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Regular Article |
a The Fels Institute for Cancer Research and Molecular Biology
b Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
c Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cloning, early development, embryo, ovum, reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The available data indicate that the most severe effects of culture media occur during the first few cell cycles [7]. In the mouse, this may reflect the dramatic changes in cellular morphology and physiology that accompany blastomere polarization, epithelialization, and compaction, which begin at the 8-cell stage [8]. These changes in cellular morphology are accompanied by significant changes in mRNA expression [9, 10] and in the expression, localization, and activity of numerous proteins, including ion transport proteins, glucose transport proteins, and other proteins that collectively may provide a greater ability for the embryo to maintain homeostasis [11–17]. Once embryos have undergone this transition, they appear to be more tolerant of their in vitro culture environment, although effects of exogenous growth factors added to the medium become apparent [18–23].
The development of cloned embryos remains poor, with respect to the number of embryos that develop to term [24–34]. Even preimplantation development is somewhat modest, and striking variability exists, for example, in development to the blastocyst stage [29]. Much recent discussion about the basis for the limitations in cloned embryo development has centered around the question of whether the donor somatic cell nucleus is reprogrammed efficiently. Comparatively little attention has been devoted to the possible effect of culture environment on clone development. Although nuclear reprogramming is of obvious importance to the success of the procedure, it is equally important that the overall physiology and metabolism of the cloned embryo operate efficiently; first to maintain viability, and second, to support the fundamental process of nuclear reprogramming. We investigated the effects of different culture media on preimplantation cloned embryo development in vitro. We report here unusual culture medium preferences for cloned mouse embryos during the preimplantation period, and discuss the possible implications of this for understanding nuclear reprogramming and for the eventual improvement of the overall efficiency of cloning.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recipient oocytes for all studies were from (B6D2)F1 females superovulated by the injection of 5 IU of eCG followed 48 h later by 5 IU of human chorionic gonadotropin (hCG). Oocytes were isolated at 13–15 h post-hCG injection in M2 medium [35], and cumulus cells were removed with 100 U/ml hyaluronidase (ICN Pharmaceuticals, Costa Mesa, CA). Oocytes were cultured in CZB medium [2] for those conditions that lacked glucose during the 1-cell stage, or in CZB medium supplemented with 5.5 mM glucose (CZB-G) for those conditions that employed glucose during the 1-cell stage or media other than CZB or CZB-G. Oocytes were cultured at 37°C in an atmosphere of 5% CO2 in air as described elsewhere [24]. Meiotic spindles, visualized using differential interference contrast optics, were removed using a blunt, piezo-driven pipette (
15 µm outside diameter) to penetrate the zona pellucida as described elsewhere [24]. Spindle removal was performed in warm (37°C) Hepes-buffered CZB medium [36] with or without 5.5 mM glucose, and supplemented with 2.5 µg/ml cytochalasin B (Sigma, St. Louis, MO). Spindles were removed within 10 min of placing oocytes in the manipulation chamber. The spindle-free oocytes were then washed extensively and returned to culture as above, for 1–2 h before injection.
Cumulus cells were removed from oocytes using hyaluronidase as described above, except that the hyaluronidase concentration was increased to 300 U/ml. The lower concentration of enzyme used for oocyte isolation is adequate for removing cumulus cells without damaging oocytes, whereas the higher concentration reduces adhesion of cumulus cells to the pipette during injection. Cumulus cells were maintained in this solution on ice until just before use, when they were collected by centrifugation, resuspended in a solution of CZB-G supplemented with 2%–3% polyvinyl pyrrolidine (PVP), and dispersed in small quantities into microdrops on the injection chamber. Previous studies employed a higher concentration (12%) of PVP [24], but in our experience this high concentration can adversely affect embryo health, and is not needed when siliconized pipettes are employed. All studies adhered to procedures that are consistent with the National Research Council Guide for the Care and Use of Laboratory Animals.
Injection of Cumulus Cell Nuclei and Oocyte Activation
Cumulus cells were used as nuclear donor cells in all studies. Cumulus cells were isolated from cumulus-oocyte complexes following superovulation of three different genotypes of females, which were employed as a part of other ongoing studies. These include (B6D2)F1 females (designated hereafter as BD), (C57BL/6 x Mus muscaris castaneus)F1 females (designated hereafter as B-CAST), and F1 progeny produced by mating females bearing a M. muscaris castaneus-derived chromosome 7 (unpublished data) to C57BL/6 males (designated hereafter as CAST7-B). No significant difference was observed among these three genotypes of donor cell with respect to preimplantation cloned embryo development, and data for different genotypes were combined where indicated (see below).
Cumulus cell nuclei were isolated from individual cells by several rounds of trituration into the injection pipette as described elsewhere [24]. The pipette was then passed through the zona pellucida using the piezo pipette driver as described in [24], and the nuclei were injected into the spindle-free oocytes. On average, approximately 80% (± 6% SD) of oocytes survived injection. Injections were performed at room temperature within 5–10 min on small groups of spindle-free oocytes in fully equilibrated CZB or CZB-G lacking BSA, but supplemented with 0.1% polyvinyl alcohol [36]. Tetraploid control embryos, produced by injecting nuclei into eggs with spindles remaining, were also obtained. These embryos provide controls for nonspecific effects of the injection process. After a recovery period of 2–4 h, injected oocytes were activated by a 6-h incubation in calcium-free CZB or calcium-free CZB-G supplemented with 10 mM SrCl2 and 5 µg/ml cytochalasin B as described in other reports [24, 29]. A small fraction (5%) of activated oocytes underwent fragmentation after removal from cytochalasin B. This most likely resulted from failure to inject nuclei into those oocytes successfully, as spindle-free oocytes without injected nuclei generally fragment in this manner (unpublished data). These fragmented embryos were therefore not counted as successfully injected oocytes. Activated oocytes were extensively washed and cultured as described below. Parthenogenetic control embryos were obtained by activation of intact oocytes in the same manner as above.
Embryo Culture
Cloned embryos and their parthenogenetic and tetraploid control embryos were cultured at 37°C in an atmosphere of 5% CO2 in air as recommended in [24]. Fertilized embryos were cultured either in 5% CO2 in air or 5% CO2, 5% O2, and 90% N2, as indicated. Media included in the study were Whitten medium (WM) supplemented with EDTA [37], CZB [2], CZB-G [38], and potassium simplex optimized medium supplemented with amino acids [3,39] (denoted here as KSOM).
Statistical Analysis
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 cell number between blastocysts cultured under different conditions were evaluated by the t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several different culture medium formulations were employed in this study. For embryos of the (B6D2)F1 strain and numerous inbred strains (including parthenogenetic embryos and uniparental embryos produced by pronuclear transfer), all of these media support highly efficient in vitro development from the 1-cell to blastocyst stage, either alone (WM or KSOM) or in sequential combinations (e.g., CZB followed by WM) [27, 29–41]. This high efficiency of development to the blastocyst stage was confirmed for all of these media (Table 1). A high efficiency of blastocyst formation was also observed for fertilized embryos under either 5% or atmospheric O2 concentration (Table 1). No significant differences were seen for fertilized control embryos between different culture media.
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Beneficial Effect of Glucose for Cloned Embryos During the 1-Cell Stage
Previous studies indicated the use of CZB-G in the production of cloned mouse embryos [38], whereas other studies specified unsupplemented CZB medium lacking glucose [24]. Because CZB medium, which lacks glucose, normally supports very efficient mouse embryo development in vitro [2], and given this variation in reported culture conditions, we wished to evaluate the effects of glucose on early development (Table 2). Parthenogenetic controls, which provide a control for the oocyte activation and culture protocols applied to the cloned embryos, develop with high efficiency to the blastocyst stage in both media (Table 2). Tetraploid control embryos, for which cumulus cell nuclei were injected without prior spindle removal, also develop efficiently to the blastocyst stage, but at a reduced rate compared with parthenogenetic controls (Table 2, P < 0.01). However, no difference was observed between CZB and CZB-G for either parthenogenetic or tetraploid control embryos. In striking contrast to parthenogenetic and tetraploid control embryos, we observed a severely reduced ability of cloned embryos to develop in either CZB or CZB-G (Table 2, P < 10-6) . We also observed a highly significant difference (P < 0.001) between these two media in the progression of cloned embryos to each cleavage stage. The fraction of cloned embryos developing from the 2-cell stage to blastocyst stage, however, was not significantly different. Thus, the difference in development observed for cloned embryos between CZB and CZB-G media was attributable primarily to an enhanced rate of development to the 2-cell stage with the CZB-G medium. Moreover, fertilized embryos do not require glucose for early cleavage development. Glucose-free CZB medium supports efficient early cleavage development (with >90% blastocyst formation employing the two-step CZB/WM culture system), whereas glucose-containing media support equally high rates of blastocyst development (Table 1). This contrasts sharply with results obtained for cloned embryos (P < 10-6). The demonstrated preference for glucose-containing medium during the 1-cell stage thus distinguishes cloned embryos from fertilized embryos, as well as parthenogenetic and tetraploid control embryos.
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Effect of Dimethyl Sulfoxide on Cloned Embryo Development
As shown above, CZB and CZB-G media supported poor overall efficiency of development of cloned embryos to the blastocyst stage when employed for continuous culture (Table 2). Other studies reported somewhat higher rates of development for diploid cloned embryos to the blastocyst stage in CZB-G [24, 28, 29]. The difference between those studies and this study appears to be related to a procedural difference, specifically the addition of dimethyl sulfoxide (DMSO) during the activation step [42]. Addition of DMSO at a concentration of 1% to the activation medium produced a significant increase (P < 0.001) in blastocyst formation in CZB-G medium (Table 2). Such an effect has also been observed for concentrations of DMSO as low as 0.1% [42]. It is clear, however, that in the absence of DMSO treatment, which may interfere with the study of the effects of ooplasm on nuclear reprogramming [42], CZB-G fails to support reliable blastocyst development. Therefore, we tested whether other culture media could improve cloned embryo development without DMSO treatment.
Cloned Embryo Development in Other Media
An obvious medium to test for a possible improvement of cloned embryo development is KSOM medium [39]. KSOM medium was specifically designed to overcome the 2-cell stage block in certain strains of embryos, and also supports efficient development to the blastocyst stage [39] (Table 1), with apparent improvements in gene expression profiles over another standard culture medium [3]. Consequently, we tested the ability of KSOM to improve cloned embryo development. Contrary to expectations, KSOM did not improve diploid cloned embryo development, and in fact, was significantly worse at supporting development than CZB-G (Table 3). The inability of cloned embryos to develop efficiently to the blastocyst stage in KSOM medium contrasts with the ability of fertilized embryos to do so at high efficiency (>90% blastocysts; Table 1, P < 10-6).
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In contrast to the poor results obtained with CZB-G and KSOM, other culture media produced significantly better results. WM supported significantly (P < 0.001) greater development to the blastocyst stage than CZB-G (Table 3). This beneficial effect of WM could be realized in embryos initially cultured in CZB-G but switched to WM at the late 4-cell/early 8-cell stage (P < 0.01; Table 3). Switching embryos cultured initially in CZB-G to KSOM at the late 4-cell/early 8-cell stage also produced a significant benefit as compared with culture in CZB-G alone (P < 0.05) (Table 3). Culturing embryos initially in WM followed by KSOM also produced an improved rate of blastocyst development relative to CZB-G alone (P < 0.001; Table 3). It should be noted, however, that development of cloned embryos in all of these culture conditions to the blastocyst stage was significantly less (P < 0.001) than for fertilized embryos (Tables 1 and 3).
There were no significant differences in the percentage of cloned embryos developing to the blastocyst stage between WM, WM/KSOM, CZB-G/WM, or CZB-G/KSOM. No significant differences in development were observed between cloned embryos produced with the three different types of donor cells and cultured in a given medium. The blastocysts developing in the WM/KSOM combination exhibited increased cell numbers per blastocyst. For example, blastocysts (120 h postactivation, BD donor cell genotype) obtained from continuous culture in WM possessed an average of 27.9 (± 10.4 SD) cells, compared with 36.2 cells (±11.5 SD) cells for embryos switched to KSOM medium at the late 4-cell/early 8-cell stage, which was a significant difference (P < 0.03). The results therefore indicate that two-step or multistep culture systems (e.g., changing culture medium at the late 4-cell/early 8-cell stage), can be beneficial for the development of cloned embryos. While CZB-G supports good development of cloned embryos to the 2-cell stage, switching to WM or KSOM at the late 4-cell/early 8-cell stage significantly improves blastocyst formation relative to continuous culture in CZB-G. Similarly, switching from WM to KSOM improves blastocyst cell number. Thus, the in vitro culture requirements of cloned embryos may change over time. The cell number values were lower for cloned embryos than those obtained for fertilized embryos fixed after a comparable developmental period (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While this study was under review, another laboratory reported similar effects of culture medium formulations, but with alternative culture media to those employed here [43]. Development was poor in CZB-G and was significantly improved in a combination culture medium system (G1/G2 or KSOM/G2), just as we observed with our combination systems. It was likewise observed that the effects of culture medium were specific to the cloned embryos and not seen with parthenogenetic embryos [43]. The effect of glucose, however, was not revealed in that study because only CZB-G was used. That other study also revealed that the effects of culture medium were most pronounced before the morula stage, as we observed. One difference between the two studies related to the 1-cell to 2-cell conversion in KSOM. This may reflect an unexpected effect of amino acid augmentation of KSOM medium, which typically has no harmful effect on early development of fertilized embryos (Table 1 and [3]).
We propose that the cellular physiology or metabolism of early cloned embryos differ from those of normal embryos due to incomplete nuclear reprogramming of gene expression before the 8-cell stage. One or more rounds of DNA replication may be required to achieve complete nuclear reprogramming, and genes previously programmed for a high rate of transcription in the donor cell nucleus may remain highly expressed, due to the transcriptionally permissive nature of the developing 1-cell embryo [10, 44, 45]. This may support the production of transcripts from the donor cell nucleus that are not normally produced during the 1-cell stage. As a consequence of oocyte activation, translational recruitment of maternally inherited mRNAs would promote the expression of proteins that support normal embryogenesis. The expression levels of other genes that are characteristic of normal 2-cell to 8-cell embryos may not be realized. As a result, during the 1-cell stage, and possibly one or two subsequent cell cycles, the gene expression repertoire of the cloned embryo would be intermediate between that of the donor cell and that of the normal embryo, leading to a physiologically or metabolically dysfunctional state.
The dysfunctional state can be at least partially overcome through the use of culture conditions that have come to be viewed as suboptimal for normal mouse embryos, whereas embryo-optimized media (e.g., CZB or KSOM) can be less suitable for cloned embryos. This effect of culture medium could relate to the energy substrate preference and ability of cloned embryos to regulate ionic composition. The beneficial effect of glucose during the 1-cell stage of cloned embryos is especially intriguing in this regard. Glucose has often been reported to inhibit early cleavage development [2, 5, 46, 47], although it appears likely that this inhibition is dependent upon other medium components, such as inorganic phosphate [1, 3, 48, 49]. Normal mouse embryos clearly do not require glucose in vitro before the 8-cell stage, and the CZB medium contains sufficient pyruvate and lactate to support early cleavage development (Table 1 and [2]). That the cloned embryos exhibit a beneficial effect of glucose distinguishes them from normal embryos, and indicates that the transcriptional activity of the donor nucleus may result in an enhanced ability to uptake and metabolize glucose, possibly through expression of glucose transporters [50–52] and other proteins involved in glucose metabolism. It is interesting that the donor cell nuclei were obtained from cumulus cells, which are specifically designed to metabolize glucose and pass metabolic intermediates to the developing oocyte at a high rate [53]. Thus, if nuclear reprogramming is incomplete immediately following oocyte activation, then a propensity for increased glucose uptake and oxidative glucose metabolism may continue to be expressed in the cloned embryo. This may in turn drive the cloned embryo to a preference for glucose as an energy substrate, in contrast to the normal 1-cell embryo. An alternative possibility for the beneficial effect of glucose could relate to a role for glucose as a signaling molecule. The GLUT1 glucose transporter localizes preferentially to the nuclei of normal 1-cell to 4-cell embryos, and it was suggested that this may affect gene transcription [13]. Other reports indicate that exogenous glucose affects gene expression in preimplantation embryos and can have long-term effects on developmental phenotype [23, 54, 55]. Metabolic defects can also negatively affect ATP production and seriously impede many fundamental processes that are essential for development, and that are especially important for nuclear reprogramming and genome stability, such as DNA replication, DNA repair, and karyokinesis.
In addition, cloned embryos may differ in expression of proteins that regulate ionic composition, which would also affect developmental capacity in different culture media [35, 56]. Embryos are sensitive to changes in medium osmolarity, and in fact, one attribute of KSOM medium is a reduced sodium content and reduced osmolarity compared to other media [3, 57, 58]. This effect of osmolarity can be modulated by other solutes, such as betaine and amino acids [58, 59], thus revealing complex interactions between inorganic ion composition, organic osmolytes, glucose, and other medium components in affecting embryo development.
The altered responses of cloned embryos demonstrated here, which are the more remarkable for having occurred with a nonblocking genetic background, indicate that traditional embryo culture conditions are likely suboptimal for cloned embryos, and in fact, the in vivo environment of the oviduct itself may also be suboptimal. Although altering medium composition can improve blastocyst development, as shown here, this alone may not necessarily translate into greater term development. The efficiency of blastocyst development demonstrated here and in most studies remains rather low for cloned embryos relative to that observed for fertilized embryos in the same culture media. In addition, the number of cells contained in cloned blastocysts is shown here to be smaller than that typically observed for cultured fertilized blastocysts. These results indicate that improvements in medium composition may be possible. Defining optimum in vitro culture conditions for cloned embryos, therefore, may eventually improve term development for cloned embryos, but likely will not be a trivial exercise. Future studies evaluating the basic physiology and metabolic properties of cloned embryos should be useful for optimizing culture media through rational design.
The ability of DMSO treatment to overcome the effects of culture medium on cloned embryo development is also intriguing. This result indicates that, although nuclear reprogramming by the ooplasm may be incomplete, some changes induced in the nucleus by DMSO treatment may accelerate or modify the reprogramming process, alter the nucleus in a way that renders the cloned embryo's gene expression pattern more embryo-like, or alter the response of the embryo to the culture environment. DMSO has a wide range of effects on cells, including induction of cellular differentiation, modulation of transcriptional programs and transcription factor content, effects on the cytoskeleton, antioxidant protective effects, and induction of thermotolerance and stress tolerance [60–66]. It will be important to define the effects of ooplasm versus DMSO on nuclear reprogramming. Awareness of the responses of cloned embryos to different culture media will be important for undertaking such studies. It will also be interesting to evaluate whether nuclei from different donor cell types (cells from different tissues or cells in different cell cycle stages) produce cloned embryos that respond differently to culture conditions than the cumulus cell-derived cloned embryos examined here. If in the absence of DMSO treatment gene expression profiles of the donor nuclei persist for a period of time in the cloned embryo, then it is possible that the physiological state of the donor nucleus may affect the response of the embryo to the culture environment, and possibly to the oviductal environment after embryo transfer.
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FOOTNOTES |
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1 This research was supported by grants HD 38381 and 5 T32 CA09214-20 from the National Institute of Child Health and Human Development and the Howard Hughes Medical Institute. M.R.W.M. was supported by a grant from The Lalor Foundation. 
2 Correspondence: Keith Latham, Temple University School of Medicine, The Fels Institute, 3307 North Broad Street, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}unix.temple.edu
Accepted: November 14, 2001.
Received: July 19, 2001.
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and EGF. Development 1991; 113:919-930[Abstract]. Biol Reprod 1997; 56:1088-1096[Abstract]-deficient mouse blastocysts. Biol Reprod 1998; 59:136-144This article has been cited by other articles:
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  Z. Han, R. Vassena, M. M. Y. Chi, S. Potireddy, M. Sutovsky, K. H. Moley, P. Sutovsky, and K. E. Latham Role of glucose in cloned mouse embryo development Am J Physiol Endocrinol Metab, October 1, 2008; 295(4): E798 - E809. [Abstract] [Full Text] [PDF]
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 N. R Mtango and K. E Latham Differential Expression of Cell Cycle Genes in Rhesus Monkey Oocytes and Embryos of Different Developmental Potentials Biol Reprod, February 1, 2008; 78(2): 254 - 266. [Abstract] [Full Text] [PDF]
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F. Wang, Z. Kou, Y. Zhang, and S. Gao Dynamic Reprogramming of Histone Acetylation and Methylation in the First Cell Cycle of Cloned Mouse Embryos Biol Reprod, December 1, 2007; 77(6): 1007 - 1016. [Abstract] [Full Text] [PDF]
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A. Rybouchkin, Y. Kato, and Y. Tsunoda Role of Histone Acetylation in Reprogramming of Somatic Nuclei Following Nuclear Transfer Biol Reprod, June 1, 2006; 74(6): 1083 - 1089. [Abstract] [Full Text] [PDF]
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K. Takeda, M. Tasai, M. Iwamoto, A. Onishi, T. Tagami, K. Nirasawa, H. Hanada, and C. A. Pinkert Microinjection of Cytoplasm or Mitochondria Derived from Somatic Cells Affects Parthenogenetic Development of Murine Oocytes Biol Reprod, June 1, 2005; 72(6): 1397 - 1404. [Abstract] [Full Text] [PDF]
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S. Gao, G. Wu, Z. Han, E. de la Casa-Esperon, C. Sapienza, and K. E. Latham Recapitulation of the Ovum Mutant (Om) Phenotype and Loss of Om Locus Polarity in Cloned Mouse Embryos Biol Reprod, February 1, 2005; 72(2): 487 - 491. [Abstract] [Full Text] [PDF]
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S. Gao, E. Czirr, Y. G. Chung, Z. Han, and K. E. Latham Genetic Variation in Oocyte Phenotype Revealed Through Parthenogenesis and Cloning: Correlation with Differences in Pronuclear Epigenetic Modification Biol Reprod, April 1, 2004; 70(4): 1162 - 1170. [Abstract] [Full Text] [PDF]
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Y.-H. Choi, L. B. Love, M. E. Westhusin, and K. Hinrichs Activation of Equine Nuclear Transfer Oocytes: Methods and Timing of Treatment in Relation to Nuclear Remodeling Biol Reprod, January 1, 2004; 70(1): 46 - 53. [Abstract] [Full Text] [PDF]
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M. R.W. Mann, Y. G. Chung, L. D. Nolen, R. I. Verona, K. E. Latham, and M. S. Bartolomei Disruption of Imprinted Gene Methylation and Expression in Cloned Preimplantation Stage Mouse Embryos Biol Reprod, September 1, 2003; 69(3): 902 - 914. [Abstract] [Full Text] [PDF]
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S. Gao, Y. G. Chung, J. W. Williams, J. Riley, K. Moley, and K. E. Latham Somatic Cell-Like Features of Cloned Mouse Embryos Prepared with Cultured Myoblast Nuclei Biol Reprod, July 1, 2003; 69(1): 48 - 56. [Abstract] [Full Text] [PDF]
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