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BOR - Papers in Press, published online ahead of print February 16, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036129
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BIOLOGY OF REPRODUCTION 72, 1397–1404 (2005)
DOI: 10.1095/biolreprod.104.036129
© 2005 by the Society for the Study of Reproduction, Inc.

Microinjection of Cytoplasm or Mitochondria Derived from Somatic Cells Affects Parthenogenetic Development of Murine Oocytes1

Kumiko Takeda 2 3,7 , Mariko Tasai 3, Masaki Iwamoto 4, Akira Onishi 5, Takahiro Tagami 3, Keijiro Nirasawa 3, Hirofumi Hanada 6, and Carl A. Pinkert 7

Department of Animal Breeding and Reproduction,3 National Institute of Livestock and Grassland Science, National Agricultural and Bio-Oriented Research Organization, Tsukuba, Ibaraki, 305-0901, Japan Prime Tech Ltd,4 Tsuchiura, Ibaraki 300-0841, Japan Developmental Biology Departments,5 National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan Department of Zootechnical Science,6 Faculty of Agriculture, Tokyo University of Agriculture, Atsugi, Kanagawa 243-0034, Japan Department of Pathology and Laboratory Medicine,7 Center for Aging and Developmental Biology, University of Rochester Medical Center, Rochester, New York 14642-8645


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloned mammals are readily obtained by nuclear transfer using cultured somatic cells; however, the rate of generating live offspring from the reconstructed embryos remains low. In nuclear transfer procedures, varying quantities of donor cell mitochondria are transferred with nuclei into recipient oocytes, and mitochondrial heteroplasmy has been observed. A mouse model was used to examine whether transferred mitochondria affect the development of the reconstructed oocytes. Cytoplasm or purified mitochondria from somatic cells derived from the external ear, skeletal muscle, and testis of Mus spretus mice or cumulus cells of Mus musculus domesticus mice were transferred into M. m. domesticus (B6SJLF1 and B6D2F1) oocytes to observe parthenogenetic development through the morula stage. All B6D2F1 oocytes injected with somatic cytoplasm or mitochondria showed delayed development when compared to oocytes injected with buffer. The developmental rates were not different among injected cell sources, with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). The developmental rate of B6D2F1 oocytes injected with buffer alone (98.8% survival) was different from those injected with somatic cytoplasm (60.8% survival) or somatic mitochondria (56.5% survival) (P < 0.01). Conversely, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100% survival). Our results indicate that injection of somatic cytoplasm or mitochondria affected parthenogenetic development of murine oocytes. These results have further implications for in vitro fertilization protocols employing ooplasmic transfer where primary oocyte failure is not confirmed.

cytoplasm, early development, mitochondrial transfer, Mus musculus domesticus, Mus spretus, ooplasm, parthenogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloning mammals by somatic cell nuclear transfer was achieved in sheep [1], cattle [2], swine [3], and rodents [4]. Yet, since initial reports, the overall efficiency of generating live offspring from the reconstructed embryos remains at less than 5% [5, 6]. Depending on the source of donor cell (e.g., embryonic blastomeres, embryonic stem cells, cumulus cells, fetal fibroblast cells, and adult fibroblast cells), preimplantation development of reconstructed embryos to the blastocyst stage varies [7, 8]. In mice and cattle, the rate of development of reconstructed oocytes to the morula or blastocyst stage is considerably higher with cumulus cells compared with other adult donor cells [7, 8]. It has been suggested that organisms have evolved a variety of epigenetic defense mechanisms such as imprinting, permutation, and gene silencing, which will inevitably compromise attempts to clone animals from somatic cell nuclei [9].

Mitochondrial function is normally controlled by a dual genome system with cooperation between nuclear- and mitochondrial-encoded genes. The potential for conflict has implications for the poor rates of success in cloning animals [10]. Additionally, the importance of mitochondrial function in oxidative phosphorylation and energy homeostasis has been well documented [11]. The reconstructed zygotes with untreated and oxidative-stressed cytoplasm obtained from pronuclear stage zygotes were destined to oxidative stress-induced apoptotic cell death, likely due to mitochondrial dysfunction [12, 13]. In addition to supporting classical mitochondrial function and nuclear transfer studies, ooplasmic transfer has been used as a relatively new assisted reproduction technique to supplement putatively defective cytoplasm of oocytes from patients with embryonic development failure [14, 15].

In nuclear transfer protocols employed at this time, donor cell cytoplasm, containing varying mitochondrial population, is routinely transferred into recipient oocytes. In some protocols, mitochondrial heteroplasmy has been detected [16]. Mitochondrial morphology and activity are different between donor cells and oocytes. Unlike mature mitochondria found in somatic cells, oocyte mitochondria are rounded or oval in appearance with a dense matrix and few arched cristae [17, 18]. This morphological appearance may reflect a functional difference within these cell types. Yet, to date, no attempts have been made to evaluate the influences of mitochondrial transfer and subsequent development of reconstructed embryos. This study was conducted to examine whether donor cell mitochondria specifically affected the development of reconstructed embryos using our transspecies model system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Oocytes and Cumulus Cells

Mus musculus domesticus strain female mice B6SJLF1/J (C57BL/6J x SJL/J F1 hybrids) were purchased from the Jackson Laboratory (Bar Harbor, ME), and B6D2F1 (C57BL/6-Cr x DBA/2-Cr F1 hybrids) were purchased from SLC Japan (Hamamatsu, Japan). To obtain oocytes, females were superovulated by intraperitoneal injection of 5 IU of eCG (A.F. Parlow, UCLA Medical Center, or Serotropin, Teikokuzoki, Tokyo, Japan) followed at 48 h with 5 IU of hCG (ICN, Costa Mesa, CA, or Mochida, Tokyo, Japan). Oocytes were collected by rupturing the oviducts at 15–17 h after hCG injection. Oocytes were treated with 0.1% bovine testicular hyaluronidase (Type I-S, 359 U/mg; Sigma, St. Louis, MO) in M2 medium (Sigma) to dissociate cumulus cells. Cumulus cells were then collected and incubated in Dulbecco Modified Eagle Medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% FBS at 37°C in 5% CO2 in air.

Preparation of Somatic Cells

Inbred M. spretus mice (SPRET/Ei) purchased from the Jackson Laboratory were used as mitochondrial donors. Species-specific mitochondrial sequences could then be used to monitor transfer efficiency and survival in reconstructed oocytes. Primary cell cultures were established from ear, testis, and skeletal muscle from male mice or ear and skeletal muscle from female mice. Tissues were rinsed in modified Dulbecco PBS (PBS–; Gibco, Invitrogen), minced, and incubated in 1 mg/ml collagenase (Type I-A; Sigma) at 4°C overnight. Digested tissues were placed in Petri dishes (Falcon, Lincoln Park, NJ) and incubated in DMEM + 10% FBS at 37°C for ~1 wk in 5% CO2 in air until sufficient cell populations were propagated from each culture for outlined experiments.

Microinjection of Cytoplasm into Oocytes

Cytoplasts were obtained as described [19, 20]. Cytoplasm injection was performed as previously described [4, 21] but without nuclei in modified CZB medium containing 5.56 mM D-glucose, 20 mM Hepes-HCl, 5 mM NaHCO3, with 0.1 mg/ml polyvinylalcohol (Mr. 100 000) replacing BSA (Hepes-mCZB) [21, 22]. Confluent cultures were incubated in DMEM + 10% FBS containing 200 nM MitoTracker GreenFM (Molecular Probes, Eugene, OR) and 10 µg/ml cytochalasin B for 20 min at 37°C. For nuclear staining, 1 µg/ml Hoechst 33258 (Molecular Probes) was added. The cells were resuspended in a mixture of 4 ml of DMEM + 10% FBS, 4 ml of Percoll (Sigma), and 20 µg/ml cytochalasin B in a 10-ml polysulfone Oakridge centrifuge tube (Nalgene, Rochester, NY), then centrifuged at 44 000 g for 70–80 min at 37°C. Cytoplasts were resuspended in the Hepes-mCZB supplemented with 10% polyvinylpyrrolidone (PVP; Mr. 360 000) (Hepes-mCZB-PVP). For introduction into oocytes, plasma membranes of intact cytoplasts were broken and withdrawn into an injection pipette, then cytoplasm was injected into oocytes using a Piezo micromanipulator (Prime-Tech, Tsuchiura, Japan) in Hepes-mCZB [22].

For ooplasm transfer, ooplasm was aspirated by injection pipette directly from B6D2F1 oocytes, then injected into recipient B6D2F1 oocytes using a Piezo micromanipulator.

Microinjection of Mitochondria into Oocytes

Mitochondria were isolated by differential centrifugation as previously described [23–25]. Confluent cultures were incubated in DMEM + 10% FBS containing 200 nM MitoTracker GreenFM for 20 min at 37°C. Approximately 5 x 106 cells were harvested and resuspended in an isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM Hepes, 1 mM KCl, 1 mM EGTA, pH 7.2 containing 0.5% fatty-acid-free bovine serum albumin; Sigma) homogenized using a Potter Elvehjem-type Teflon homogenizer. The homogenate was centrifuged at 750 g for 10 min, and the supernatant was decanted and centrifuged at 9800 g for 15 min at 4°C. The mitochondrial pellet was resuspended in respiration buffer (225 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris-HCl, 5 mM KH2PO4, pH 7.2) to 1–4 mg/ml (total protein concentration). The mitochondrial suspensions were placed on the working plate, and settled mitochondria were picked by pipette. Approximately 1–2 pl of the mitochondrial suspension were injected into oocytes using a Piezo micromanipulator (Prime-Tech). Fluorescence-labeled mitochondria were detected after injections using fluorescence microscopy (TE300; Nikon, Tokyo, Japan) with the B-2A filter (excitation 520 nm, emission 450–490). Relative fluorescence intensity was calculated using an image processing and analysis program (ImageJ; National Institutes of Health, http://rsb.info.nih.gov/ij/index.html). The data were analyzed by SAS/STAT with the GLM procedure (SAS Institute, Inc., Cary, NC).

Oocyte Activation

Surviving oocytes, 1 h after injection, were cultured in Ca2+-free CZB medium supplemented with 5.56 mM D-glucose containing both 10 mM SrCl2 and 5 µg/ml cytochalasin B [22] for 5 h at 37°C under 5% CO2 in air. Oocytes were washed and cultured in modified CZB medium [21, 22] for 4 days at 37°C under 5% CO2 in air.

Microinjection of Mitochondria into Zygotes

For collection of zygotes, B6D2F1 females were superovulated and mated with B6D2F1 males. Zygotes were collected from the oviducts at 15–17 h after hCG injection. Mitochondrial injection was performed as previously described. Surviving zygotes were incubated in modified CZB medium for 3 days at 37°C under 5% CO2 in air.

Detection of Exogenous M. spretus mtDNA in Embryos

Individual ova were used for PCR analysis after microinjection, activation, and 4-day incubation. Eggs were pretreated with 0.5% pronase to remove zonae pellucidae and washed in PBS– five times. Each egg was placed into 10 µl of distilled water in a 0.2-ml PCR tube, then frozen (–80°C for 5 min)/thawed (37°C for 5 min) three times. Nested PCR was performed as described [26]; however, FastStart Taq Polymerase (Roche Diagnostics GmbH, Mannheim, Germany) was used to improve specificity. Products from the nested PCR were visualized on 3% agarose gels.

Statistical Analysis

Differences were calculated using chi-square analysis. The critical threshold value was derived by application of a Bonferroni correction. Significant differences were defined as P < 0.05.

Animal Use

All procedures followed the AVMA or NILGS Laboratory Animal Experimental Guides. Procedures and experiments were performed according to protocols approved by the U.S. Office of Laboratory Animal Welfare (URMC assurance A3292-01) or the Japanese Prime Minister's Office of Laboratory Animal Welfare (No. 6, 1980).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Parthenogenetic Development of Cytoplasm-Injected Oocytes

Cytoplasts from cells obtained from M. spretus external ear, testis, and skeletal muscle; M. m. domesticus cumulus cells; and ooplasm from M. m. domesticus oocytes were microinjected into oocytes to compare influences on parthenogenetic development. Cytoplasts obtained by the method included some nuclei (Fig. 1). Fluorescence-labeled mitochondria were observed in oocytes (n = 62) after cytoplasm injection (Fig. 2, A and B), and the fluorescence intensity is shown in Table 1.



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  		FIG. 1. Cytoplasm fractions obtained from the cells derived from testis (A), skeletal muscle (B), and external ear (C) of a M. spretus male mouse. Nuclei are stained using Hoechst 33258. Original magnifications x200



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  		FIG. 2. Mitochondria observed after microinjection into B6D2F1 oocytes. A and A') Oocytes were injected with cytoplasm derived from B6D2F1 cumulus cells. B and B') Oocytes were injected with cytoplasm derived from M. spretus external ear cells. C and C') Oocytes were injected with mitochondria derived from B6D2F1 cumulus cells. D and D') Oocytes were injected with mitochondria derived from M. spretus external ear epithelium cells. A–D) Fluorescence images of mitochondria stained using MitoTracker Green FM. A'–D') Phase contrast images of A– D. Original magnifications x200


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  		TABLE 1. Relative fluorescence intensities of labeled mitochondria intro duced into B6D2F1 oocytes

A total of 1783 oocytes were microinjected, and significant model effects were identified (Table 2). All B6D2F1 oocytes injected with somatic cytoplasm showed lower rates of morula formation development when compared to oocytes injected with Hepes-mCZB-PVP (P < 0.01). The developmental rates were not different among injected cell sources including M. m. domesticus cumulus (P > 0.05), with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01). In contrast, injection of ooplasm into B6D2F1 oocytes did not affect parthenogenetic development (100%). Uninjected oocytes showed lower developmental rate than oocytes injected with either ooplasm or medium.


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  		TABLE 2. Effects of cell source and stage of parthenogenetic development after cytoplasm injection into oocytes

Parthenogenetic Development of Mitochondrial-Injected Oocytes

To study influences on parthenogenetic development of mouse oocytes with exogenous mitochondria, we microinjected mitochondrial fractions from cells obtained from M. spretus ear and testis and M. m. domesticus cumulus cells into M. m. domesticus oocytes. Fluorescence-labeled mitochondria were observed in the oocytes (n = 56) after mitochondrial injection (Fig. 2, C and D), and fluorescence intensity is shown in Table 1. The fluorescence intensities were similar between mitochondrial injection and cytoplasm injection experiments (P > 0.05).

A total of 1485 oocytes were injected, and significant model effects were observed (Table 3). All B6D2F1 oocytes injected with somatic mitochondria showed delayed development when compared to oocytes injected with buffer (P < 0.01). The developmental rates were not different among injected cell sources, including M. m. domesticus cumulus (P > 0.05), with the exception of testis-derived donor cells injected into B6SJLF1 oocytes (P < 0.01).


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  		TABLE 3. Effects of cell source and stage of parthenogenetic development after mitochondria injection into oocytes

Cytoplasm and mitochondria injection affected development of B6D2F1 oocytes as shown in Table 4. The rates of cleavage and morula formation were similar (P > 0.05) following injection with somatic cytoplasm (87.3% and 60.8%) and somatic mitochondria (81.5% and 56.5%) but lower when both groups were compared to oocytes injected with buffer (100% and 98.8% survival) (P < 0.01). In contrast, injection of ooplasm did not affect parthenogenetic development (100%).


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  		TABLE 4. Effects of cytoplasm and mitochondria injection on parthenogenetic development of B6D2F1 oocytes (overall data)

Development of Mitochondria-Injected Zygotes

Early development of mouse zygotes following transfer of exogenous mitochondria was investigated (Table 5). Mitochondrial fractions extracted from M. spretus ear (male) cells were injected into B6D2F1 zygotes (n = 149), and the developmental rate through morula formation (80.0%) was not different from buffer-injected controls (85.2%) (P = 0.2). Additionally, the rate of morula formation per cleaved eggs was lower in mitochondria-injected zygotes than buffer-injected controls (91.3% vs. 100%; P = 0.02). While this result was anticipated, the relative survival rate in both groups was high and illustrated a minimal effect of manipulation on ova.


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  		TABLE 5. Early development of B6D2F1 zygotes after injection of somatic mitochondria obtained from M. spretus ear cells

Detection of Exogenous M. spretus mtDNA in Embryos

The detection limit for exogenous M. spretus mtDNA was 0.05 pg of genomic DNA including mtDNA extracted from ear cells (Fig. 3A). Injected embryos were cultured for 4 days and then subjected to PCR analysis of mtDNA-specific sequences.



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  		FIG. 3. A) M. spretus mtDNA detection limits. The genomic DNAs extracted from M. spretus ear cells were used as the templates, 1, 0.05 fg: 2, 0.5 fg; 3, 5.0 fg; 4, 0.05 pg; 5, 0.5 pg; 6, 5.0 pg; 7, 50 pg; 8, 500 pg; 9, 5 ng; 10, 50 ng. B) M. spretus mtDNA detection of the microinjected B6D2F1 eggs incubated for 4 days. The eggs were injected with M. spretus cytoplasm derived from male (M1–4) or female (F1–9) external ear cells. Negative controls; B6D2F1 cumulus (C1–2), uninjected egg (E1), and PBS-added (N1–2). The white arrows show the specific M. spretus mtDNA bands

M. spretus mtDNA could be detected in some embryos after 4 days of incubation (Table 3 and Fig. 3B). A total of 194 B6SJLF1 embryos injected with cytoplasm were analyzed, and 52% harbored detectable concentrations of M. spretus mtDNA. A total of 122 B6D2F1 ova were injected with cytoplasm; 66% had a detectable presence of the M. spretus mtDNA amplicon (Table 6). Finally, a total of 306 B6D2F1 ova were injected with mitochondria and then analyzed; 93% of these ova harbored the introduced M. spretus mtDNA.


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  		TABLE 6. Detection of exogenous M. spretus mtDNA in the manipulated embryos by PCR.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
While nuclear transfer technology has been employed since the mid-1990s to either clone or modify the genetic composition of mammals, a parallel genetic mechanism involves mitochondrial genetics using recently described techniques for mitochondrial transfer into embryos [11, 23, 27, 28]. Development of cloned animals by nuclear transfer resulted in conflicting consequences when retrospective studies on mitochondrial transmission were reported [16, 23, 27]. Depending on the specific methodology employed for nuclear transfer and cytoplasm/ooplasm transfer to rescue low-quality embryos, additional models of heteroplasmy (where two or more populations of mitochondria exist) may have been created [14, 16, 29, 30]. In the aggregate, such techniques may illustrate mechanisms underlying the dynamics related to persistence of foreign mitochondria and maintenance of heteroplasmy in various experimental protocols.

We examined whether the exogenous mitochondria originating from donor cells directly affected parthenogenetic development. The overall results showed that injection of somatic cytoplasm or mitochondria influenced parthenogenetic development of murine oocytes, whereas injection of ooplasm or buffer did not effect a similar response. The rates of cleavage or morula formation did not differ between cytoplasm and mitochondria injection. Some of the inhibition seen following cytoplast injection could be due to nuclei that were inadvertently transferred. Nonetheless, it is likely that the mitochondrial introduction was a contributing factor influencing the subsequent rate of parthenogenetic development. Yet somatic mitochondria injection did not affect early development of zygotes. Perhaps, zygotes were less susceptible to manipulation because of their postfertilization development beyond a critical developmental juncture at the time exogenous mitochondria were injected. Indeed, newborn mice were previously obtained from zygotes injected with cytoplasm or mitochondria derived from M. spretus spermatids or liver [24, 26, 31]. Therefore, somatic mitochondria were implicated in the inefficient development of oocytes following various transfer protocols. For mouse cloning using Piezo methodology, donor nuclei were largely devoid of visible cytoplasmic material. To reduce injection volume, generally very little cytoplasm and hence very few mitochondria were transferred during the injection procedure [4, 7]. As such, at this time, it is unknown if transfer of nuclei with accompanying cytoplasm is a superior or an inferior practice when compared to more routine methods employed for mouse cloning.

Exogenous mitochondria were detected postinjection. However, M. spretus mtDNA was detected in approximately half the cytoplasm-injected embryos and most of the mitochondria-injected embryos after a 4-day incubation. Unfortunately, quantification of the number of mitochondria in each cytoplast or within individual injections was not calculated. The reason underlying the inability to routinely detect M. spretus mtDNA was unclear. The finding may reflect the low concentrations of exogenous (and viable) mtDNA introduced into the oocyte. Alternatively, exogenous mtDNA may have been regulated in some as yet undefined mechanism, resulting in the decreases seen here and elsewhere [31–34]. The later scenario is of particular interest, as there have also been two reports where exogenous xenomitochondrial populations actually increased after introduction [35, 36]. A mechanism regulating competing mitochondrial populations in a coordinated fashion through embryo development is appealing in targeting future studies where mutant mitochondrial populations result in dysfunctional phenotype or in addressing human genetic disorders.

In light of past reports where either heteroplasmy or homoplasmy for oocyte-specific mitochondria were observed, injection of purified mitochondria appeared less detrimental to subsequent embryo survival. However, based on these analyses, both methods limit the overall survivability and developmental capacity of oocytes.

In evaluating parthenogenetic development, different sources of donor cells, including region specificity (i.e., ear, skeletal muscle, testis, cumulus), sex, and mouse species (M. spretus or M. m. domesticus), resulted in comparable embryo survival rates. The efficiency of mouse and bovine cloning using adult somatic cells has been reported as highest when cumulus cells were employed as donors [4, 7, 8, 37]. In contrast, there were no differences detected when using either cumulus- or adult fibroblast-derived donor cytoplasm or purified mitochondria. The relative success of cumulus cell NT could be a function of embryo culture media chosen for cloned embryos [38]. Interestingly, testis-derived donor cytoplasm elicited a lower developmental rate of injected embryos than others when introduced into B6SLJF1 oocytes (P < 0.01). Mitochondria injection into B6SLJF1 oocytes was performed, and a low rate of development was observed. This result corresponds to our cytoplasm injection data. Yet this finding appeared to be a strain-specific phenomenon, as there were no differences noted for donor cytoplasm derived from testis cells or other cell types when introduced into B6D2F1 oocytes. In contrast to zygotic injections, there were no reports to date detailing subsequent embryonic development following mitochondrial injection into oocytes. However, B6D2F1 oocytes developed to morula or blastocyst stages with 55% survival efficiency following fertilization with spermatids (injection of spermatids harboring endogenous mitochondria) [22].

Unlike cytoplasmic or mitochondrial injections, ooplasm injection did not affect embryo development. Ooplasmic transfer has been purported to increase pregnancy maintenance in women who have previously failed to conceive via conventional methods of in vitro fertilization [14, 15]. Such success appears more striking in older women with poor-quality oocytes [30]. Mitochondria are dynamic organelles, interconnected and functionally homogeneous within the cytoplasm. Recent studies showed that mitochondrial fusion occurs rapidly and frequently in mammalian cells [39, 40]. Ooplasm-derived mitochondria were similar in morphology and more closely synchronized with those of recipient mitochondria; therefore, they may be more easily accommodated following transfer. Perhaps the origin of mitochondria derived from various somatic cells may be critical; the transferred mitochondria may be recognized as foreign and inhibit embryo development. Alternatively, transferred mitochondria may not be easily intercalated into the endogenous mitochondrial network in the recipient oocytes.

The production efficiency in development of nuclear-transfer (NT)-derived mice in a majority of laboratories continues to be suboptimal, particularly when donor cells are derived from inbred ES cell lines [41, 42]. Identification of specific media components affecting overall NT efficiency was reported by a number of laboratories [25, 38, 43, 44] and has led to marginal gains in experimental efficiencies. Independent of media modifications, parthenogenetic embryos produced by the injection of cumulus cell nuclei, diploid NT mouse embryos, and normal fertilized embryos have exhibited developmental differences. Such differences appeared most pronounced before the eight-cell stage. It was proposed that the variation in cellular metabolism observed between cloned and normal embryos might have been the result of incomplete nuclear reprogramming before the eight-cell stage. Additionally, NT embryos appeared more sensitive than parthenogenetic embryos to in vitro culture conditions. It is possible that modifications of culture conditions exerted a significant effect on the in vitro development of cloned embryos [38, 41, 45] and that the effects of mitochondria on embryo development may be due to an alteration in cell physiology giving rise to an effect on culture medium preference.

The results reported here demonstrate that injection of somatic cytoplasm or mitochondria affects parthenogenetic development of murine oocytes. Survival of oocytes after mitochondrial or ooplasm injections did not differ and illustrate developmental consequences of current nuclear transfer protocols. In a similar fashion, nuclear transfer protocols for livestock animals are performed by cell fusion with a donor cell and a recipient enucleated oocyte. Our results suggest that the somatic cytoplasm introduction accompanying nuclei has the capacity to affect reconstructed embryo development and that mitochondrial transfer is implicated in this deficiency. These results have further implications 1) associated with nuclear transfer to preserve endangered species using enucleated domestic animal oocytes as hosts and 2) in enhancing efficiency of ooplasmic transfer as a component of in vitro fertilization protocols where primary oocyte failure is not confirmed.


   ACKNOWLEDGMENTS
 
We thank R.L. Howell, C.A. Cassar, C.A. Donegan, C.A. Ingraham, D.A. Dunn, C.A. Lerner, K. Shibasaki, and L. Seung (University of Rochester); S. Takahashi and O. Sasaki (NILGS); and A. Mimatsu (Prime-Tech) for their critical comments and valuable assistance.


   FOOTNOTES
 
1 Supported by funds from the MAFF Japan (K.T.), the NIH (RR-16286 and DE-12634) (C.A.P.), the National Agricultural and Bio-Oriented Research Organization, and the University of Rochester Medical Center. Back

2 Correspondence: Kumiko Takeda, Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, National Agricultural and Bio-Oriented Research Organization, Ikenodai 2, Tsukuba, Ibaraki, 305-0901, Japan. FAX: 81 298 388606; kumiko{at}affrc.go.jp Back

Received: 8 September 2004.

First decision: 8 October 2004.

Accepted: 10 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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