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Regular Article |
a Oregon Regional Primate Research Center, Beaverton, Oregon 97006
b Departments of Obstetrics and Gynecology and of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, developmental biology, early development, embryo, fertilization, implantation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The success of NT depends on several parameters that impact the ability of the cytoplast to reprogram the nucleus of the donor cell or to reverse the epigenetic changes that occur during development. Synchronization of the cell cycle stages of the donor nucleus and recipient cytoplast also appears to be critical when differentiated cells are employed as the source of donor nuclei for somatic cell cloning. During initial experimentation with sheep, donor cells were subjected to serum starvation in an effort to force them out of an active cell cycle and into a quiescent stage, called G0 [6, 7]. The chromatin of quiescent cells was thought to be more amendable to the structural changes required for reprogramming due to dramatically reduced transcriptional activity [13]. However, somatic cell cloning has also been achieved with donor cells in G1 and G2/M [8, 14, 15].
Freshly isolated [9] or cultured embryonic, fetal, and adult cells [6–8] as well as established embryonic stem (ES) cells [14] have been used successfully as donor nuclei for NT; however, it has not yet been determined which donor cell types are the most efficient for somatic cell NT. Epithelial cells from mammary gland [7] and oviduct [16] as well as cumulus or mural granulosa cells [9, 17] have been employed along with fetal, newborn, and adult fibroblasts isolated from various tissues [7, 8, 11, 15, 18, 19]. The failure to generate viable clones from Sertoli and neuronal cells in the mouse may reflect limitations in the NT technique and/or irreversible differentiation of these cell types.
Despite the successful production of rhesus monkeys by NT from embryonic blastomeres [20], somatic cell cloning has not yet been accomplished in primates. In this study, we describe the production of monkey NT embryos derived from both embryonic blastomeres and fetal fibroblasts. We demonstrate in vitro and in vivo development of embryonic cell NT embryos along with limitations in the development of monkey somatic cell NT embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature rhesus macaque males and females housed in individual cages were used in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee at the Oregon Regional Primate Research Center/Oregon Health & Science University.
Establishment and Culture of Donor Fetal Fibroblasts
Skin biopsy specimens derived from a 130-day-old rhesus macaque fetus were washed in 0.5 mM EDTA (Invitrogen, Carlsbad, CA) in Ca2+- and Mg2+-free Dulbecco PBS (PBS; Invitrogen) and minced into pieces before incubation in Dulbecco modified Eagle medium (DMEM; Invitrogen) containing 1 mg/ml of collagenase IV (Invitrogen) at 37°C in 5% (w/v) CO2 for 20 min. Tissue pieces were then vortexed, washed, and seeded into 75-cm3 cell culture flasks (Corning, Acton, MA) containing DMEM supplemented with 100 IU/ml of penicillin, 100 µg/ml of streptomycin (Invitrogen), and 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT) and cultured at 37°C in 5% CO2. After reaching 100% confluency, monolayers of cells with fibroblast-like morphology were disaggregated using PBS containing 0.15% (w/v) trypsin and 1.8 mM EDTA and passaged two more times before being frozen in DMEM with 25% FBS and 10% (w/v) dimethyl sulfoxide and stored in liquid nitrogen. Unless indicated otherwise, all reagents were from Sigma-Aldrich Co. (St. Louis, MO), and all hormones were from Ares Advanced Technologies, Inc. (Norwell, MA).
Donor Cell Treatments and Flow Cytometric Analysis of the Cell Cycle
The cell cycle comparisons of primary fetal fibroblasts were made between cycling, serum-starved cells and cells that were cultured to confluency. Cell culture flasks (volume, 75 cm3) were plated with frozen/thawed fetal fibroblasts at a concentration of 1 x 106 cells/flask. After reaching 70%–80% confluency, cycling cells were fixed in ethanol as described below. These cells were used as controls for comparison purposes. Other cells were grown to 100% confluency and then allocated to one of the following treatments before being fixed: replacement of growth medium with DMEM + 0.5% FBS and culture for an additional 2 or 5 days (serum starvation), or changing of regular growth medium every 2–3 days for an additional 2 or 5 days of culture (contact inhibition). For fixation, cells from each treatment were disaggregated as described above, pelleted by centrifugation (5 min at 130 x g), resuspended in 0.5 ml of PBS, and slowly mixed with 4.5 ml of cold, 70% (v/v) ethanol. After at least 12 h of ethanol fixation at 4°C, cells were pelleted, washed twice with PBS, and stained in PBS containing 0.1% (v/v) Triton X-100, 0.2 mg/ml of RNase A, and 20 µg/ml of propidium iodide for 15 min at 37°C. Stained cells were then filtered through a 30-µm nylon mesh (Spectrum, Los Angeles, CA) and analyzed with a fluorescence-activated cell sorting (FACS) Calibur flow analyzer (Becton-Dickinson, Rutherford, NJ). The cells were first analyzed in a width-versus-FL1 area scattergram. A region was then drawn on the scattergram to gate out aggregates, and the gated populations were displayed on a FL1-area histogram. Percentages of cells existing within the G0/G1, S, and G2/M phases of the cell cycle were calculated using ModFit LT software (Verity Software House, Inc., Topsham, ME).
Ovarian Stimulation, Recovery of Rhesus Macaque Oocytes, Fertilization by Intracytoplasmic Sperm Injection, and Embryo Culture
Controlled ovarian stimulation and oocyte recovery have been described previously [21]. Briefly, cycling females were subjected to follicular stimulation using twice-daily i.m. injections of recombinant human FSH as well as concurrent treatment with Antide, a GnRH antagonist, for 9 days. Females received recombinant human LH on Days 7–9 and recombinant hCG on Day 10.
Cumulus-oocyte complexes were collected from anesthetized animals by laparoscopic follicular aspiration (28–29 h after hCG administration) and placed in Hepes-buffered TALP (modified Tyrode solution with albumin, lactate, and pyruvate) medium [22] containing 0.3% BSA (TH3) at 37°C. Oocytes stripped of cumulus cells by mechanical pipetting after brief exposure (<1 min) to hyaluronidase (0.5 mg/ml) were placed in CMRL (Connaught Medical Research Laboratories) medium (Invitrogen) containing 10% FBS, 10 mM L-glutamine (Invitrogen), 5 mM sodium pyruvate, 1 mM sodium lactate, 100 µg/ml of penicillin, and 100 µg/ml of streptomycin at 37°C in 5% CO2 until further use.
Fertilization by intracytoplasmic sperm injection (ICSI) and embryo culture were performed as described previously [23]. Briefly, sperm were diluted with 10% polyvinyl pyrrolidone (1:4 ;obv/v;cb; Irvine Scientific, Santa Ana, CA), and a 5-µl drop was placed in a micromanipulation chamber. A 30-µl drop of TH3 was placed in the same micromanipulation chamber next to the sperm droplet and covered with paraffin oil (Zander IVF, Vero Beach, FL). The micromanipulation chamber was mounted on an inverted microscope equipped with DIC or Hoffman optics (Olympus, Melville, NY) and micromanipulators. An individual sperm was immobilized, aspirated into an ICSI pipette (Humagen, Charlottesville, VA), and injected into the cytoplasm, away from the polar body, of a MII oocyte. After ICSI, injected oocytes were placed in four-well dishes (Nalge Nunc International Co., Naperville, IL) and cocultured at 37°C in 5% CO2 on buffalo rat liver (BRL) cell monolayers in CMRL medium. Cultures were maintained under paraffin oil. Pronuclear formation was recorded 16–20 h post-ICSI, and progression of embryo growth was recorded daily. Development to 8-cell, morula, compact morula, and blastocyst stages was determined and expressed relative to the number of nucleated 2-cell stage embryos. Pronuclear formation and initial cleavage rates were similar within each treatment; therefore, percentages of pronuclear stage and cleaved (i.e., 2-cell stage) embryos were pooled. Embryos were transferred to fresh plates of BRL cells every other day for a maximum of 8 days.
NT Procedures
Activation and NT manipulations were performed as described before [23]. Briefly, recipient MII oocytes were incubated for 5 min with 5 µg/ml of Hoechst 33342, transferred to 30 µl of TH3 containing 3.5 µg/ml of cytochalasin D, and incubated for 10–15 min before enucleation. The first polar body and approximately 10% of the underlying cytoplast were drawn into an enucleation pipette (outer diameter, 25–28 µm), with subsequent confirmation of removal of the metaphase spindle by epifluorescent microscopy. The time of exposure to ultraviolet light was restricted to less than 10 sec.
Cytoplasts were activated either 4 h before fusion (preactivation), immediately after fusion (instant activation), or 3 h after fusion (postactivation). The activation stimulus consisted of an exposure to 5 µM ionomycin (Calbiochem, La Jolla, CA) for 2 min, followed by (unless indicated otherwise) 4 h of incubation in either 2 mM dimethylaminopurine (I/DMAP) or 50 µM roscovitine (Calbiochem) combined with 5 µg/ml of cytochalasin B (I/roscovitine).
Donor fetal fibroblasts were synchronized at the G0/G1 phase of the cell cycle by 5-day contact inhibition or serum-starvation treatments. Blastomeres of Day 3 (Day 0 = day of fertilization) ICSI-produced embryos were also used as the source of donor nuclei. A disaggregated donor fibroblast or blastomere was aspirated into a pipette and transferred into the perivitelline space of the cytoplast. Fusion of NT pairs was induced by two 50-µsec DC pulses of 2.7 kV/cm (Electro Square Porator T-820; BTX, Inc., San Diego, CA) in 0.25 M D-sorbitol buffer containing 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.5 mM Hepes, and 1 mg/ml of fatty acid-free BSA. Fusion was evaluated visually 45–60 min after electroporation by confirming the presence or absence of donor cells in the perivitelline space. Fused and activated NT embryos were placed in CMRL medium and cocultured with BRL cells as described above. Pronuclear formation was monitored and recorded 12 h postactivation, and progression of embryo growth was recorded daily, as outlined for the ICSI controls, for up to 8 days.
Embryo Transfer
Adult, multiparous females were used as recipients and monitored for menses. Daily blood samples were collected beginning on Day 8 of the menstrual cycle, and serum levels of estradiol were quantitated by RIA. The day following the peak in serum estradiol was considered to be the day of ovulation (Day 0). Within 0–5 days of ovulation, recipient females were anesthetized with ketamine (10 mg/kg body weight i.m.; Fort Dodge Laboratories, Fort Dodge, IA) and prepared for embryo transfer. Typically, three to five NT embryos were transferred at minilaparotomy to the oviducts of recipients [24]. Embryos were transferred to TH3 medium and aspirated into a prerinsed, silicon-tubing catheter controlled by a 1-cc syringe. The tip of the catheter was inserted 2- to 3-cm deep into the oviduct, and the embryos were expelled. To detect pregnancy, serum levels of estrogen and progesterone were monitored every third day after embryo transfer. Pregnancy was confirmed by ultrasound approximately 25 days posttransfer and monitored periodically throughout gestation.
Statistical Analysis
Results, expressed as means ± SEM, were analyzed using one-way ANOVA and the Fisher protected least significant difference test within Statview software (SAS Institute Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With the objective of conducting NT with donor cells in the G0/G1 stage of the cell cycle, FACS was used to separate and to quantitate the number of fetal fibroblasts in G0/G1 (2C DNA content), S (between 2C and 4C), and G2/M (4C) phases based on cellular DNA content (Table 1). The majority of cycling fetal fibroblasts (at 70%–80% confluency) were detected in G0 or G1. This portion of the population was significantly increased after serum starvation (2 or 5 days of starvation) or with cells cultured to confluency (contact inhibition for 5 days). Conversely, cycling cultures contained a significantly higher percentage of cells in the S and G2/M phases compared to those cultures subjected to starvation or inhibition for 5 days. However, this distribution of cycling cells in G0/G1, S, and G2/M did not change significantly when cells were sampled 2 days after reaching 100% confluency (contact inhibition, 2 days). Based on these results and the absence of a marked difference between donor cells synchronized at G0/G1 phase of the cell cycle by contact inhibition or starvation for 5 days, the two approaches were considered to be interchangeable for use in subsequent NT experimentation.
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Experiment II: Timing of Cytoplast Activation
Oocyte quality is an important variable in any experiment involving gametes, and it seems to be donor or even cycle dependent [25]. Thus, assessment of oocyte quality is appropriate, and we have used the ability of ICSI-fertilized embryos to develop in vitro to the blastocyst stage as a relevant measure. In the present study, despite high fertilization and initial cleavage rates, development of ICSI-fertilized embryos to blastocysts varied dramatically (0%–80%) between individual oocyte cohorts. Of 33 NT experiments, nine were discarded, because none of the control, sperm-fertilized embryos developed to the blastocyst stage.
Initially, a series of experiments was conducted regarding the timing of cytoplast activation relative to NT (Table 2). To this end, the efficiency of activation, pronuclear formation, and in vitro development was determined. Fetal fibroblasts as nuclear donor cells were synchronized at the G0/G1 stage of the cell cycle by contact inhibition (confluent culture maintained for 5 days). Cytoplast activation induced by I/DMAP was applied either 4 h before donor cell-cytoplast fusion (preactivation, n = 47), immediately after fusion (instant activation, n = 68), or 3 h after fusion (postactivation, n = 165). Similar fusion rates were observed with all treatments (87 ± 12%, 83 ± 2%, and 71 ± 7% for preactivation, instant activation, and postactivation treatments, respectively; P > 0.05). Immediately after fusion, the disappearance of donor nuclei was noted in all groups, indicative of nuclear envelope breakdown (NEBD). However, NEBD was unexpected in the preactivation group, because the low levels of maturation-promoting factor (MPF) presumed under these conditions should preclude NEBD. In subsequent experiments, the duration of DMAP treatment in the preactivation group was extended for up to 6 h to prevent the possible reactivation of MPF. In these cases, after initial treatment with ionomycin followed by 4 h of incubation in DMAP, cytoplasts were exposed to DMAP during the transfer of donor cells into the perivitelline space, fusion, and for up to 1 h after fusion. However, even this extended DMAP treatment did not preclude NEBD. The presence of intact nuclei after fusion was observed only when enucleated pronuclear-stage zygotes were used as the cytoplast source (results not shown). Hoechst staining of selected NT embryos immediately after fusion and NEBD indicated that chromosome condensation had occurred in all treatments (i.e., premature chromosome condensation [PCC]; results not shown).
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Pronuclear formation, cleavage, and development to the 8-cell stage for NT embryos produced by these activation protocols were comparable to those of the ISCI control (P > 0.05) (Table 2). The majority of NT embryos formed a single, large pronucleus containing multiple nucleoli (Fig. 1A). An increased rate of development to the 8-cell stage in all groups of NT embryos was noticed when compared to sperm-fertilized controls; NT embryos formed pronuclei and subsequently cleaved approximately 6 h earlier than controls. However, a high rate of developmental arrest beyond the 8-cell stage occurred in all groups of NT embryos.
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Experiment III: Cytoplast Activation
Electrofusion alone does not result in cytoplast activation in the monkey, necessitating a chemical exposure step [23]. Therefore, the in vitro developmental potential of somatic cell NT embryos activated with I/DMAP or I/roscovitine (roscovitine is a specific MPF inhibitor) combined with cytochalasin B was studied (Table 3). Unlike exposure to DMAP, exposure to roscovitine does not interfere with cytoskeletal reorganization and second polar body extrusion after parthenogenetic activation of monkey MII oocytes or NT embryos [23]. To avoid chromatin segregation into the second polar body and to maintain correct ploidy of the resulting NT embryos, roscovitine was combined with cytochalasin B treatment in this study. Also included in Table 3, for comparative purposes, are results of the in vitro development of parthenotes activated by I/DMAP or I/roscovitine [23]. In this chemical activation series, based on the result that some blastocyst formation was observed in the postactivation group (Table 2), all NT embryos were activated 3 h after fusion (postactivation). The pronuclear formation and cleavage rates were similar between the two NT groups (P > 0.05) (Table 3) and were comparable to those of their parthenogenetic counterparts. However, the majority of NT embryos activated with I/roscovitine, unlike those activated with I/DMAP, contained two or three pronuclei. Also, unlike parthenotes, a significant portion of embryos in both NT treatments again failed to progress beyond the 8-cell stage, and only 1% of I/DMAP-activated embryos reached the blastocyst stage. The ICSI-fertilized control embryos (results not reported) developed to the blastocyst stage at a rate similar to that reported in Table 2.
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Experiment IV: Effect of Donor Cell Treatments
To test the equivalency of fetal fibroblasts arrested in G0/G1 by contact inhibition (5 days) versus serum starvation (5 days), the in vitro developmental potential of NT embryos derived from each donor cell treatment was measured. The NT embryos were activated postfusion with I/DMAP. Despite similar fusion rates (71%), significantly lower pronuclear formation and initial cleavage rates were documented in the serum-starvation group (41 ± 14% and 87 ± 5% for starvation and inhibition group, respectively; P < 0.05). However, subsequent in vitro development of 2-cell stage embryos was not statistically different (72 ± 19% and 96 ± 2% reached the 8-cell stage in starvation and inhibition groups, respectively; P > 0.05), with most NT embryos in both groups arresting between the 8-cell and the morula stages. A few embryos derived from fibroblasts synchronized by contact inhibition developed to compact morulae and blastocysts.
Experiment V: Somatic and Embryonic Cell NT
To determine if developmental arrest of NT embryos observed in experiments II–IV was due to cytoplast/NT embryo manipulation, the in vitro development of NT embryos derived from embryonic blastomeres was included as a control. In this experimental series, the NT parameters that yielded the highest in vitro development in previous experiments (i.e., contact inhibition of donor cells and postactivation with I/DMAP) were used for somatic cell NT. In initial studies, in vitro developmental rates to the blastocyst stage for embryonic cell NT embryos were higher when unsynchronized blastomeres were fused with preactivation compared to instant activation or postactivation cytoplasts (results not shown). Therefore, the parameters for embryonic cell NT were cytoplast preactivation with I/DMAP exposure (6 h of exposure to DMAP) and fusion with unsynchronized blastomeres obtained from ICSI-produced, 8- to 16-cell stage embryos (Days 2–3). Fusion rates were similar between somatic and embryonic cell NT groups (P > 0.05) (Table 4), despite the higher rates expected for blastomeres based on their larger size. Additionally, NEBD was documented in both somatic and embryonic NT groups immediately after fusion. Whereas lower pronuclear formation and cleavage rates were observed in the embryonic cell NT group compared to the ICSI control (67 ± 5% and 86 ± 3%, respectively; P< 0.05) (Table 4), subsequent development of embryos to the 8-cell stage was similar for all groups (P > 0.05) (Table 4). However, significantly lower rates of development to morula, compact morula, and blastocyst stages were observed with somatic cell NT embryos, whereas embryonic cell NT embryos progressed to the blastocyst stage at rates comparable to those of ICSI controls (Table 4).
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To evaluate the in vivo developmental potential of embryonic and somatic cell NT embryos produced in these experiments, early cleavage stage (Days 2–3) NT embryos (Fig. 1B) were transferred into the oviducts of synchronized recipients during spontaneous menstrual cycles. Thirty embryonic cell NT and 14 somatic cell NT embryos were transferred into 11 and 3 recipients, respectively. Recipients were monitored for pregnancy by measuring circulating levels of estradiol and progesterone as well as by ultrasonography. One pregnancy resulting from an embryonic cell NT embryo (derived from a 16-cell stage blastomere) was detected and maintained to term (pregnancy rate, 9%; implantation rate, 3%). Fetal heart beat rates and body size measurements taken monthly for up to 130 days were within the range expected for normal rhesus monkey fetal development. However, a single stillborn female infant (body weight, 290 g) was recovered by caesarean section 155 days after embryo transfer. Necropsy showed no gross abnormalities, and fetal asphyxia due to multiple umbilical cord wraps around the body and limbs was considered to be the most likely cause of death.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental approaches to evaluate individual steps in the NT procedure were conducted; however, their optimization was not associated with a marked improvement in the developmental potential of NT embryos produced from somatic cells. Major variables between somatic and embryonic cell NT include donor cell type and reprogramming requirements as well as cell cycle stage and synchrony.
Nuclear exposure to the cytoplasm is critical to reprogramming, and the timing of these events can be varied. No significant differences in the development of sheep NT embryos was associated with timing of cytoplast activation [6], although improved developmental rates were observed when activation, in relation to fusion, was delayed in cattle and mice [9, 17, 26]. In the present series with preactivation, instant activation, and postactivation protocols, similar outcomes, including development rates to the 8-cell stage, were observed. However, the poor development beyond the 8-cell stage is suggestive of a reprogramming problem; indeed, the embryonic genome likely is not even activated or functional at this stage.
Morphological changes occur in the donor nucleus following transfer into an enucleated MII oocyte, including NEBD followed by PCC [27–29]. Both NEBD and PCC, which occur when the nucleus is introduced into the nonactivated cytoplast, do not take place when the cytoplast is preactivated [28, 30]. Additionally, both NEBD and PCC are associated with high MPF activity levels in the cytoplasm of matured MII oocytes, and cytoplast activation induces a decline in MPF activity to basal levels incapable of NEBD and PCC. In the present study, NEBD was noted immediately after fusion, regardless of the timing of activation. In fact, NEBD observed after NT into preactivated cytoplasts suggests that young monkey cytoplasts may still contain high residual MPF activity capable of dissolving the nuclear membrane, which is in agreement with the slow decline of MPF activity seen in bovine cytoplasts over a period of 9 h after activation [29]. Despite the presumptive high residual MPF activity, the I/DMAP activation treatment employed here induced pronuclear formation and cleavage of monkey NT embryos (Table 2) and parthenotes [23]. Moreover, direct exposure of the cytoplast to DMAP during fusion did not prevent NEBD. The absence of NEBD was observed only when enucleated, pronuclear-stage zygotes were used as cytoplasts (results not shown).
The cell cycle synchrony between donor nucleus and recipient cytoplast is important for normal ploidy and successful NT. A high percentage of cycling monkey fetal fibroblasts were detected at G0/G1 phase of the cell cycle, with small but significant increases in the percentages of G0/G1 cells associated with serum deprivation and growth to confluence. These results are in agreement with those of recent studies of porcine fetal fibroblasts, in which nearly 80% of cells were at G0/G1 phase after 24 h of starvation [31].
As noted above, the relatively high rate of development of embryonic cell NT embryos implies that developmental arrest of somatic NT monkey embryos in vitro is due to improper or incomplete reprogramming of donor nuclei. After fertilization, little or no transcriptional activity occurs in the embryonic nucleus, and development is primarily supported by maternally inherited proteins and mRNAs present in the cytoplasm of the unfertilized oocyte. The onset of embryonic transcription at the maternal-embryonic transition (MET) is species dependent, starting as early as the 1- to 2-cell stage in mice [32], the 8- to 16-cell stage in cattle [33], and the 4- to 8-cell stage in rhesus monkeys [34, 35]. In the absence of embryonic genome activation, the embryo arrests. Indeed, studies using embryonic blastomeres as nuclear donor cells correlate development of the NT embryo to the donor blastomere stage relative to the MET. The later the onset of the MET, the later the stage from which successful NT development could be obtained (for review, see [13]). Because embryonic cell NT embryos produced in this study successfully passed through the MET and developed to the blastocyst stage at rates comparable to control ICSI-fertilized embryos, reprogramming after NT may not have been required. Our current NT procedures apparently do not reprogram the differentiated, somatic nuclei of fetal fibroblasts; therefore, NT with donor cells expressing, for example, green fluorescence protein driven by exogenous or endogenous preimplantation, embryo-specific promoters could be useful in allowing assessment of embryonic genome activation [36].
Parenthetically, preactivation of the cytoplast was beneficial for the unsynchronized embryonic cell NT embryos in this study (Table 4), which is in agreement with previous results in monkeys [20], cattle [4, 26], and rabbits [37]. We show here a high blastocyst formation rate based on a significant number of NT embryos (n = 240) derived from 8- to 16-cell stage blastomeres. To our knowledge, this is the first report that monkey embryonic cell NT embryos develop to blastocysts in vitro at a rate similar to that of sperm-fertilized controls.
The in vivo developmental potential of rhesus monkey embryonic cell NT embryos reconstructed from 2- to 4-cell stage blastomeres has been demonstrated (pregnancy rate, 3%) and the birth of two infants reported [20]. The pregnancy established in this study occurred after the transfer of 30 NT embryos derived from 8- to 16-cell stage blastomeres into 11 recipients. This pregnancy rate of 9% is significantly lower than that observed following the oviductal transfer of in vitro fertilization-produced intact embryos (66%) in our laboratory [38].
Future research on somatic cell cloning in the monkey will address the effect of various cell types (e.g., younger fetal fibroblasts as well as cumulus, oviductal epithelial, and ES cells) on NT development in efforts to screen for the best source of donor nuclei. Reprogramming of the donor nucleus is obviously of importance, both as implied here and as shown by successful somatic cell cloning in several mammalian species; even highly differentiated donor cell nuclei can be reprogrammed in egg cytoplasm, acquiring the capacity to support full-term development [7, 9, 12, 16, 18]. However, only a few NT embryos develop to term, and of those, many die shortly after birth in all species in which cloning has been successful. The most likely explanation for the poor survival of NT offspring is the inability of the cytoplast to reprogram the epigenetic profile of the somatic donor nucleus to that of the fertilized zygote [39].
In summary, we demonstrated high rates of in vitro development to blastocysts by monkey embryonic cell NT embryos reconstructed from 8- to 16-cell stage blastomeres. Further, the in vivo developmental potential of these embryos was confirmed by embryo transfer and establishment of one term pregnancy. These NT procedures were not efficient in reprogramming somatic nuclei of fetal fibroblasts, however, because the developmental capacity of somatic cell NT embryos was restricted to the 8- to 16-cell stage.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Institutes of Health grants RR12804, RR00163, A135365, and A-T Children's Project to D.P.W. 
2 Correspondence: Don Wolf, Oregon Regional Primate Research Center, Oregon Health & Science University, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690 5384; wolfd{at}ohsu.edu
3 Current address: Department of Otolaryngology, Oregon Health & Science University, Portland, OR 97201
Accepted: November 30, 2001.
Received: October 12, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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