Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print June 28, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.053330
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
75/4/524    most recent
biolreprod.106.053330v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Popova, E.
Right arrow Articles by Krivokharchenko, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Popova, E.
Right arrow Articles by Krivokharchenko, A.
Agricola
Right arrow Articles by Popova, E.
Right arrow Articles by Krivokharchenko, A.
BIOLOGY OF REPRODUCTION 75, 524–530 (2006)
DOI: 10.1095/biolreprod.106.053330
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Full-Term Development of Rat after Transfer of Nuclei from Two-Cell Stage Embryos1

Elena Popova , Michael Bader 2, and Alexander Krivokharchenko 

Max-Delbrück Center for Molecular Medicine (MDC), D-13092 Berlin-Buch, Germany

ABSTRACT

Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%–2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.

embryo, oocyte development

INTRODUCTION

Historically, the first attempts to produce cloned animals by nuclear transfer (NT) were undertaken using nuclei from blastomeres of various preimplantation-stage embryos, and full-term development has been reported in the sheep [1], mouse [2], cattle [3], rabbit [4], pig [5], rhesus monkey [6], and goat [7]. Results of many subsequent studies have shown that the combination of different cell cycle stages of donor nuclei with various types of recipient cytoplasm, such as ovulated oocytes at metaphase stage (MII oocytes), parthenogenetically preactivated oocytes, zygotes, and two-cell embryos, is very important for the success of cloning [7–10].

Further development of cloning technology led to success in somatic cell nuclear transfer in a large number of species that were cloned previously using nuclei from blastomeres as karyoplasts. Cloned animals have been generated by combining enucleated oocytes and nuclei of various somatic cells in the sheep, cattle, mouse, goat, pig, rabbit, cat, and horse. However, the developmental ability of reconstructed embryos and the overall efficiency of somatic cloning was dramatically lower compared with embryonic cell cloning [11–15].

The rat is an important model species for physiology, pathophysiology, and toxicology. Nevertheless, there are no methods for the targeted manipulation of the rat genome available due to the lack of germline-competent embryonic stem cells [16]. Since cloning has been successfully applied already for gene targeting in other mammalian species [17, 18], we aimed to optimize the methods for the construction of cloned rat embryos by nuclear transfer, which has been achieved recently [19]. This initial success of rat cloning using somatic cells as karyoplasts showed the importance of coordination between nuclear transfer and timing of oocyte activation for the outcome in overall efficiency. However, this study remained singular, and rat cloning has not yet become a routine method. Moreover, the production of cloned rats by transfer of nuclei from embryonic blastomeres used as donor karyoplasts has not yet been reported, and all attempts to generate cloned rats in this way have been unsuccessful until now [20, 21].

In the present study we tested the developmental competence of embryos reconstructed with MII oocytes, zygotes, and preactivated oocytes as recipient cytoplasm receiving nuclei from two-cell embryos at different stages of the cell cycle.

MATERIALS AND METHODS

Animals

Female Sprague-Dawley rats (28–35 days old) were obtained from a commercial animal breeder (Taconic), as were Black Hooded Wistar rats (Harlan). Transgenic rats carrying a green fluorescent protein (GFP) gene under the control of the ubiquitously active cytomegalovirus-enhancer/ß-actin promoter were generated using recently published methods [22]. The rats were kept at a temperature of 21°C ± 2°C in a 12L:12D cycle (lights-on 0600 h to 1800 h) with a humidity of 65% ± 5%. All experimental protocols were performed in accordance with the guidelines for the human use of laboratory animals by the Max-Delbrück Center for Molecular Medicine and were approved by the local ethics committee.

Isolation of Oocytes and Zygotes

Immature rats of Sprague-Dawley, Black Hooded, and GFP-transgenic strains were induced to superovulate by intraperitoneal injection of 15 IU eCG (Intervet) followed by the injection of 30 IU hCG (Sigma) as described previously [22]. To obtain zygotes after hCG administration, female rats were mated overnight with male rats of the same strain. On the following morning, the rats were examined for the presence of vaginal plugs. Superovulated rats were killed by cervical dislocation. Ovulated oocytes (14–16 h after the hCG injection) and zygotes (at 1200 h on Day 1) were recovered from the excised oviducts into M2 medium (Sigma) containing 0.1% (w/v) hyaluronidase (Sigma) to remove cumulus cells. The ova then were washed in M16 medium (Sigma).

Parthenogenetic Activation

Parthenogenetic activation was performed as described previously [23]. MII oocytes or embryos reconstructed using nonactivated oocytes as recipient cytoplasts were incubated during 15 min in Ca2+-free and Mg2+-free M16 medium containing 2 mM Sr2+ at 37°C in a CO2 incubator. After treatment the ova were washed carefully and cultured again in M16 medium. MII oocytes after pronucleus formation (about 5 h) were used for reconstruction.

Preparation of Blastomeres for Nuclear Transfer

The cell cycle stage of donor karyoplasts was classified as early, middle, or late. For the production of two-cell embryos for donor karyoplasts of early stage, zygotes were cultured in vitro in M16 medium supplemented with 0.025 µg/ml (0.083 µM) nocodazole (Sigma) for 14–15 h under 5% CO2 in air at 37°C. Nocodazole was dissolved in dimethyl sulfoxide (Sigma, stock solution 1 mg/ml) and stored at –20°C. After removal from nocodazole embryos were carefully washed and used for NT within 2 to 3 h immediately after cleavage. Donor karyoplasts of middle stage were produced from two-cell embryos recovered from oviducts of donor animals 45 to 48 h after hCG injection and were used immediately for NT. Donor karyoplasts of late stage were produced from two-cell embryos recovered from oviducts of donor animals 45 to 48 h after hCG injection and then cultured in vitro overnight for 24 to 26 h in mR1ECM medium before NT. Zona-free two-cell embryos were obtained by removing the zona pellucida with an acid Tyrode's solution (pH 2.5; Sigma). Blastomeres from two-cell embryos were isolated by pipetting the embryos in Ca2+-free and Mg2+-free PBS (Sigma).

Enucleation and Nuclear Transfer

All microsurgery procedures, including enucleation and nuclear transfer, were performed in M2 medium containing 5 µg/ml cytochalasin B (Sigma) at room temperature. Zona pellucida of oocytes and zygotes were opened with a fine glass pipette. Nonactivated MII oocytes were enucleated by removing the MII plate in the region of the cytoplasmic bulge using a glass pipette (~25 µm external diameter). Successful enucleation was confirmed by Hoechst 33342 (Sigma) staining and inspection of the cytoplasts under ultraviolet light. Zygotes at the two-pronuclei stage or preactivated parthenogenetic oocytes with visibly formed nucleoli were enucleated using a glass pipette and were used immediately for NT. A karyoplast from a donor two-cell embryo was introduced into the perivitelline space of the enucleated oocyte or zygote using the same pipette as for enucleation.

Electrofusion of Reconstructed Embryos

Electrofusion was performed using the method developed for the production of viable tetraploid embryos [24]. For electrofusion, embryos were preequilibrated in fusion medium consisting of 0.3 M mannitol solution containing 0.1 mM MgCl2 and 0.1 mM CaCl2 (1–2 min). Embryos were then placed in fusion medium between the electrodes of a fusion chamber connected to a pulse generator (GI-2; Pushchino, Russia). An alternating current (AC) field (8 V, 500 kHz, 10 sec) was used to orientate the agglutination plane of cytoplast and karyoplast parallel to the electrode. Fusion was induced with the aid of two direct current (DC) pulses (60V, 20 msec, 100 msec apart). The treated embryos were washed carefully. Most oocytes or zygotes were fused with the nuclear karyoplast within 30 min.

Embryo Culture

For in vitro cultivation, the ova (10–20 embryos) were cultured during the day in M16 medium, and at 1900–2000 h they were transferred into 700 µl mR1ECM medium [25] in four-well culture dishes (Nunc) and cultured overnight under 5% CO2 in air at 37°C [24]. For in vitro cultivation of blastomeres until blastocyst stage, the single blastomeres were transferred into small wells formed on the bottom of four-well dishes (Well of the Well [WOW] system) [26]. Previously, the culture medium was equilibrated with the gas phase and temperature in a CO2 incubator for 2–3 h.

Embryo Transfer

Two hours after reconstruction the surviving fused embryos in M2 medium were transferred into the oviducts of Day 1 (the day the vaginal plug was detected) pseudopregnant Sprague-Dawley recipients (20–30 embryos per recipient). The pseudopregnancy of the females was induced by mating with vasectomized males with proven sterility. For embryo transfer, 4- to 5-month-old rats (200–280 g) were anesthetized with a mixture of 0.25 ml Ketavet (100 mg/ml; Pharmacia & Upjohn GmbH) and 0.05 ml Rompun (2%; Bayer AG, Leverkusen) per animal. To count the number of implantation sites and fetuses the recipient rats were killed on Day 12 of gestation. In other cases, Cesarean section was performed at 22–24 days of gestation to examine the development of fetuses and to recover live pups.

Fluorescence Detection of Expressed GFP in Cloned Fetuses and Offspring

The expression of the GFP gene in live fetuses with beating hearts and offspring was assessed under UV light (489 nm; fluorescent microscope [Leica]).

Statistical Analysis

The comparisons for multigroup and multifactorial analyses were done with a two-way ANOVA and one-way ANOVA on ranks for multiple group comparisons. A value of P < 0.05 was chosen as an indication of statistical significance.

RESULTS

In Vitro Development of Rat Embryos, Single Blastomeres, and Zygotes Reconstructed by Pronuclei Exchange

We investigated the possible effects of the micromanipulations, including enucleation and electrofusion procedures, on in vitro preimplantation development using pronuclear exchange in zygotes as a model. To study the developmental potential of single blastomeres, we isolated them from two-cell embryos and cultured them until blastocyst stage in vitro using the WOW system. As a control of development we used nonmanipulated zygotes and two-cell stage embryos produced in vivo (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. In vitro development of rat embryos and single blastomeres

Development to the two-cell stage was significantly lower for single blastomeres (80.3%) compared with the control group of zygotes (94.6%) (P < 0.01) and was similar to reconstructed zygotes after pronuclear transfer from one zygote to another (82.1%). A significantly higher number of two-cell embryos developed to the blastocyst stage compared with all other groups (P < 0.001). However, there was no difference in blastocyst development between blastomeres and intact and reconstructed zygotes (9.1%, 21.6%, and 25.0%, respectively).

Effect of Nocodazole Treatment on In Vitro Development of the Rat Zygotes

The minimal working concentration of nocodazole, 0.025 µg/ml or 0.083 µM, was determined in preliminary experiments (data not shown). At this concentration of nocodazole 100% (94 of 94) of zygotes were blocked at the one-cell stage after 14 h of incubation, and 92.6% (87 of 94) reached the two-cell stage within 3 h after washing and transfer into nocodazole-free medium, a rate comparable with control intact embryos 90.0% (27 of 30). After further in vitro cultivation, 31.3% (21 of 67) of nocodazole-treated embryos developed to morula and 10.5% (7 of 67) developed to blastocyst stages. In the control group, developmental rates to morula and blastocyst stages were significantly higher (56.7% [17 of 30], P < 0.01; and 30.0% [9 of 30], P < 0.05, respectively).

Effect of Recipient Cytoplasm and Cell Cycle Stage of Karyoplast on In Vitro Development of Reconstructed Embryos

Three different cell cycle stages of donor karyoplasts—early, middle, and late—were tested as described in Materials and Methods. The survival and cleavage rates after reconstruction, including enucleation and injection of the karyoplast under the zona pellucida, were not dependent on the cell cycle stage of the karyoplasts transferred into ovulated nonactivated MII oocytes (Table 2). At the same time, the fusion rate was significantly higher when middle-stage karyoplasts were used (76.9%) compared with the transfer of late-stage karyoplasts (60.9%; P < 0.05).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Efficiency of the generation of reconstructed embryos using nonactivated MII oocytes as recipient cytoplasm

The cell cycle stage of donor karyoplasts had no effect on survival and fusion rates when preactivated oocytes were used as recipients (Table 3). However, the ability of the reconstructed embryos to develop to the two-cell stage was affected by the stage of donor nuclei at transfer. The developmental capacity of the reconstructed embryos that received donor nuclei at the early stage was markedly higher (72.1%) (P < 0.001) compared with the other groups comprising middle-stage (19.4%) or late-stage (18.4%) karyoplasts.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Efficiency of the generation of reconstructed embryos using preactivated oocytes as recipient cytoplasm

When zygotes were used as recipient cytoplasm, no differences were obtained in rates of survival and fusion in reconstructed embryos that received donor nuclei at different stages of the second cell cycle (Table 4). Of the reconstructed embryos, 67.9% with early and 67.4% with middle nuclei developed to the two-cell stage. In contrast, the embryos reconstructed using nuclei from late two-cell embryos had significantly less competence to develop to the two-cell stage compared with the other groups (P < 0.001).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Efficiency of the generation of reconstructed embryos using zygotes as recipient cytoplasm

Neither cytoplasm from nonactivated oocytes nor that from preactivated oocytes or zygotes combined with all stages of donor karyoplasts supported in vitro development of reconstructed embryos to the blastocyst stage (data not shown).

In Vivo Development of Reconstructed Embryos Following Embryo Transfer to Foster Mothers

To examine the ability of embryos to develop in vivo until implantation, only the most successful schemes of nuclear transfer that showed the highest in vitro cleavage rates of reconstructed embryos (>65%) were used (Table 5). Embryo transfer of reconstructed, preactivated, enucleated oocytes of Sprague-Dawley strain rats that had received karyoplasts from two-cell embryos in early cell cycle stage resulted in pregnancy in 64.7% of the recipients. When enucleated zygotes were used as recipient cytoplasts combined with nuclei from two-cell stage embryos of early and middle stages, the pregnancy rates were 37.5% and 20.0%, respectively, thus showing no significant differences between the groups.


View this table:
[in this window]
[in a new window]
  		TABLE 5. Development of reconstructed SD embryos following transfer to foster mothers

However, the implantation rate on Day 12 after embryo transfer was significantly higher when preactivated, enucleated oocytes received nuclei from two-cell embryos of early stage (7.1%) compared with the other groups (5.9% and 4.0%) (P < 0.05) (Table 5). Moreover, of the 253 embryos reconstructed by this scheme and transferred to pseudopregnant recipients, 13 (5.1%) were capable of developing into live fetuses with beating hearts. In addition, two live offspring developed from 92 reconstructed embryos transferred to pseudopregnant recipients were removed at 24 days of gestation by Cesarean section. However, embryos reconstructed using the other two NT schemes were able only to implant, and they developed robust decidual swellings but not live embryos.

To confirm the origin of the cloned embryos and examine their ability to develop to term, Sprague-Dawley enucleated oocytes receiving nuclei from two-cell embryos of early cell cycle stage from donor females of Black Hooded Wistar and GFP-transgenic Sprague-Dawley rats were transferred to the Sprague-Dawley foster mothers. The transfer of these embryos resulted in quite a low pregnancy rate of foster mothers (10.0%–18.2%), without any differences between the groups. The overall efficiency of live offspring production was very low, without any differences between the groups (0.4%–0.7%) (Table 6). One pregnant foster that had received embryos reconstructed using preactivated, enucleated ooplasm with early-stage nuclei from GFP-transgenic donors was killed on Day 12 of gestation. The only recovered live fetus with a beating heart was analyzed under UV light showing a positive signal for GFP (data not shown). In a separate experiment, all live pups of such a foster were removed at 23–24 days of gestation by Cesarean section. Unfortunately, all pups died 1 or 2 days after birth.


View this table:
[in this window]
[in a new window]
  		TABLE 6. Development of reconstructed embryos to term receiving donor nuclei from different rat strains

In the next series of experiments we tried to increase the survival rate to term by transplanting into the same foster mother reconstructed embryos together with nonmanipulated embryos from Sprague-Dawley rats. Two fosters from each experimental group were employed. A total of 40 cloned embryos receiving nuclei from donor females of GFP-transgenic rats and 19 nonmanipulated Sprague-Dawley embryos were transferred. From these 59 transferred embryos, 10 developed to term. Unfortunately, none of these were cloned. After transfer of 36 reconstructed embryos receiving nuclei from females of Black Hooded Wistar together with 17 intact embryos, 9 pups were born. One of them resulted from cloning, showing the corresponding fur color phenotype (Fig. 1). Thus, the pregnancy rates (100%) and total number of pups born (16.9%) were improved by adding 8 or 10 nonmanipulated embryos per transfer for both groups of reconstructed embryos. However, only one foster mother contained a viable female cloned rat, which developed from a reconstructed embryo with a blastomere nucleus from a Black Hooded donor (Fig. 1).


Figure 1
View larger version (138K):
[in this window]
[in a new window]
  		FIG. 1. Offspring of cloning experiment. One of the pups resulted from the transfer of a blastomere nucleus from a two-cell Black Hooded Wistar embryo into a preactivated, enucleated Sprague-Dawley oocyte and the subsequent transfer of this embryo to a Sprague-Dawley foster mother (also shown). The other pups resulted from the parallel transfer of normal Sprague-Dawley embryos

DISCUSSION

In the early phases of animal cloning many studies have been performed to optimize the procedure and improve the efficiency of nuclear transfer, including pronuclear transplantation in mice [2, 27–32]. The first publication concerning nuclear transplantation of rat embryos showed that enucleated zygotes that received pronuclear karyoplasts can reach the two-cell stage after overnight in vitro culture (89%) and can develop to term (22%) after transfer to recipient rats [20]. Recently reported results have demonstrated that enucleated zygotes that received pronuclear karyoplast from another zygote can develop in vitro until the blastocyst stage or in vivo to term in mice (68.5% and 34.1%, respectively) [12] and in rats (34.3% and 4.2%, respectively) [33]. In the latter report, full-term development of reconstructed rat embryos following transfer of nuclei from two-cell stage embryos into enucleated two-cell stage embryos also was achieved [33]. In order to check the effect of various manipulations such as enucleation, electrofusion, and culture, we also evaluated the capability of zygotes reconstructed by pronuclear exchange to develop in vitro until blastocyst stage and obtained an efficiency comparable to the one reported by others.

Despite successful production of identical twins in rat by separating two-cell embryos and transfer of the single blastomeres to fosters [34], the capability of rat blastomeres to develop in vitro has not been reported thus far. The results of our experiments showed that using the WOW culture system, 9.1% of single blastomeres from two-cell embryos can develop in vitro until the blastocyst stage. To the best of our knowledge this is the first report about successful in vitro culture of single blastomeres of rats in contrast to other species such as mouse, rabbit, sheep, and cow, in which in vitro and in vivo development of single blastomeres has been achieved previously [35–37].

The influence of the cell cycle stage of the donor nucleus and the condition of the recipient cytoplasm on the development of reconstructed embryos have been studied in various mammalian species. However, the published results are contradictory, sometimes even for the same species. For the mouse the best results were obtained after transfer of G1-phase nuclei from 2- to 16-cell embryos into nonactivated MII cytoplasts [9, 38, 39], and the use of S-phase nuclei [38] impaired the development of reconstructed embryos. Others reported optimal success rates after transfer into enucleated telophase I oocytes of donor nuclei from late-stage two-cell mouse embryos which may have been in middle or late G2 phase [40]. Interestingly, live cloned mice were produced by the transfer of metaphase nuclei from four-cell embryos into MII ooplasts and subsequent re-cloning [41]. Studies in the rabbit using donor blastomeres at defined cell cycle stages have shown that the development to blastocyst is more efficient when donor nuclei in G1 or early S phase compared with late S or G2 phase are transferred [42]. Blastomere nuclei at these cell cycle stages also were used for successful NT in sheep [8] and goat [7].

The state of the cytoplast also can have substantial effects on the development of reconstructed embryos. Preactivation of the recipient cytoplast is one way to improve the efficiency of embryo cloning. The positive effect of oocyte preactivation was demonstrated after transfer of nuclei at presumptive S phase in sheep [8], at G1/S phase in goat [7], at G1 phase in rabbit [10], and at morula/blastocyst stage without cell cycle determination of donor nuclei in cattle [43]. However, in mouse, the preactivation of cytoplast before transfer of G1 nuclei decreased the rate of future development [39].

The first attempt to produce cloned rats using a nuclear transplantation method described for the mouse [31] has been performed with embryos reconstructed using as recipients enucleated zygotes and donor nuclei from blastomeres of two-, four-, or eight-cell embryos [20]. In this study, the ability of the reconstructed embryos to develop to blastocysts in vitro was not examined, since no in vitro culture system for rat embryos had been established at that time. The authors reported that the reconstructed embryos failed to develop to term after embryo transfer to foster mothers.

The second attempt to generate cloned rats by transfer of nuclei from embryonic cells was undertaken only 15 years later [21]. In this study, enucleated zygotes as recipient cytoplasm that received nuclei from two-cell embryos after overnight in vitro culture allowed development to blastocysts in vitro. However, the rate of in vitro development still was low (2.6%), and no fetus was observed after embryo transfer. On the other hand, the development of embryos reconstituted with enucleated MII oocytes and parthenogenetically activated oocytes at pronuclear stages as recipient cytoplasm was arrested at the two-cell stage.

Interestingly, both reports used enucleated zygotes for the transfer of blastomere nuclei, meaning that ooplasts already were preactivated by fertilization. Furthermore, both groups did not synchronize and determine the cell cycle stage of the donor nuclei. In the present work, we for the first time studied the interaction of donor nuclei in various cell cycle phases with various recipient cytoplasts to find the most effective combination for NT in the rat.

The first step of our work was to develop a method for blastomere synchronization of early rat embryos. There are two possible methods for the production of synchronized donor nuclei at various stages of the cell cycle. The first is to calculate the time from hCG injection [40], and the second comprises the use of various cell cycle arrest agents [9, 44–48]. Nocodazole is a very popular agent for arrest and synchronization of cell division but can exert a detrimental effect on mammalian preimplantation embryos [38, 49]. Since there were no reports about the use of this drug in early rat embryos, we had to find the optimal conditions. There are numerous protocols for nocodazole treatment of preimplantation embryos from other mammalian species: 0.4 µg/ml for 8-cell bovine embryos [46] and for 4-cell rabbit embryos [10]; 7.5 µg/ml for 16-cell goat embryos [7]; 10 µM for 16-cell bovine embryos [48]; 1.2 µM for bovine morulae [50]; 3 µg/ml for 2-cell mouse embryos [44]; 2.5–5.0 µM for 2-, 4-, 8-, and 16-cell mouse embryos [38]; 0.33 µM for 4-cell mouse embryos [45]; or 1 µg/ml for 4-cell mouse embryos [41]. The time of incubation in nocodazole reported by these authors varied from 4 to 20 h.

In our experiments, we defined the minimal effective concentration of nocodazole suitable for rat zygotes as 0.025 µg/ml (0.083 µM) for a 14-h incubation time. Practically complete cell cycle arrest was observed during incubation in nocodazole-containing medium, and 92.6% of the treated embryos reached the two-cell stage after removal of the drug. This concentration was markedly below those used by other authors but was comparable with recently published protocols for cell cycle synchronization in two- and four-cell stage mouse embryos (0.05 µM) [49]. However, even this very low effective concentration significantly decreased further development of treated rat zygotes, indicating a greater sensitivity of early rat embryos to nocodazole.

In the present study no direct determination of the cell cycle stage of donor nuclei was performed, but results obtained in mice showed that the majority of nuclei of two- and four-cell embryos stayed in G1 for about 2 h [38, 49]. Therefore, it is likely that nuclei isolated in our experiments from two-cell embryos and described as early, middle, and late corresponded to G1/early S, S, and late S/G2 stages, respectively.

In our experiments, embryos reconstructed using nuclei of early stage with enucleated, preactivated oocytes or enucleated zygotes and nuclei of middle stage with enucleated zygotes demonstrated the highest cleavage rates. In other words, the best results were obtained by transfer of nuclei at the G1/S border into preactivated ooplasm, in agreement with results obtained in rabbit, sheep, and goat. Much lower cleavage rates were achieved when embryos were reconstructed using as recipient cytoplasm nonactivated oocytes regardless of the cell cycle of donor nuclei, preactivated oocytes with donor nuclei in the S and late S/G2 phase, and zygotes with donor nuclei in the late S/G2 phase of the cell cycle. However, none of the reconstructed embryos developed in vitro beyond early cleavage stages. Interestingly, in the same culture conditions, parthenogenetically activated oocytes, single blastomeres, nocodazole-treated embryos, and zygotes after pronuclear exchange developed until blastocyst stage in vitro.

These results are in contrast to those in a recent study showing that enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts in vitro [21]. However, in this work the nuclei were transferred after overnight in vitro culture and without cell cycle synchronization and determination. Successful in vitro development until blastocyst stage of reconstructed embryos using nuclei from embryonic blastomere as karyoplasts also has been reported in various species such as mouse [12, 39, 41, 51], rabbit [10, 42], rhesus monkey [52], sheep [8], goat [7], and cattle [43, 47]. The reasons why in our hands the fusion of enucleated oocytes or zygotes with blastomere nuclei blocked further in vitro development to late preimplantation stages remain unclear.

Until now, live offspring after transfer of embryonic nuclei in rat have been obtained only after the exchange of either pronuclei between zygotes or nuclei between two-cell embryos [20, 33]. However, the transfer of blastomere nuclei into enucleated zygotes did not result in live offspring [20]. Moreover, other authors showed that some recipients of such embryos showed robust decidual swellings but no postimplantation structures [21]. However, we obtained live fetuses (5.1%) after transfer of embryos reconstructed by fusion of early blastomere nuclei with preactivated enucleated oocytes as recipient ooplasm. None of the embryos reconstructed with zygote cytoplasm developed in vivo into live fetuses, in agreement with reports mentioned above. Possible reasons for this difference between zygotes and oocytes may be the disappearance of reprogramming factors and/or the occurrence of factors interfering with nuclear remodeling after fertilization.

In order to confirm the origin of the cloned embryos, we used nuclei and oocytes from different rat strains for the production of NT embryos. In these experiments, embryos were reconstituted by transfer of nuclei of GFP-transgenic and Black Hooded Wistar rats into enucleated oocytes of Sprague-Dawley rats and than transferred into Sprague-Dawley foster mothers. Live fetuses and offspring with corresponding phenotypes were produced in both groups without any difference in birth rates (0.4%–0.7%). At the same time, in our study the pregnancy rate and total number of pups born were improved by adding nonmanipulated embryos each transfer. Attempts to help maintain pregnancy and improve the efficiency of embryo transfer by combining normal with manipulated embryos during transfer were reported already in the literature [53–54]. In the mouse, the pregnancy rate, the total number of pups born, and the number and percentage of pups from microinjected embryos were improved by this procedure [53]. In pigs, it was shown that pregnancies can be established with a mixture of fertilized and parthenote embryos and that fertilized embryos can develop to term [55]. This method also has been used to maintain pregnancies of cloned pig embryos [54].

In general, the efficiency of NT in our experiments was very low for all studied karyoplast–ooplast combinations and was comparable to that of somatic cell cloning. The reasons for this low efficiency of rat cloning even after transfer of nuclei from early preimplantation embryos still are not clear and may be related to peculiarities of rat genome reprogramming.

ACKNOWLEDGMENTS

We would like to thank Rosemarie Barnow for skillful technical assistance.

FOOTNOTES

1 Supported through EURATools (European Commission contract no. LSHG-CT-2005–019015). Back

2 Correspondence. FAX: 49 30 9406 2110; mbader{at}mdc-berlin.de Back

Received: 21 April 2006.

First decision: 10 May 2006.

Accepted: 22 June 2006.

REFERENCES

  1. Willadsen SM, Nuclear transplantation in sheep embryos. Nature 1986 320:63-65[CrossRef][Medline]
  2. Tsunoda Y, Yasui T, Shioda Y, Nakamura K, Uchida T, Sugie T, Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos. J Exp Zool 1987 242:147-151[CrossRef][Medline]
  3. Prather RS, Barnes FL, Sims MM, Robl JM, Eyestone WH, First NL, Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte. Biol Reprod 1987 37:859-866[Abstract]
  4. Stice SL, Robl JM, Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod 1988 39:657-664[Abstract]
  5. Prather RS, Sim MM, First NL, Nuclear transplantation in early pig embryos. Biol Reprod 1989 41:414-418[Abstract]
  6. Meng L, Ely JJ, Stouffer RL, Wolf DP, Rhesus monkeys produced by nuclear transfer. Biol Reprod 1997 57:454-459[Abstract]
  7. Yong Z, Yuqiang L, Nuclear-cytoplasmic interaction and development of goat embryos reconstructed by nuclear transplantation: production of goats by serially cloning embryos. Biol Reprod 1998 58:266-269[Abstract/Free Full Text]
  8. Campbell KH, Loi P, Cappai P, Wilmut I, Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes. Biol Reprod 1994 50:1385-1393[Abstract]
  9. Cheong HT, Takahashi Y, Kanagawa H, Birth of mice after transplantation of early cell-cycle-stage embryonic nuclei into enucleated oocytes. Biol Reprod 1993 48:958-963[Abstract]
  10. Piotrowska K, Modlinski JA, Korwin-Kossakowski M, Karasiewicz J, Effect of preactivation of ooplast or synchronization of blastomere nuclei in G1 on preimplantation development of rabbit serial nuclear transfer embryos. Biol Reprod 2000 63:677-682[Abstract/Free Full Text]
  11. Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP, Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol 2002 20:366-369[CrossRef][Medline]
  12. Hiiragi T, Solter D, Reprogramming is essential in nuclear transfer. Mol Reprod Dev 2005 70:417-421[CrossRef][Medline]
  13. Perry AC, Wakayama T, Untimely ends and new beginnings in mouse cloning. Nat Genet 2002 30:243-244[CrossRef][Medline]
  14. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KH, Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000 407:86-90[CrossRef][Medline]
  15. 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]
  16. Fändrich F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DS, Parwaresch R, Zavazava N, Binas B, Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat Med 2002 8:171-178[CrossRef][Medline]
  17. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, et al Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002 295:1089-1092[Abstract/Free Full Text]
  18. Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, Fletcher J, King T, Ritchie M, Ritchie WA, Rollo M, de Sousa P, Travers A, et al Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep. Nat Biotechnol 2001 19:559-562[CrossRef][Medline]
  19. Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, Fraichard A, Cozzi J, Generation of fertile cloned rats by regulation oocyte activation. Science 2003 302:1179[Free Full Text]
  20. Kono T, Shioda Y, Tsunoda Y, Nuclear transplantation of rat embryos. J Exp Zool 1988 248:303-305[CrossRef]
  21. Shinozawa T, Mizutani E, Tomioka I, Kawahara M, Sasada H, Matsumoto H, Sato E, Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer. Mol Reprod Dev 2004 68:313-318[CrossRef][Medline]
  22. Popova E, Krivokharchenko A, Ganten D, Bader M, Comparison between PMSG-and FSH-induced superovulation for the generation of transgenic rats. Mol Reprod Dev 2002 63:177-182[CrossRef][Medline]
  23. Krivokharchenko A, Popova E, Zaitseva I, Vil'ianovich L, Ganten D, Bader M, Development of parthenogenetic rat embryos. Biol Reprod 2003 68:829-836[Abstract/Free Full Text]
  24. Krivokharchenko A, Galat V, Ganten D, Bader M, In vitro formation of tetraploid rat blastocysts after fusion of two-cell embryos. Mol Reprod Dev 2002 61:460-465[CrossRef][Medline]
  25. Miyoshi K, Kono T, Niwa K, Stage-dependent development of rat 1-cell embryos in a chemically defined medium after fertilization in vivo and in vitro. Biol Reprod 1997 56:180-185[Abstract]
  26. Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, Callesen H, New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system. Mol Reprod Dev 2000 55:256-264[CrossRef][Medline]
  27. Cheong HT, Takahashi Y, Kanagawa H, Developmental capacity of reconstituted mouse embryos: influences of nucleus and cytoplasm sources. J Vet Med Sci 1992 54:1099-1103[Medline]
  28. Kono T, Tsunoda Y, Effects of induction current and other factors on large-scale electrofusion for pronuclear transplantation of mouse eggs. Gamete Res 1988 19:349-357[CrossRef][Medline]
  29. Tsunoda Y, Kato Y, Shioda Y, Electrofusion for the pronuclear transplantation of mouse eggs. Gamete Res 1987 17:15-20[CrossRef][Medline]
  30. McGrath J, Solter D, Nuclear transplantation in mouse embryos. J Exp Zool 1983 228:355-362[CrossRef][Medline]
  31. McGrath J, Solter D, Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 1983 220:1300-1302[Abstract/Free Full Text]
  32. McGrath J, Solter D, Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science 1984 226:1317-1319[Abstract/Free Full Text]
  33. Roh S, Guo J, Malakooti N, Morrison JR, Trounson AO, Du ZT, Birth of rats following nuclear exchange at the 2-cell stage. Zygote 2003 11:317-321[CrossRef][Medline]
  34. Matsumoto K, Miyake M, Utsumi K, Iritani A, Production of identical twins by separating two-cell rat embryos. Gamete Res 1989 22:257-263[CrossRef][Medline]
  35. Tsunoda Y, McLaren A, Effect of various procedures on the viability of mouse embryos containing half the normal number of blastomeres. J Reprod Fert 1983 69:315-322[Abstract/Free Full Text]
  36. Moore NW, Adams CE, Rowson LE, Developmental potential of single blastomeres of the rabbit egg. J Reprod Fertil 1968 17:527-531[Abstract/Free Full Text]
  37. Willadsen SM, The development capacity of blastomeres from 4- and 8-cell sheep embryos. J Embryol Exp Morphol 1981 65:165-172[Medline]
  38. Otaegui PJ, O'Neill GT, Campbell KH, Wilmut I, Transfer of nuclei from 8-cell stage mouse embryos following use of nocodazole to control the cell cycle. Mol Reprod Dev 1994 39:147-152[CrossRef][Medline]
  39. Tsunoda Y, Kato Y, Full-term development after transfer of nuclei from 4-cell and compacted morula stage embryos to enucleated oocytes in the mouse. J Exp Zool 1997 278:250-254[CrossRef][Medline]
  40. Kono T, Kwon OY, Watanabe T, Nakahara T, Development of mouse enucleated oocytes receiving a nucleus from different stages of the second cell cycle. J Reprod Fertil 1992 94:481-487[Abstract/Free Full Text]
  41. Kwon OY, Kono T, Production of identical sextuplet mice by transferring metaphase nuclei from four-cell embryos. Proc Natl Acad Sci U S A 1996 93:13010-13013[Abstract/Free Full Text]
  42. Collas P, Balise JJ, Robl JM, Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. Biol Reprod 1992 46:492-500[Abstract]
  43. Du F, Sung LY, Tian XC, Yang X, Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle. Mol Reprod Dev 2002 63:183-191[CrossRef][Medline]
  44. Kato Y, Tsunoda Y, Synchronous division of mouse two-cell embryos with nocodazole in vitro. J Reprod Fertil 1992 95:39-43[Abstract/Free Full Text]
  45. Samake S, Smith LC, Effects of cell-cycle-arrest agents on cleavage and development of mouse embryos. J Exp Zool 1996 274:111-120[CrossRef][Medline]
  46. Samake S, Smith LC, Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of nocodazole. Mol Reprod Dev 1996 44:486-492[CrossRef][Medline]
  47. Tanaka H, Takahashi Y, Hishinuma M, Kanagawa H, Influence of time after the removal of nocodazole from nuclear donors on the development of reconstituted embryos in bovine nuclear transplantation. Jpn J Vet Res 1995 43:135-143[Medline]
  48. Tanaka H, Influence of the timing of blastomere isolation after the removal of nocodazole in bovine nuclear transfer. Theriogenology 1999 51:1225-1237[CrossRef][Medline]
  49. Yu Y, Xia P, Li S, Yan Y, Tan J, Determination and synchronisation of G1-phase of the cell cycle in 2- and 4-cell mouse embryos. Zygote 2002 10:245-251[CrossRef][Medline]
  50. Alberio R, Motlik J, Stojkovic M, Wolf E, Zakhartchenko V, Behavior of M-phase synchronized blastomeres after nuclear transfer in cattle. Mol Reprod Dev 2000 57:37-47[CrossRef][Medline]
  51. Kono T, Kwon OY, Nakahara T, Development of enucleated mouse oocytes reconstituted with embryonic nuclei. J Reprod Fertil 1991 93:165-172[Abstract/Free Full Text]
  52. Mitalipov SM, Yeoman RR, Nusser KD, Wolf DP, Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells. Biol Reprod 2002 66:1367-1373[Abstract/Free Full Text]
  53. Canseco RS, Sparks AE, Page RL, Russell CG, Johnson JL, Velander WH, Pearson RE, Drohan WN, Gwazdauskas FC, Gene transfer efficiency during gestation and the influence of co-transfer of non-manipulated embryos on production of transgenic mice. Transgenic Res 1994 3:20-25[CrossRef][Medline]
  54. King TJ, Dobrinsky JR, Zhu J, Finlayson HA, Bosma W, Harkness L, Ritchie WA, Travers A, McCorquodale C, Day BN, Dinnyes A, De Sousa PA, et al Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos. Reproduction 2002 123:507-515[Abstract]
  55. De Sousa PA, Dobrinsky JR, Zhu J, Archibald AL, Ainslie A, Bosma W, Bowering J, Bracken J, Ferrier PM, Fletcher J, Gasparrini B, Harkness L, et al Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. Biol Reprod 2002 66:642-650[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
75/4/524    most recent
biolreprod.106.053330v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Popova, E.
Right arrow Articles by Krivokharchenko, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Popova, E.
Right arrow Articles by Krivokharchenko, A.
Agricola
Right arrow Articles by Popova, E.
Right arrow Articles by Krivokharchenko, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS