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BOR - Papers in Press, published online ahead of print December 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.024216
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BIOLOGY OF REPRODUCTION 70, 1162–1170 (2004)
DOI: 10.1095/biolreprod.103.024216
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Genetic Variation in Oocyte Phenotype Revealed Through Parthenogenesis and Cloning: Correlation with Differences in Pronuclear Epigenetic Modification1

Shaorong Gao5, Eva Czirr3,5, Young Gie Chung4,5, Zhiming Han5, and Keith E. Latham2,5,6

The Fels Institute for Cancer Research and Molecular Biology5 the Department of Biochemistry,6 Temple University School of Medicine, Philadelphia, Pennsylvania, 19140


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies revealed that oocytes of different genetic strains (e.g, C57BL/6 and DBA/2) modify maternal and paternal pronuclei differently, affecting early preimplantation development. To determine whether these strain-dependent effects would also apply to oocyte modifications of somatic cell nuclei introduced during cloning procedures, we compared the efficiency of development of parthenogenetic and cloned embryos made with DBA/2, C57BL/6, and (B6D2)F1 oocytes. Our results reveal significant differences in the ability of oocytes of different genetic backgrounds to support parthenogenetic development in different culture media. Additionally, our results reveal oocyte strain-dependent differences in the ability to support cloned embryo development beyond what can be accounted for on the basis of differences in parthenogenesis. Thus, the previously documented differences in oocyte-directed parental genome modification are accompanied in the same strains by differences in the ability of oocytes to modify somatic cell nuclei and support clonal development, raising the possibility that these oocyte functions may be mediated by related mechanisms. These results provide a genetic basis for further studies seeking to identify specific genes that determine oocyte phenotype, as well as genes that determine the success of nuclear reprogramming and clonal development.

embryo, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The oocyte possesses the unique capacity to direct essential, unique processes in the early embryo, including embryonic genome formation, transcriptional activation, and correct embryo-specific modes of gene regulation. Deficiencies in these processes have obvious undesirable consequences, and failure of these processes to occur are attributable to poor oocyte quality. Genetic differences in oocyte phenotype are poorly understood, but are of relevance to basic, applied, and clinical reproductive biology. Earlier studies have revealed genetic differences in how oocytes modify maternal chromosomes during oogenesis using maternal pronuclear exchanges between strains [1, 2], differences in how oocytes modify paternal genomes after fertilization using androgenetic embryos [3–5], and differences in how oocytes modify the methylation patterns and embryonic expression of transgenes [6, 7]. Ooplasmic factors can also interact with paternal factors to cause embryo lethality [8–10] or blastomere fragmentation [11, 12] and can act upon exogenous nuclei to repress the expression of an array of gene products [13] or to promote development of cloned embryos [14–18]. These observations reveal important early roles for ooplasm in epigenetic gene regulation through such processes as imprinting modifications and stable alterations in chromatin structure. Additionally, numerous clinical studies point to ooplasmic effects on apoptosis and other interactions with paternal factors [19–21]. Efforts to improve oocyte quality have involved such procedures as germinal vesicle transfer and ooplasm donation [22, 23], but it remains unclear what effect, if any, such procedures actually exert on genome function.

The paternal genome exerts significant effects on early embryo development. Studies from a variety of species (some employing in vivo-produced embryos, and others employing in vitro-produced embryos) have reported paternal genome transcription soon after fertilization [24–33]. In fact, in the mouse it appears that the paternal genome is transcriptionally more active than the maternal genome [25, 34–36], and thus may exert a disproportionate effect on early embryo phenotype. Sperm-derived nuclear factors and organelles may provide essential functions and could affect early development [37–39].

Because the paternal genome and sperm-derived factors can exert such pronounced effects on embryo phenotype, the study of genetic determinants of oocyte phenotype can be facilitated in situations that do not involve fertilization. In this study, we have compared oocyte phenotypes between several different genotypes of female mice, as related to parthenogenesis and the ability to support development of cloned embryos, neither of which involves fertilization. We observed strain-dependent differences in the ability of oocytes to support parthenogenetic development in different culture media. Additionally, our results reveal oocyte strain-dependent differences in the ability to support cloned embryo development beyond what can be accounted for on the basis of differences in parthenogenesis. Thus, the previously documented differences in oocyte-directed parental genome modification are accompanied in the same strains by differences in the ability of oocytes to modify somatic cell nuclei and support clonal development. The modifications of parental genomes and modifications of somatic cell nuclei may, therefore, be mediated by related mechanisms. These results provide a genetic basis for further studies seeking to identify specific genes that determine oocyte phenotype, as well as genes that determine nuclear reprogramming and clonal development. Understanding such genetic effects on oocyte quality may also be relevant to clinical treatments of infertility involving oocyte and embryo manipulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte Collection and Embryo Culture

Adult female mice at 8–12 weeks of age were superovulated by sequential administration of 5 IU equine chorionic gonadotropin (eCG; Calbiochem, San Diego, CA) and human chorionic gonadotropin (hCG; Sigma-Aldrich, St. Louis, MO) 48 h apart. Mice were of the following genotypes: (C57BL/6 x DBA/2)F1, denoted as (B6D2)F1 (Taconic, Germantown, NY); C57BL/6 (Harlan Sprague-Dawley, Indianapolis, IN); and DBA/2 (Taconic). To obtain fertilized embryos, superovulated (B6D2)F1 females were mated to (B6D2)F1 males. For parthenogenesis and cloning studies, oocytes were isolated at approximately 14–15 h post-hCG injection (hphCG), and cumulus cells were removed by hyaluronidase treatment in HEPES-buffered M2 medium as described [40]. Fertilized embryos were isolated at approximately 20 hphCG. All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals.

Oocytes were cultured in CZB medium [41] supplemented with 5.5 mM glucose (CZB-G) [42]. Activated parthenotes, fertilized embryos, and cloned constructs were cultured under an atmosphere of 5% CO2 and 21% O2 in nitrogen at 37°C in a humidified modular incubator (Billups-Rothenberg, Del Mar, CA). Three culture systems were employed for parthenotes and cloned embryos based on our previous cloning studies [40, 43] and unpublished studies: MEM{alpha} supplemented with 1 mM glutamine and 5 mg/ml BSA, which has produced a high rate of preimplantation development and also term development (2% of activated constructs; S.G. and K.E.L., unpublished data) in the absence of dimethylsulfoxide (DMSO, which can by itself alter cloned embryo phenotype [40]). Whitten medium with EDTA [44] switching to KSOM medium with amino acids (KSOMaa) [45] at the eight-cell stage (abbreviated below as WK), which also produces a high rate of development of cloned embryos to the blastocyst stage [40], and KSOM without amino acids switching to KSOMaa (abbreviated below as KK), which produces a slightly lower rate of blastocyst formation for cloned embryos [43, 46]. All three culture systems produce efficient development to blastocyst stage for control fertilized (B6D2)F1 embryos [40, and unpublished data]. Fertilized embryos for TRC expression analysis (see below) were cultured in MEM{alpha}.

Somatic Cell Nuclear Transfer and Oocyte Activation

Cloned embryos were produced as described [14] and later modified in our laboratory [40] using cumulus cell donor nuclei of the genotypes indicated below. The use of DMSO as a solvent for cytochalasin B was avoided by preparing a 1000x stock solution in ethanol. The concentration of polyvinylpyrrolidone in the cell suspension medium was reduced to 3% to avoid toxic effects of higher concentrations. Embryos were injected in either bicarbonate-buffered CZB-G medium or in HEPES-buffered CZB-G, washed extensively, and then cultured in the media described above. Oocyte activation for parthenotes and clones was achieved by 6-h incubation in calcium-free CZB-G supplemented with 10 mM SrCl2 and 5 µg/ml cytochalasin B [14].

Analysis of Transcription Requiring Complex Synthesis

To visualize synthesis of the two-cell, stage-specific 70 kDa transcription requiring complex (TRC), embryos were labeled at the mid two-cell stage with L-[35S]methionine (>1000 Ci/mmol) at a concentration of 0.5 mCi/ml in CZB medium for 2 h. At the end of the labeling period, embryos were treated for 10 min at room temperature in a solution of 50 mM Tris-HCl, pH 7.4 containing 2% Triton-X100, and 0.3M KCl [47]. The 70-kDa TRC remains insoluble under these conditions while most cellular proteins are extracted, thus enriching for the TRC and improving its visualization. Treated embryos were then solubilized in Laemmli sample buffer [48] and subjected to polyacrylamide gel electrophoresis on 10% gels. Samples of cloned or parthenogenetic embryos were coelectrophoresed with four to five samples of fertilized embryos on the same gel. The radiolabeled proteins were visualized using a Fuji PhosphorImager. The TRC expression for each cloned or parthenogenetic embryo was expressed as a fraction of the mean TRC signal obtained for the fertilized embryos on the same gel.

Statistical Analysis

The statistical significance of differences in development was evaluated using a chi-squared test of independence to determine whether the proportion of embryos attaining a specific stage was affected by culture condition. Differences in TRC expression were evaluated by the t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Oocyte Genotype on Parthenogenetic Development

Previous studies revealed that oocytes of the C57BL/6 and DBA/2 strains impose different epigenetic modifications on both maternal (oocytic) and paternal (sperm-derived) genomes, indicating significant differences in oocyte phenotype [1–5]. The effect on the paternal genome occurs postfertilization, indicating differences in how the oocytes of these two genotypes modify incoming chromosomes [3–5]. Differences in oocyte phenotype between these strains have not been examined, however, in the absence of fertilization. The response of parthenogenetic embryos to different culture systems provides one way of evaluating genetic effects on oocyte and embryo quality in the absence of fertilization.

The KK culture system was developed to enhance successful development of fertilized embryos of strains that display the "two-cell block" in other culture systems [45, 49] and appears to be a highly optimized system for mouse embryo culture. Parthenotes produced by activating eggs from (B6D2)F1 females developed at 97% efficiency to the blastocyst stage in this system (Fig. 1A). Parthenotes produced by activating C57BL/6 oocytes, however, developed at a high rate to the eight-cell and morula stages, but then formed blastocysts at a rate of only 45%, significantly less than (B6D2)F1 parthenotes (P < 0.001). Parthenotes produced with DBA/2 oocytes displayed a small but significant increase in the rate of arrest at the two-cell stage, and then increasingly diminished development from the four-cell stage onward (P < 0.001) with only 27% blastocyst formation, significantly less than C57BL/6 or (B6D2)F1 parthenotes (P < 0.05 and 0.001, respectively).



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  		FIG. 1. Development of parthenogenetic embryos prepared with different strains of oocytes and cultured in different media. Embryos were cultured in (A) KSOM/KSOMaa media (KK), (B) Whitten/KSOMaa media (WK), or (C) MEM{alpha} medium. The number of activated embryos assayed for each strain and culture system is indicated above the bars shown for the two-cell stage. Height of bars depicts the fraction (%) of activated embryos reaching the designated stage and represents the total for embryos obtained from at least two different experiments for each strain and medium. B, C57BL/6; D, DBA/2; BD, (B6D2)F1. Different letters denote statistically significant differences within a given stage

Two other culture systems that we have employed for cloning studies are the WK and the MEM{alpha} systems. Because cloning requires in vitro activation of oocytes, as is performed in the production of parthenotes, we examined the effects of oocyte genotype on parthenote development in these culture systems. As with the KK system, we observed efficient (90%) development of (B6D2)F1 parthenotes to the blastocyst stage in the WK system (Fig. 1B). The C57BL/6 parthenotes developed significantly more efficiently in the WK system than in the KK system (P < 0.001). Development of DBA/2 parthenotes was essentially equal between KK and WK, and significantly less than observed for either C57BL/6 or (B6D2)F1 parthenotes. The incidence of two-cell arrest for DBA/2 parthenotes was significantly (P < 0.05) greater in the WK system than in the KK system. For the MEM{alpha} system, efficient development (91%) was again seen for (B6D2)F1 parthenotes (Fig. 1C). The C57BL/6 parthenotes displayed efficient development to the eight-cell/morula stage, but blastocyst formation was significantly reduced to 24% (P < 0.001). DBA/2 parthenotes displayed a severe two-cell block in this culture medium.

Effect of Oocyte Genotype on Cloned Embryo Development

The ability of the oocyte to support cloned embryo development is a unique property of the oocyte and constitutes a useful indicator of genetic differences in oocyte quality, specifically in ooplasmic reprogramming of nuclei. With the current widespread interest in improving the success of cloning, it is imperative to understand the effects of oocyte genotype on clone development. Additionally, the cloning method provides an opportunity to determine whether strain-dependent differences in epigenetic modification of parental genomes in fertilized embryos correlate with differences in somatic cell nuclear modification after nuclear transfer. We therefore evaluated the ability of oocytes of these three genotypes to support cloned embryo development in the three different culture systems using (B6D2)F1 cumulus cell donor nuclei as a constant. A total of 3991 cloned constructs were produced for the studies shown below.

We previously reported that clones prepared with (B6D2)F1 oocytes can form blastocysts at a rate of approximately 22% in the KK system [43], and those data are reproduced in Figure 2A for comparative purposes. Surprisingly, we observed that the medium employed for nuclear transfer can significantly affect development (P < 0.001) at subsequent stages in the KK system, with a sharp reduction in progression past the two-cell stage and only 6% blastocyst formation observed using HEPES-buffered nuclear transfer medium for clones made with (B6D2)F1 oocytes. Clones made with both C57BL/6 and DBA/2 oocytes displayed a high incidence of two-cell arrest in KK, and little or no blastocyst formation. Development of clones made with DBA/2 oocytes was more severely affected at the two-cell to four-cell stage transition using the HEPES-buffered as compared with the bicarbonate-buffered nuclear transfer medium (P < 0.001).



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  		FIG. 2. Effect of recipient oocyte genotype on cloned embryo development in different media. Cloned embryos were prepared with (B6D2)F1 cumulus cell nuclei and then cultured in (A) KSOM/KSOMaa media (KK), (B) Whitten/KSOMaa media (WK), or (C) MEM{alpha} medium. The number of activated embryos assayed for each strain and culture system is indicated above the bars shown for the two-cell stage. Height of bars depicts the fraction (%) of activated embryos reaching the designated stage and represents the total for embryos obtained from at least two different experiments for each strain and medium. Samples are designated in the accompanying keys according to the formula: donor/recipient–nuclear transfer medium-culture system. B, C57BL/6; D, DBA/2; BD, (B6D2)F1. Different letters denote statistically significant differences within a given stage

In the WK system (Fig. 2B), we also observed the highest rate of blastocyst formation among clones made with (B6D2)F1 oocytes. In this culture system, the HEPES-buffered nuclear transfer medium produced better results than the bicarbonate-buffered medium (P < 0.001) for clones made with (B6D2)F1 oocytes, which was opposite the results obtained with the KK system. There was no significant difference between clones made with (B6D2)F1 and C57BL/6 oocytes using the bicarbonate-buffered nuclear transfer medium, but there was a significant difference when using the HEPES-buffered nuclear transfer medium (P < 0.001). No difference was seen between HEPES- and bicarbonate-buffered nuclear transfer media using C57BL/6 or DBA/2 recipient oocytes (i.e., HEPES-buffered nuclear transfer medium did not confer any advantage). Development of clones made with DBA/2 oocytes was significantly reduced relative to the other genotypes, with a pronounced arrest at the two-cell stage in both sets of conditions, as was observed for the KK system.

We achieved the highest rate of blastocyst formation (60%) using (B6D2)F1 oocytes and culturing the embryos in MEM{alpha} (Fig. 2C). The HEPES-buffered medium for nuclear transfer also performed better for this culture system (P < 0.001). Clones made with C57BL/6 oocytes produced fewer blastocysts than (B6D2)F1 oocytes (P < 0.001), but development was still appreciable at 37%. Clones made with DBA/2 oocytes once again displayed a severe arrest at the two-cell stage and no blastocyst formation.

These data permit an assessment of the effect of oocyte strain on clone development under conditions where non-clone-specific effects of oocyte genotype, as revealed by parthenote development, are minimal. To facilitate this, the data shown in Figure 2 were normalized to the fraction of parthenotes attaining each stage (Fig. 3). We observed the lowest amount of two-cell arrest among DBA/2 parthenotes in the KK culture system. The efficiency of development of clones made with DBA/2 oocytes was significantly lower than those made with (B6D2)F1 oocytes going from the two-cell to four-cell stages and again from the four-cell to eight-cell/morula stages using either nuclear transfer medium. The DBA/2 clones performed somewhat better when the bicarbonate-buffered nuclear transfer medium was used. Interestingly, the rate of blastocyst development, corrected for parthenote development, was similar between clones made with (B6D2)F1 and DBA/2 oocytes, a result that reflected developmental arrest between the morula and blastocyst stages in many of the clones made with (B6D2)F1 oocytes. Thus, there remained a difference between the clones made with DBA/2 and (B6D2) F1 oocytes, in that the DBA/2 clones arrested earlier (i.e., at the two-cell stage). For comparisons between C57BL/6 and (B6D2)F1 oocytes, the highest rate of parthenote development to blastocyst stage for C57BL/6 oocytes was in the WK system, in which there was no difference relative to (B6D2)F1 oocytes (Figure 1B). There was no difference between C57BL/6 and (B6D2)F1 clone development in this culture system when the bicarbonate-buffered nuclear transfer medium was present, but clone development was significantly greater in the (B6D2)F1 clones when the HEPES-buffered nuclear transfer medium was used (Figure 3B). This difference in the group employing the HEPES-buffered nuclear transfer medium was also evident in the fraction of four-cell stage embryos progressing to the blastocyst stage. This indicates that the overall efficiency of reprogramming may be similar between these two strains, but that there is an effect of nuclear transfer medium wherein the efficiency of reprogramming may be selectively improved in the (B6D2)F1 clones by the use of the HEPES-buffered medium. Overall, although it is difficult to compare directly the efficiency of clone development between C57BL/6 and DBA/2 oocytes (because parthenotes of these two strains displayed different culture medium preferences), comparisons within the context of the medium preferred by parthenotes of the same two strains reveal clear differences. Clones made with C57BL/6 oocytes did not display a significantly elevated incidence of two-cell arrest in the culture system (WK) preferred by C57BL/6 parthenotes. In contrast, clones made with DBA/2 oocytes displayed a significant two-cell arrest in the culture system (KK) preferred by DBA/2 parthenotes, which did not display a severe two-cell arrest (Figure 1A). Additionally, development of the C57BL/6 clones, when normalized to parthenote development, was elevated in the MEM{alpha} medium, reminiscent of the enhancement seen for (B6D2)F1 clones, but no such enhancement was seen for DBA/2 clones.



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  		FIG. 3. Cloned embryos developmental data normalized to parthenote development. The data in Figure 2 were divided by the fraction of parthenotes attaining each stage. For the MEM{alpha} data (C), data for clones made with DBA/2 oocytes were not normalized, because nearly all parthenotes arrested development at the two-cell stage. Samples are designated as in Figure 2. B, C57BL/6; D, DBA/2; BD, (B6D2)F1. Different letters denote statistically significant differences within a given stage

Effect of Donor Cell Genotype on Cloned Embryo Development

The above results indicated that oocytes produce strain-specific effects on the response of cloned embryos to different culture systems. Previous studies indicated that the donor cell nucleus can profoundly alter cloned embryo phenotype, even as early as the one-cell stage [40, 43]. We therefore examined whether the donor cell genotype affected cloned embryo development using (B6D2)F1 oocytes in two different culture systems (Fig. 4). The effect of donor cell genotype was similar in both media, with both C57BL/6 and DBA/2 donor cell nuclei producing a modest reduction in efficiency of preimplantation development (P < 0.05 and 0.01, respectively, for KK; P < 0.01 and 0.001, respectively, for MEM{alpha}). Clones prepared with DBA/2 nuclei developed at a significantly reduced rate relative to nuclei of the other donor genotypes and exhibited a greater tendency to arrest at the two-cell stage.



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  		FIG. 4. Effect of donor cell genotype on cloned embryo development in different media. Cumulus cell nuclei of the three genotypes indicated were transferred to (B6D2)F1 oocytes and cultured in the indicated systems. Samples are described in the accompanying keys as in Figure 2. The number of activated embryos assayed for each strain and culture system is indicated above the bars shown for the two-cell stage. Height of bars depicts the fraction (%) of activated embryos reaching the designated stage and represents the total for embryos obtained from at least two different experiments for each strain and medium. Some of the clones prepared with C57BL/6 cumulus nuclei and cultured in MEM{alpha} were transferred at the morula stage to foster mothers, leaving the indicated number of embryos to be assayed for blastocyst formation. Different letters denote statistically significant differences within a given stage

Effect of Oocyte Genotype on TRC Expression in Cloned Embryos

The severe arrest at the two-cell stage observed for clones made with DBA/2 oocytes, and to a lesser extent for clones made with C57BL/6 oocytes, indicated a possible defect in embryonic genome activation. Synthesis of the 70 kDa TRC provides a convenient marker of genome activation that can be assayed at the single embryo level [47]. We therefore compared TRC synthesis between cloned embryos prepared with different types of oocytes and cultured in different media, and between clones, parthenotes, and fertilized embryos cultured in MEM{alpha}. For these studies, all clones were prepared using the HEPES-buffered nuclear transfer medium. The average rate of TRC synthesis was quite low in DBA/2 parthenotes compared with fertilized embryos, C57BL/6 parthenotes, or (B6D2)F1 parthenotes cultured in MEM{alpha} (Fig. 5). Thus, for parthenogenetic embryos a reduced rate of TRC synthesis was associated with a high rate of two-cell arrest. The overall expression of the TRC was reduced in cloned embryos under most conditions. Cloned embryos made with (B6D2)F1 oocytes, which displayed a comparatively high rate of blastocyst formation in all media, displayed significantly reduced (P < 1 x 10-4) TRC synthesis that was approximately 40% that of fertilized two-cell embryos in either MEM{alpha} or KK culture systems (Fig. 5). The degree of reduction among groups of cloned embryos, however, did not correlate consistently with the degree of two-cell stage arrest. The rate of TRC synthesis was reduced to a greater degree (P < 0.05) in clones made with (B6D2)F1 oocytes and cultured in WM, which contrasted with the lower rate of two-cell arrest for these clones in WM (Fig. 2). Clones made with C57BL/6 oocytes and cultured in the KK system displayed a reduced rate of TRC synthesis relative to fertilized embryos, but TRC synthesis was similar to that evident in clones made with (B6D2)F1 oocytes and grown in MEM{alpha}. The rate of TRC synthesis in clones made with C57BL/6 oocytes was significantly reduced in the MEM{alpha} medium relative to those made with (B6D2)F1 oocytes. Thus, the reduction in TRC synthesis in clones made with C57BL/6 oocytes was more severe in the MEM{alpha} system, whereas the rate of two-cell arrest was more severe in the KK system (Fig. 2). Interestingly, clones made with C57BL/6 oocytes and cultured in WM displayed a high rate of TRC synthesis similar to fertilized embryos and C57BL/6 parthenotes cultured in MEM{alpha}, and significantly greater than the mean values for other groups of clones. Clones made with DBA/2 oocytes synthesized the TRC at a rate that was reduced relative to fertilized embryos, but not statistically different from that observed for clones made with (B6D2)F1 oocytes in the KK system. The rate of TRC synthesis in clones made with DBA/2 oocytes and cultured in MEM{alpha} medium was significantly reduced relative to clones made with (B6D2)F1 oocytes and cultured in MEM{alpha}, which agreed with the difference in the rate of two-cell arrest between these types of clones in MEM{alpha} (Fig. 2C). The rate of TRC synthesis was not different, however, between clones made with C57BL/6 and DBA/2 oocytes and cultured in MEM{alpha}, despite a large difference in the incidence of two-cell arrest (Fig. 2C).



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  		FIG. 5. Relative rates of synthesis of the 70 kDa transcription requiring complex (TRC) in clones produced in different media. Fertilized embryos were cultured in MEM{alpha} and then labeled in CZB medium with L-[35S]methionine for 2 h at 44 h post-hCG injection. Cloned embryos were prepared with cumulus cell nuclei using HEPES-buffered injection medium and then cultured in the media indicated. Cloned embryos and parthenotes were then labeled in CZB medium for 2 h at 24.5 h postactivation. Embryos were then treated with triton X-100 as described in Materials and Methods and lysed individually in sample buffer. Groups of each type of cloned embryo sample were coelectrophoresed with four to five fertilized embryo samples, the radioactivity in the TRC bands quantified, and the values for the cloned embryos expressed as the percent of the mean value for fertilized embryos (hatched bar) for that gel. Abbreviations are as in Figure 2. Different letters denote statistically significant differences


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Oocytes of different strains display important genetic differences that affect the overall physiology and health of parthenogenetic embryos. The oocyte strain of origin also affects cloned embryo development. This effect on clone development exceeds the differences observed in parthenogenetic embryos. Specifically, even when the data are normalized to correct for the efficiency of parthenote development (i.e., non-clone-specific effects of the ooplasm), clones made with DBA/2 oocytes display a severe arrest at the two-cell stage, thus distinguishing them from clones made with either C57BL/6 or (B6D2)F1 oocytes. Clones made with C57BL/6 oocytes generally displayed developmental potentials similar to those made with (B6D2)F1 oocytes (with some effects of culture system evident), and generally displayed better development than clones made with DBA/2 oocytes. These differences in clone development are consistent with oocyte strain-dependent differences in nuclear reprogramming, specifically in the cloned embryos.

Androgenetic embryos constructed from fertilized DBA/2 oocytes to contain two sets of paternal chromosomes display limited developmental potential, whereas those prepared with C57BL/6 oocytes develop to the blastocyst stage at a significantly greater rate [3–5]. The C57BL/6 phenotype is dominant, and two controlling loci (Egm1 and Egm2) have been mapped [5]. This phenotypic difference is attributable to differential modification of the paternal genome during pronucleus formation [3–5]. The deficiency in somatic cell nuclear reprogramming reflected in the pronounced two-cell arrest among clones made with DBA/2 oocytes is thus associated with this previously documented deficiency in programming of paternal pronuclei to support blastocyst formation. This is consistent with the hypothesis that pronuclear programming and somatic cell nuclear reprogramming are mediated by related mechanisms. Additionally, the data for parthenogenetic development reveal for the first time differences between oocytes of these two strains in the absence of fertilization. The genetic differences in oocyte quality revealed here provide a basis for future studies to address the molecular determinants of oocyte quality and factors that participate in nuclear reprogramming during cloning.

The effect of oocyte strain of origin on parthenogenesis may reflect a combination of factors related either to ooplasmic components that support early embryo metabolism, or strain-dependent effects of the oocyte activation protocol. We previously showed that embryos of different strains differ in their ability to progress beyond the two-cell stage in the presence of {alpha}-amanitin [50], indicating a difference in ooplasm composition. Other studies have revealed that the number of calcium oscillations can produce different degrees of oocyte activation that affect development [51]. Oocytes of different genotypes may possess different thresholds of responses to such oscillations, creating a situation in which the activation protocol may require optimization for different strains. Such a situation would be of obvious importance for improving cloning procedures, wherein standard protocols are commonly applied to material of diverse genetic origin.

Several studies have addressed the effects of donor cell type and genotype on the success of cloning [52, 53]. Inoue et al. [52] reported no significant effect of donor cell genotype on preimplantation clone development, but significant effects on term development, which were affected by donor cell type (cumulus versus Sertoli cell). Wakayama and Yanagimachi [53] also reported an effect of donor cell type. Additionally, donor cells of most homozygous inbred genotypes were less effective than those of hybrid genotypes with respect to term development [53]. With respect to preimplantation development, we observed a similar advantage of the hybrid genotype over homozygous genotypes. Wakayama and Yanagimachi [53] also reported that C57BL/6 donor cumulus cell nuclei were less effective than DBA/2 cumulus cell nuclei, whereas we observe the opposite relationship. This difference in observations most likely reflects differences in micromanipulation and culture systems employed.

Few data exist in the literature about the effects of the oocyte genotype [53, 54]. The data related to oocyte genotype effects have been limited in their scope, and no study has been presented that takes into account genotype-dependent responses to the culture environment. Our data reveal a partial correlation between the ability of a given oocyte genotype to support parthenogenetic development and its ability to support cloned embryo development. Qualitatively, DBA/2 oocytes supported the lowest rate of parthenogenetic development and the lowest rate of clone development. However, the severe two-cell arrest among DBA/2 cloned embryos clearly exceeded the modest reduction in progression of parthenotes to the four-cell stage in the KK or WK systems. The greatest rate of blastocyst formation for clones made with C57BL/6 oocytes was seen in MEM{alpha}, whereas this medium supported the lowest rate of C57BL/6 parthenogenetic development. Thus, parthenogenetic development may not provide a clear indicator of the ability of oocytes to support clone development.

Cloned embryos generally displayed a deficiency in the expression of the 70 kDa TRC, a frequently used marker of embryonic genome activation, the expression of which is affected in other nuclear transfer embryos that undergo developmental arrest [13]. The degree of reduction in TRC synthesis, however, was not consistently correlated with the fraction of cloned embryos arresting at the two-cell stage. TRC synthesis was generally reduced for clones made with DBA/2 oocytes, most of which underwent two-cell stage arrest. However, TRC synthesis was also reduced in cases where two-cell stage arrest was not prominent. These data indicate that the TRC is not a reliable marker for evaluating the suitability of experimental conditions for supporting the long-term developmental potential of cloned embryos. Interestingly, the WK system supported the most efficient production of blastocysts for parthenogenetic C57BL/6 embryos and also supported the highest average rate of TRC synthesis observed among the groups of cloned embryos. This indicates a possible interaction between ooplasmic factors, culture medium, and TRC induction. The overall reduction TRC synthesis in clones under most conditions suggests that the activation of stage-specific genes like the TRC gene(s) may be either delayed or inhibited in a majority of cloned embryos in most situations. Such defects in the expression of two-cell, stage-specific genes may reflect broader deficiencies that could reduce the long-term developmental potential of cloned embryos.

Our data also show that embryo culture media that have been optimized for one instance are not necessarily optimum in other cases. In particular, the KK culture system, which was optimized to avoid the two-cell block in embryos of outbred strains [45, 49] was inferior to the WK system for C57BL/6 parthenotes and did not support efficient development of the DBA/2 parthenotes.

Last, it is important to note the effect of nuclear transfer medium on subsequent development. We observed that this effect differed depending on the culture system employed postactivation and the strain of oocyte employed. As a result, one cannot conclude simply that a particular kind of nuclear transfer medium is inherently good or bad, but rather that the choice of the best nuclear transfer medium is dependent upon the selection of media used at later stages. This reveals an important and previously unappreciated interaction between media employed at different stages of the cloning procedure. This has obvious and important ramifications with respect to improving cloning conditions. Additionally, this observation provides an example of how procedures that are undertaken at an early stage of development can have significant effects during subsequent development. The recent reports describing possible effects of embryo/oocyte culture or assisted reproduction methods on the incidence of imprinting disorders among children [55–58], as well as the effects of embryo culture on imprinting [59, 60], also make this point. The surprising feature shown here is that even a brief residence period of 10–20 min in the nuclear transfer medium and/or nearly negligible quantities of medium that might enter the oocyte during injection can exert such an effect. This observation provides an important reminder of the potential pitfall of assuming that seemingly minor steps in a procedure, even those that have appeared to be innocuous previously, can have significant consequences, particularly if later parts of the procedure are altered. Further studies in suitable animal models and exploiting a variety of different experimental and microsurgical approaches remain necessary in order to evaluate fully the consequences of such procedures.


   ACKNOWLEDGMENTS
 
We thank Bela Patel and Malgorzata McMenamin for their technical assistance.


   FOOTNOTES
 
1 Supported by grants from the NIH/NICHD (HD38381 and HD43092) to K.E.L. and a grant from the Fulbright Commission, Germany, to E.C. Back

2 Correspondence: Keith E. Latham, 3307 North Broad Street, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}temple.edu Back

3 Current address: Department of Zoology and Developmental Biology, University of Göttingen, 37077 Göttingen, Germany Back

4 Current address: Advanced Cell Technology, One Innovation Drive, Biotech Three, Worcester, MA 01605 Back

Received: 15 October 2003.

First decision: 5 November 2003.

Accepted: 3 December 2003.


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