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Biology of Reproduction 67, 928-934 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Germinal Vesicle Material Is Essential for Nucleus Remodeling after Nuclear Transfer1

Shaorong Gaoa, Bianca Gasparrinia, Michelle McGarrya, Tricia Ferriera, Judy Fletchera, Linda Harknessa, Paul De Sousaa, and Ian Wilmut2,a

a Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, Scotland, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.

early development, embryo, gamete biology, implantation, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloned mammals have been generated through introducing somatic or embryonic stem cell nuclei into enucleated metaphase II (MII) oocytes. Thus far, only oocytes enucleated at MII or soon after preactivation have been shown to fully reprogram transferred nuclei [1–7], including serial nuclear transfer [8]. Zygote cytoplasm is not considered suitable for nuclear transfer [9]. So the question arose, is it possible to enucleate early stage oocytes such as germinal vesicle (GV) or pro-metaphase I (pro-MI) stage oocytes before they mature to the MII stage and use them as recipients for nuclear transfer?

In mammals, oocytes are arrested naturally in the first prophase with a very large nucleus called the GV. Following hormone stimulation, GV breakdown (GVBD) allows the mixing of GV material with cytoplasm and is thought to be necessary for sperm chromatin decondensation and cleavage in fertilized eggs [10, 11]. A number of classic studies have shown that enucleated amphibian oocytes and anucleate mouse oocyte fragments undergo maturation promoting factor (MPF) activation soon after release from prophase I arrest, as semiquantitatively indicated by their cytoplasmic activities, to induce GVBD in recipient immature oocytes [12–16]. In a recent study, GV material was essential for MPF reactivation in Xenopus oocytes [17]. Besides MPF, another protein kinase, mitogen-activated protein kinase (MAPK), may be important in regulating meiotic maturation of mammalian oocytes [18, 19]. However, GV material was not necessary for the activation of MAPK in porcine oocytes [20]. In another study, the mixing of GV material with cytoplasm was not essential for the initiation of protein reprogramming process during maturation; no differences in protein synthesis were apparent between oocytes and enucleated oocytes after release from prophase I [21, 22].

The purpose of this study was to determine whether oocytes enucleated at GV or pro-MI stage can be used as recipients for nuclear transfer, thus answering the question of whether GV material is essential for nuclear remodeling. The oocytes were enucleated at GV or pro-MI stage and cultured to MII as recipients for nuclear transfer. Immunocytochemical staining was employed to observe cytoskeleton organization and DNA changes of reconstructed oocytes following injection of embryonic stem (ES) cells and activation. The results indicate that GV material is essential for nucleus remodeling; no pseudopronuclei were formed in GV stage enucleated oocytes after nuclear transfer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

All inorganic and organic compounds were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.

ES Cell and Culture Conditions

The ES cell line R1 [23] was provided by Dr. Lesley Forrester at passage 14 and was grown for another five passages at the institute. Thus, the R1 cells used in the present experiments were at passage 19. The ES cells were cultured in Dulbecco modified Eagle medium (MEM; Gibco BRL [Life Sciences Ltd.], Paisley, Scotland, U.K.) supplemented with 15% heat-inactivated fetal calf serum (FCS), 1000 units/ml of leukemia inhibitory factor, 2 mM L-glutamine, 1% MEM nonessential amino acids solution (Gibco BRL), and 1% ß-mercaptoethanol. One night before the experiment, the serum concentration was reduced to 5%. Small (<10 µm) ES cells presumably at G1 stage were selected for nuclear transfer.

Collection of Oocytes

The experiments were conducted following approval by the Roslin Institute Animal Ethics Committee and within a project license issued under the Animal (Scientific Procedures) Act of 1986.

B6D2F1 female mice, 8–10 wk old, were used in all experiments. For collection of GV oocytes, the ovaries were removed from the female mice 42–44 h after eCG injection. Antral follicles were punctured by 30-ga needles, and cumulus-enclosed GV oocytes were released into Hepes-buffered CZB medium (HCZB) [24] containing 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) to inhibit GVBD. GV oocytes were alternatively precultured in MEM containing IBMX for 1 h to increase perivitelline space (PVS) or cultured in MEM for 2 h to allow GVBD [25]. Cumulus cells were removed by pipetting, and cumulus-free oocytes were selected for enucleation. After 2 h of culture in MEM, pro-MI oocytes were freed of cumulus cells and selected for enucleation. For collection of mature oocytes, oviducts were removed from the female mice 13–15 h after hCG injection. Cumulus-oocyte complexes were released into HCZB containing 0.1% bovine testicular hyaluronidase (300 USP units/mg; ICN Biomedicals Inc., Aurora, OH). Cumulus cells were dispersed by a 5-min treatment with hyaluronidase, and MII oocytes were kept in CZB before enucleation.

Enucleation

Enucleation was performed under an inverted microscope equipped with Eppendorf TransferMan NK micromanipulator. The method used to remove GV was described previously [26]. A group of GV oocytes were transferred to a droplet of HCZB containing 0.2 mM IBMX and 5 µg/ml of cytochalasin B. The PVS-enlarged oocyte was held by a holding pipette and the zona pellucida was cut by a sharp glass needle (Fig. 1A). An enucleation pipette with an inner diameter of 25 µm was used to go through the same cut and to remove the GV with a minimal volume of cytoplasm. Enucleation of pro-MI and MII oocytes was carried out as previously reported [2]. A group of oocytes was transferred to a drop of HCZB containing 5 µg/ml cytochalasin B (CB). A small pipette with an inner diameter of 6–8 µm was attached to a Piezo drill miromanipulator controller (PMM 150; Prime Tech Ltd., Ibaraki, Japan). After applying several Piezo pulses to the tip of the pipette, a hole was drilled in the zona. Pro-MI or MII spindle, distinguished as a translucent spot in the cytoplasm (Fig. 1C, arrow), was drawn into the pipette with a small amount of cytoplasm and gently pulled away from the oocyte until a stretched cytoplasmic bridge was pinched off. After enucleation, the GV and pro-MI cytoplasm were cultured in MEM containing 10% FCS for another 14 and 13 h, respectively. MII cytoplasm was kept in CZB before nuclear transfer.



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  		FIG. 1. Enucleation of oocytes at the GV and MII stages. A) Zona of GV oocyte is cut by a sharp needle. B) GV is removed by a pipette. C) MII spindle is visible, and zona is drilled by Piezo pulses. D) MII spindle with small amount of cytoplasm is removed by a pipette. Bar = 50 µm

Nuclear Transfer

Small drops of ES cell suspension were mixed well with 10% polyvinylpyrrolidone (360 kDa; ICN) in HCZB. A small (<10 µm) R1 ES cell was drawn in and out of the injection pipette (inner diameter 5 µm) until the cell membrane was broken and separated from the nucleus. After an ES cell nucleus was drawn deep into the pipette, the same pipette was used to pick up several cell nuclei. Within a few minutes, four to six nuclei were lined up inside the pipette. These nuclei were injected one by one into the three kinds of cytoplasm. After injection, the reconstructed oocytes were kept in CZB for 1–3 h before activation.

Activation and Embryo Culture

Reconstructed oocytes were activated for 5–6 h in Ca2+-free CZB containing 10 mM Sr2+ and 5 µg/ml CB to inhibit polar body release [2, 27]. After activation, the surviving embryos with pseudopronuclei were collected and cultured in CZB to the morula/blastocyst stage for embryo transfer.

Embryo Transfer and Cesarean Section

Morula/blastocyst embryos (72–74 h after activation) were transferred into uteri of pseudopregnant surrogate mothers that had been mated with vasectomized male mice 2.5 days previously. Five to 10 morula/blastocyst embryos were transferred into each uterus horn. Recipient mothers were killed at 19.5 days postcoitum, and the pups were quickly removed from the uteri. After cleaning of fluid from their air passages, pups were kept in a warm box supplied with oxygen. Surviving pups were raised by lactating mothers.

Immunocytochemical Staining of Reconstructed Oocytes

At different time points following injection and activation, reconstructed oocytes were processed for immunocytochemical staining to observe cytoskeletal organization and DNA configuration. Oocytes were fixed and extracted for 30 min at 37°C in a microtubule stabilization buffer (0.1 M Pipes, pH 6.9, 5 mM MgCl2·6H2O, 2.5 mM EGTA) containing 2% formaldehyde, 0.5% Triton X-100, 50% deuterium oxide, and 1 mM dithiothreitol. After washing three times in a blocking solution of PBS containing 10% normal goat serum, 0.1% Triton X-100, and 0.02% sodium azide, the oocytes were stored at 4°C until processed. To evaluate microtubule and microfilament dynamics and chromatin configuration, multiple fluorescence labeling using triple-stain analysis was performed. Oocytes were incubated with fluorescein isothiocyantate-conjugated anti-{alpha}-tubulin antibody and rhodamine phalloidin (1:4000; Molecular Probes, Eugene, OR) in a blocking solution of PBS containing 5% normal goat serum in the dark at 37°C for 1 h. After washing three times in the blocking solution, the oocytes were mounted in vectashield containing 5 µg/ml of 4',6'-diamidino-2-phenylindole (DAPI). Labeled oocytes were viewed using an Axiovert S 100 photomicroscope, Zeiss (Imaging Associates Ltd., Thame, U.K.) equipped with fluorescein, Zeiss 487709; Texas red, Zeiss 487714; and Hoechst, Zeiss 487702 (Imaging Associates Ltd.) selective filter sets and a 50-W mercury arc lamp using a 40x Neofluar objective. Images were acquired using the Kinetic Imaging System (Imaging Associates Ltd.).

Statistical Analysis

The results were evaluated using a chi-square test. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Microtubule Patterns and Nucleus Changes after Nuclear Transfer

Microtubule patterns and nucleus changes following ES cell injection into mature cytoplasm enucleated at the GV stage are shown in Figure 2, A–D. The nucleus remained intact 5 min after injection, and no spindle started to organize (Fig. 2A). A spindle started to organize 30 min after injection, and condensed chromosomes formed (Fig. 2B). A well-organized spindle that resembled an MI oocyte spindle formed 1 h after injection, but the chromosomes appeared disorganized (Fig. 2C). Chromosome distribution throughout the cytoplasm was observed 3 h after injection (Fig. 2D, arrow). In contrast to GV cytoplasm, microtubule organization and nucleus changes in pro-MI cytoplasm were comparable to those of MII cytoplasm injected with ES cell nuclei. The nucleus remained intact after injection for 5 min before a spindle started to organize and associate with condensed chromosomes 30 min after injection (Fig. 2, E–H). A well-organized spindle that resembled an MII oocyte spindle formed and chromosomes aligned on the metaphase plate 1 h after injection. Microtubule organization and DNA configuration in MII cytoplasm injected with ES cell nuclei are shown in Figure 2, I–L.



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  		FIG. 2. Microtubule patterns and DNA configuration in nuclear transfer oocytes. A–D) Mature GV stage enucleated oocytes injected with ES cell nuclei. A) Nucleus remained intact 5 min after injection. A) Spindle started to organize 30 min after injection. C and D) Well-organized spindle with disorganized chromosomes in the cytoplasm 1–3 h after injection. Arrowheads show the chromosomes distributed throughout the cytoplasm. E–H) Microtubule and DNA organization in the mature pro-MI stage enucleated oocytes after injection with ES cells. I–L) Microtubule and DNA organization in enucleated MII oocytes injected with ES cells. Well-organized spindle and chromosomes aligned on the metaphase plate are visible in both types of cytoplasm. Bar = 30 µm

Microtubule Patterns and DNA Configuration During Activation

Calcium-free CZB containing 10 mM Sr2+ and 5 µg/ml CB was used as activation medium. Microtubule patterns and DNA configuration of reconstructed oocytes with GV cytoplasm are shown in Figure 3, A–C. The spindle remained intact and some scattered chromosomes could be detected in the cytoplasm 1 h after activation (Fig. 3A, arrow). Chromosomes separated into two groups and moved toward their respective poles 3 h after activation (Fig. 3B), and the oocyte was at the telophase stage. The two groups of chromosomes remained condensed and a spindle continued to be assembled 5 h after activation, with no pseudopronuclei formation observed (Fig. 3C). In contrast to the process in GV cytoplasm, DNA and microtubule dynamics in pro-MI cytoplasm injected with ES cells was very different. Chromosomes were moving toward their respective poles and the reconstructed oocyte was at the anaphase stage 1 h after onset of activation (Fig. 3D). Two sets of separated chromosomes were starting to decondense 3 h after activation (Fig. 3E). Two large decondensed pseudopronuclei formed 5 h after activation, and the spindle remained visible (Fig. 3F). Microtubule and DNA changes in MII cytoplasm injected with ES cells during activation are shown in Figure 3, G–I. The reconstructed oocyte was at the telophase stage and two groups of chromosomes were at their respective poles 1 h after activation (Fig. 3G). Thereafter, DNA and microtubule patterns resembled a pro-MI cytoplasm reconstructed oocyte (Fig. 3, H and I). In MII reconstructed oocytes, more microfilaments were distributed in the cortex than in the GV and pro-MI reconstructed oocytes (Fig. 3, A–E).



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  		FIG. 3. Cytoskeleton organization and DNA configuration in reconstructed oocytes during activation. A–C) Enucleated GV oocytes reconstructed with ES cells during activation. No pronuclei formation is visible, and chromosomes are segregated into two groups. D–F) Enucleated pro-MI oocytes reconstructed with ES cells during activation. G–I) Reconstructed MII oocytes. Pseudopronuclei formation is similar in both pro-MI and MII reconstructed oocytes after activation. MII reconstructed oocytes showed more microfilament distributed in the cortex than do the other two types of reconstructed oocytes. Bar = 30 µm

Developmental Potential of Nuclear Transfer Embryos

Approximately 85%–95% of enucleated oocytes survived after injection of ES cells into all three types of cytoplasm. However, the survival rate using MII cytoplasm was slightly higher than that for the other two types of cytoplasm. All reconstructed oocytes from MII cytoplasm survived activation; however, only 74% and 73% of reconstructed oocytes from GV and pro-MI cytoplasm, respectively, survived activation (Table 1). No reconstructed oocytes from GV cytoplasm were activated after strontium treatment. There were no differences in pseudopronuclei formation between MII and pro-MI reconstructed oocytes after activation. Among the embryos that underwent the first cleavage, MII reconstructed embryos showed a much higher subsequent development rate than did pro-MI reconstructed embryos (Table 1). After further culture, only 8.1% of pro-MI reconstructed embryos developed to morulae and then degenerated. In contrast, 53.5% of MII reconstructed embryos developed to the morula/blastocyst stage. After embryo transfer, six pups were recovered from the surrogate mothers at 19.5 days postcoitum.


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  		TABLE 1. Development of embryos reconstructed by injection of R1 ES cell nuclei into mature cytoplasm enucleated at different stages


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, GV material was essential for nuclear remodeling after nuclear transfer. When mature GV stage enucleated oocytes were used as recipients for nuclear transfer, reconstructed oocytes could not form pseudopronuclei after activation treatment, and chromosomes remained condensed. Although reconstructed embryos from mature pro-MI stage enucleated oocytes could form pseudopronuclei normally, very few embryos cleaved and developed to morulae. In contrast, a very high percentage of embryos reconstructed by using MII cytoplasm developed to the blastocyst stage, and 5% of transferred embryos developed to term.

In animal cloning experiments, nuclear transfer has been performed mostly with enucleated MII oocytes that were subsequently activated to initiate embryo development. The transferred nucleus undergoes disassembly and reassembly in the MII cytoplasm [28]. Disassembly involves nuclear envelope and lamina breakdown and chromatin condensation [29–33]. Following activation of the reconstructed oocyte, nuclear reassembly involves decondensation of the chromatin, formation of a new nuclear envelope, polymerization of a new lamina, formation of nucleoli and expansion of the remodeled nucleus [34]. In the present study, when a mature GV stage enucleated oocyte was used as a recipient for nuclear transfer the transferred ES cell nucleus could be disassembled, as indicated by chromatin condensation. However, following activation the nucleus could not be fully remodeled; no nucleolus formation and no expansion of the nucleus was observed in the reconstructed oocyte. Disassembly of the transferred nucleus is related to the level of MPF activity, and others have demonstrated that the anucleate oocyte can undergo MPF activation [12–16]. Our results also indicate that GV material is not required for MPF activation because the spindle can organize and the nucleus can condense into chromosomes 1–3 h after injection of the ES cell nucleus into a mature GV stage enucleated oocyte. However, following activation treatment no pseudopronuclei formation was observed in the reconstructed oocyte. These results suggest that the GV material is responsible for the reassembly of the transferred nucleus. Results from previous studies on amphibians suggest that nuclear components stored in the oocyte GV facilitate remodeling of somatic nuclei, and the candidate molecules include nuceloplasmin and N1/N2 [35–38]. Both of these molecules can mediate the transfer of the core histones to DNA and the assembly of nucleosomes [39]. Nucleoplasmin is important in removing sperm protamines and decondensing the sperm chromatin to allow the assembly of the paternal pronucleus [40, 41]. To elucidate the mechanism of reprogramming, we must determine which molecules, such as nucleoplasmin and others, are released from the GV at GVBD in mammal oocytes

In contrast to GV stage enucleated oocytes, the transferred nucleus can undergo disassembly and reassembly in the mature pro-MI stage enucleated oocyte. Similar to the MII enucleated oocyte, a bipolar spindle formed and condensed chromosomes aligned on the metaphase plate 1–3 h after nuclear transfer. This situation is different from that in a previous report [7], in which the authors suggested that the nucleus condensed into a disorganized chromosome array when a small ES cell (G1/G0 stage) nucleus was transferred into MII cytoplasm. Only a large cell (G2) nucleus could condense to form an orderly chromosome array resembling an MII plate. A different staining method used in this study showed the association of chromosomes and spindle much more directly.

Compared with MII reconstructed embryos, the developmental potential of reconstructed embryos with mature pro-MI cytoplasm is very limited. Microfilament distribution in the pro-MI reconstructed embryos was very different from that of MII reconstructed embryos, and less microfilament distribution was observed in pro-MI reconstructed embryos. Actin is a major component of the cytoskeleton. Early studies suggested that actin filaments were responsible for maintenance of the meiotic spindle, spindle rotation, polar body release, pronuclei migration, and mitotic cleavage [42–46]. The decreased microfilament distribution may be one important reason for the poor development of reconstructed embryos with mature pro-MI cytoplasm. In previous studies, embryos produced by in vitro fertilization of in vitro-matured oocytes showed very different microfilament distribution and limited developmental ability compared with in vivo-produced embryos [47, 48]. Another possible reason for the poor development in pro-MI reconstructed embryos could be that enucleated oocytes were matured without nuclei. Nuclear-cytoplasm coordinated maturation is believed to be necessary for full maturation of oocytes [49]. Cumulus cell could also be a factor in the poor development of pro-MI reconstructed embryos; the pro-MI cytoplasts were matured in vitro without cumulus cells. In previous studies, cumulus cells were important for oocyte maturation and further development after fertilization [50–53].

The results of the present study demonstrated that GV material is essential for nucleus remodeling after nuclear transfer. More work is needed to determine what factors are released from the GV during GVBD, as this information will enable us to better understand the molecular basis of nuclear reprogramming.


   ACKNOWLEDGMENTS
 
We thank Miss Heather A. Warnock and Louise V. Rahr from the small animal unit of Roslin Institute for assistance with cesarean sections and pup care.


   FOOTNOTES
 
First decision: 4 March 2002.

1 This project was funded by Geron Biomed. Back

2 Correspondence. FAX: 44 0 131 527 4493; ian.wilmut{at}bbsrc.ac.uk Back

Accepted: April 15, 2002.

Received: February 14, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wilmut I, Schnieke AE, McWhirl J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997 385:810-813[CrossRef][Medline]
  2. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998 394:369-374[CrossRef][Medline]
  3. Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda Y. Eight calves cloned from somatic cells of a single adult. Science 1998 282:2096-2098
  4. Baguisi A, Behboodi E, Melican D, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y. Production of goats by somatic nuclear transfer. Nat Biotechnol 1999 17:456-461[CrossRef][Medline]
  5. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC. Pig cloning by microinjection of fetal fibroblast nuclei. Science 2000 289:1188-1190[Abstract/Free Full Text]
  6. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walke S, Ayares DL, Colman A, Campbell KH. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000 407:505-509
  7. Wakayama T, Rodriguez I, Perry AC, Yanagimachi R, Mombaerts P. Mice cloned from embryonic stem cells. Proc Natl Acad Sci U S A 1999 96:14984-14989[Abstract/Free Full Text]
  8. Ono Y, Shimozawa N, Ito M, Kono T. Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol Reprod 2001 64:44-50[Abstract/Free Full Text]
  9. Wakayama T, Tateno H, Mombaerts P, Yanagimachi R. Nuclear transfer into mouse zygotes. Nat Genet 2000 24:108-109[CrossRef][Medline]
  10. Katagiri C, Moriya M. Spermatozoan response to the toad egg matured after removal of germinal vesicle. Dev Biol 1976 50:235-241[Medline]
  11. Balakier H, Tarkowski AK. The role of germinal vesicle karyoplasms in the development of the male pronucleus in the mouse. Exp Cell Res 1980 128:79-85[CrossRef][Medline]
  12. Masui Y, Markert CL. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zool 1971 177:129-145[CrossRef][Medline]
  13. Schorderet-Slatkine S, Drury KC. Progesterone induced maturation in oocytes of Xenopus laevis. Appearance of a ‘maturation promoting factor’ in enucleated oocytes. Cell Differ 1973 2:247-254[CrossRef][Medline]
  14. Reynhout JK, Smith LD. Studies on the appearance and nature of a maturation-inducing factors in the cytoplasm of amphibian oocytes exposed to progesterone. Dev Biol 1974 38:394-400[CrossRef][Medline]
  15. Masui Y, Meyerhof PG, Ziegler DH. Control of chromosome behavior during progesterone induced maturation of amphibian oocytes. J Steroid Biochem 1979 11:715-722[CrossRef][Medline]
  16. Skoblina MN, Pivnitsky KK, Kondratieva OT. The role of germinal vesicle in maturation of Pleurodeles waltlii oocytes induced by steroids. Cell Differ 1984 14:153-157[CrossRef][Medline]
  17. Iwashita J, Hayano Y, Sagata N. Essential role of germinal vesicle in the meiotic cell cycle of Xenopus oocytes. Proc Natl Acad Sci U S A 1998 95:4392-4397[Abstract/Free Full Text]
  18. Choi T, Rulong S, Resau J, Fukasawa K, Matten W, Kuriyama R, Mansour S, Ahn N, Vande Woude CF. Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analysing spindle formation during meiosis I. Proc Natl Acad Sci U S A 1996 93:4730-4735[Abstract/Free Full Text]
  19. Inoue M, Naito K, Nakayama T, Sato E. Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in porcine oocytes. Biol Reprod 1998 58:130-136[Abstract/Free Full Text]
  20. Sugiura K, Naito K, Iwanori N, Kagii H, Goto S, Ohashi S, Yamanouchi K, Tojo H. Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturation. Mol Reprod Dev 2001 59:215-220[CrossRef][Medline]
  21. Sun FZ, Moor RM. Nuclear-cytoplasmic interactions during ovine oocyte maturation. Development 1991 11:171-180
  22. Meng L, Rutledge J, Zhu Y, Kidder GM, Khamsi F, Armstrong DT. Role of germinal vesicle in protein synthesis in rat oocyte during in vitro maturation. Mol Reprod Dev 1996 43:228-235[CrossRef][Medline]
  23. Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Rodder JC. Derivation of completely cell-culture derived mice from early-passage embryonic stem cell. Proc Natl Acad Sci U S A 1993 90:8424-8428[Abstract/Free Full Text]
  24. Chatot CL, Ziomet A, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989 86:679-688[Abstract]
  25. Schroeder AC, Eppig JJ. The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev Biol 1984 102:493-497[CrossRef][Medline]
  26. Tsunoda Y, Yasui T, Nakamura K, Uchida T, Sugie T. Effects of cutting the zona pellucida on the pronuclear transplantation in the mouse. J Exp Zool 1986 240:119-125[CrossRef][Medline]
  27. Eggan K, Akutsu H, Loring J, Jacksom-Grusby L, Klemm M, Rideout III WM, Yanagimachi R, Jaenisch R. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 2001 98:6209-6214[Abstract/Free Full Text]
  28. Collas P. Cytoplasmic control of nuclear assembly. Reprod Fertil Dev 1998 10:581-592[CrossRef][Medline]
  29. Szolloisi S, Czolawska R, Soltynska MS, Tarkowski AK. Remodelling of thymocyte nuclei in activated oocytes: an ultrastructural study. Eur J Cell Biol 1986 42:140-151[Medline]
  30. Szolloisi S, Czolawska R, Szolloisi MS, Tarkowski AK. Remodelling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes. J Cell Biol 1988 91:603-613
  31. Collas P, Robl JM. Relationship between nuclear remodelling and development in nuclear transplant rabbit embryos. Biol Reprod 1991 45:455-465[Abstract]
  32. Barnes FL, Collas O, Powell R, King WA, Westhusin M, Sheperd D. Influence of recipient oocyte cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution and development in nuclear transplant bovine embryos. Mol Reprod Dev 1993 36:33-41[CrossRef][Medline]
  33. Campbell KH, Ritchie WA, Wilmut I. Nuclear-cytoplasmic interactions during the first cell cycle of nuclear transfer reconstituted bovine embryos: implantation for deoxyribonucleic acid replication and development. Biol Reprod 1993 49:933-942[Abstract]
  34. Stice SL, Robl JM. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod 1988 39:657-664[Abstract]
  35. Gurdon JB. Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J Embryol Exp Morphol 1968 20:401-414[Medline]
  36. Gurdon JB, Laskey RA, De Robertis EM, Partington GA. Reprogramming of transplanted nuclei in amphibia. Int Rev Cytol Suppl 1979 9:161-178[Medline]
  37. Laskey RA, Honda BM, Mills AD, Finch JT. Nucleosomes are assembled by an acidic protein which binds histones and transfer them to DNA. Nature 1978 275:416-420[CrossRef][Medline]
  38. Kleinschmidt JA, Dingwall C, Maier G, Franke WW. Molecular characterization of a karophilic histone-binding protein: cDNA cloning, amino acid sequence and expression of nuclear protein N1/N2 of Xenopus laevis. EMBO J 1986 5:3547-3552[Medline]
  39. Kikyo N, Wolfe AP. Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons. J Cell Sci 2000 113:11-20[Abstract]
  40. Hiyoshi H, Yokota T, Katagiri C, Nishida H, Takai M, Agata K, Eguchi G, Abe S. Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes. Exp Cell Res 1991 194:95-99[CrossRef][Medline]
  41. Leno GH, Mills AD, Philpott A, Laskey RA. Hyperphosphorylation of nucleoplasmin facilitates Xenopus sperm decondensation at fertilization. J Biol Chem 1996 271:7253-7256[Abstract/Free Full Text]
  42. Maro B, Howlett SK, Houliston E. Cytoskeletal dynamics in the mouse egg. J Cell Sci Suppl 1986 5:343-359[Medline]
  43. Maro B, Johnson MH, Pickering SJ, Flack G. Changes in actin distribution during fertilization of the mouse egg. J Embryol Exp Morphol 1984 81:211-237[Medline]
  44. Kim NH, Funahashi H, Prather RS, Schattern G, Day BN. Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation. Mol Reprod Dev 1996 43:248-255[CrossRef][Medline]
  45. Kim NH, Funahashi H, Prather RS, Schattern G, Day BN. The distribution and requirements of microtubules and microfilaments during fertilization and parthenogenesis in pig oocytes. J Reprod Fertil 1997 111:143-149[Abstract]
  46. Matsumoto Shoji N, Umezu M, Sato E. Microtubule and microfilament dynamics during the two-cell block in vitro. J Exp Zool 1998 281:149-153[CrossRef][Medline]
  47. Wang WH, Abeydeera LR, Han YM, Prather RS, Day BN. Morhpologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo. Biol Reprod 1999 60:1020-1028[Abstract/Free Full Text]
  48. Schatten G, Schatten H, Spector I, Cline C, Paweletz N, Simerly C, Pezelt C. Latrunculin inhibits the microfilament-mediated process during fertilization, cleavage and early development in sea urchins and mice. Exp Cell Res 1986 166:191-208[CrossRef][Medline]
  49. Fulka J Jr, First NL, Moor RM. Nuclear and cytoplasmic determinants involved in the regulation of mammalian oocyte maturation. Mol Hum Reprod 1998 4:41-49[Abstract/Free Full Text]
  50. Yamazaki Y, Wakayama T, Yanagimachi R. Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic injection (ICSI). Zygote 2001 9:277-282[Medline]
  51. Downs SM. A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic maturation in isolated mouse oocytes. Zygote 2001 9:71-82[CrossRef][Medline]
  52. Cecconi S, D'Aurizio R, Colonna R. Role of antral follicle development and cumulus cells on in vitro fertilization of mouse oocytes. J Reprod Fertil 1996 107:207-214[Abstract]
  53. Downs SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocyte. Dev Biol 1995 167:502-512[CrossRef][Medline]



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