%–5%). The ability of nuclear-transferred oocytes to develop into blastocysts is a reliable index of nuclear reprogramming in bovine. Moreover, activated bovine oocytes that receive somatic cells do not exclude the polar body, leading to tetraploid chromosome constitution when nuclei at the M phase are used. Therefore, the present study was performed based on blastocyst development. We propose that two steps are required for the nuclear reprogramming of somatic cells: first, modification of chromosomes by reprogramming factor(s), and second, modification by swelling factor(s).

The ability to reprogram somatic cell nuclei in nonactivated oocyte cytoplasm disappears suddenly 6 h after parthenogenetic activation with ionomycin and CHY. The developmental potential of oocytes receiving somatic cells at the G0/G1 phase decreases significantly 1 h after activation. This decline is not due to a lack of reprogramming factors in oocytes but rather to low MPF activity. MPF activity in oocytes with or without maternal chromosomes decreases to basal levels 1 h after activation; therefore, the nuclear membrane of most somatic cells is not broken down and donor chromosomes are not exposed directly to the oocyte cytoplasm. Because enucleated oocytes receiving somatic cells at the M phase developed into blastocysts even 5 h after activation, by which time MPF activity is low enough for pronuclear formation (Fig. 1A), MPF is not necessary for reprogramming somatic cells. For donor cells not at the M phase, MPF is necessary for nuclear membrane breakdown. When somatic cells at the G0/G1 phase are fused with enucleated oocytes activated 1–5 h before, a small proportion of nuclear-transferred oocytes develop into blastocysts. This might be due to variations in the sensitivity of oocytes to activation stimuli rather than to the possibility that the reprogramming factors passed the nuclear membrane of donor cells as does the replication factor [18]. The data in the present study, however, do not exclude the possibility that essential events that occur before 1 h after activation might be required for reprogramming of G0/G1 nuclei, whereas other events that occur before 6 h might be required for M phase nuclei.

For nuclear reprogramming, the donor cells of chromosomes modified by reprogramming factors should be further modified during nuclear swelling. Although the presence of nucleoplasmin has not been reported in mammalian oocytes, it is a candidate swelling factor that moves into the donor nucleus. Previous studies of chromatin remodeling by frog egg extract demonstrated that nucleoplasmin is required for the loss of the somatic linker histone H1, its replacement by oocyte-specific histone B4 and chromatin structural protein HMG [19], and release of TATA-binding protein from somatic cell nuclei [20].

The significant decline in the ability of nuclear-transferred oocytes to develop into blastocysts occurs at a slightly different time course when different parthenogenetic activation procedures are used (Tables 1 and 2). When enucleated oocytes are activated with ionomycin and CHY, the developmental potential decreases 6 h after treatment. When oocytes are treated with ionomycin following treatment with either CHY and U0126 or 6-DMAP, the developmental potential decreases at 5 and 4 h after treatment, respectively. These results indicate that MAPK activity is related to nuclear reprogramming. Nuclear reprogramming cannot be directly regulated by MAPK activity, however, because nuclear-transferred oocytes in which MEK 1/2, ERK 1/2, and p90RSK1 were active did not develop into blastocysts. The possibility that other kinases might be involved, however, is not excluded. The fact that the dephosphorylation of MAPK in oocytes without maternal chromosomes was delayed indicates that the delay might have been due to a spindle-dependent dephosphorylation deficiency [21].

The present study clearly demonstrates that reprogramming of bovine somatic cell nuclei is not directly regulated by MPF or MAPK activity in oocytes. To identify unknown reprogramming factors, further molecular studies on the differences between oocytes with and without reprogramming potential of somatic cells are necessary.

	ACKNOWLEDGMENTS

 
We thank Dr. Anne McLaren at the Wellcome/CRC Institute in Cambridge, England, for critical review of the manuscript.

	FOOTNOTES

 
¹ This work was supported by grants from the Ministry of Education, Science, and Culture (13308054, 14034259, 15039233) and the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

² Correspondence: Yukio Tsunoda, Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204, Nakamachi, Nara, 631-8505 Japan. FAX: 81 742 43 1155; tsunoda{at}nara.kindai.ac.jp

Received: 6 May 2003.

First decision: 19 May 2003.

Accepted: 28 July 2003.

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This Article Abstract Full Text (PDF) All Versions of this Article: 69/6/1890    most recent biolreprod.103.018945v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tani, T. Articles by Tsunoda, Y. Search for Related Content PubMed PubMed Citation Articles by Tani, T. Articles by Tsunoda, Y. Agricola Articles by Tani, T. Articles by Tsunoda, Y.