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a Department of Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Efficiency of the NT procedure, however, remains low despite numerous improvements made during the past decade, and the percentage of live offspring obtained after transfer of embryos created by NT, regardless of the species, usually does not exceed 3% [16, 18]. A better understanding of cell cycle compatibilities between the recipient cytoplasm and donor cell [19–25], as well as the development of new techniques for the introduction of nuclei and activation of newly created zygotes [18], holds promise for future improvements. One of the many problems that accompany the success of the NT outcome is the availability of species-specific competent recipient cytoplasm. Incomplete understanding of oocyte maturation, limited availability, and high cost of recipient cytoplasts (e.g., monkeys) could be overcome if a common cytoplast donor could be used successfully. Development of such a common model for investigation of interactions between recipient cytoplasm and the introduced diploid nucleus during an NT procedure would greatly benefit ongoing research efforts. In the present paper we examine the ability of bovine oocyte cytoplasm to support proliferation of somatic cell nuclei from several mammalian species. We ask questions about whether chromosome number, donor nucleus species, and age of a differentiated nucleus influence the ability of bovine oocyte cytoplasm to follow the directions of the introduced nucleus. We examine fusion efficiency, rate of the first embryonic cleavage, rate of blastocyst development, and pregnancy initiation of embryos derived by combining rat, pig, monkey, sheep, and bovine fibroblasts with bovine recipient cytoplasm.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recipient bovine oocytes were matured according to procedures previously shown to produce developmentally competent oocytes [26, 27]. Cow oocytes were obtained by aspiration of small antral follicles. Immature cumulus-oocyte complexes were cultured in Tissue Culture Medium 199 supplemented with 10% fetal calf serum (FCS), 0.2 mM pyruvate, 25 µg/ml gentamicin, 0.5 µg/ml LH (NIH, Bethesda, MD), and 1 µg/ml estradiol-17ß for 16 h at 39°C with 5% CO2 in air. Sixteen hours after the start of maturation, cumulus cells were removed by manual pipetting in the presence of 2.5 mg/ml hyaluronidase, and oocytes with extruded first polar body (metaphase II arrest, MII) were selected for enucleation. Early-maturing oocytes that extruded their first polar body by 16 h after the initiation of culture were selected as recipients. The oocytes were labeled with 0.5 µg/ml DNA fluorochrome (Hoechst 33342) for 15 min at room temperature in TALP-Hepes medium (Hepes buffered-Tyrode's containing lactate, 0.2 mM pyruvate, and 3 mg/ml BSA), washed, and placed in a manipulation drop of TALP-Hepes supplemented with 7.5 µg/ml cytochalasin B. All manipulations were performed on a Nikon Diaphot (Garden City, NY) microscope equipped with Hoffman (Greenvale, NY) optics and Narishige (Tokyo, Japan) micromanipulators. The first polar body and MII plate were removed by aspiration with a 25-µm inner diameter enucleation pipette. To ensure that oocyte chromatin was removed, the aspirated cytoplasm was exposed to UV light and examined for the presence of the removed polar body and metaphase plate (Fig. 1A). All chemicals were purchased from Sigma Chemical Company (St. Louis, MO) unless noted otherwise.
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Culture of Donor Cells
Ear skin samples were obtained by biopsy from an adult cow (7 yr), a pig (4 wk), a ram (5 yr), a cynomolgus monkey (9 yr), and a rat (4 wk), and all the samples were processed identically. Tissue was manually cut into small pieces and enzymatically digested with 0.5% trypsin-EDTA in PBS for 30 min at 30°C with occasional stirring. Digested tissue was washed in PBS, and the procedure was repeated several times. Disaggregated cells were separated from larger pieces of tissue by centrifugation at 100 x g for 5 min. Supernatants were washed several times in Ca2+-, Mg2+-free PBS, and the final supernatant was centrifuged at 700 x g for 10 min to obtain a cell pellet. The pellet was diluted with 
-MEM (Minimum Essential Medium), supplemented with 10% FCS and placed in culture at 37°C with 5% CO2 in air. After 10–14 days in culture, confluent fibroblast monolayers were obtained (Fig. 1B). Cells were passaged approximately once a week. Samples from progressively growing cell lines that were established were frozen for future studies. Three to ten days prior to NT procedure, fibroblasts were placed into serum-free -MEM in order to exit the cell cycle and accumulate in the G0/G1 phase. The cells were detached from the dishes by brief exposure to 0.025% trypsin-EDTA, washed in PBS, and placed into a manipulation drop.
NT, Fusion, and Activation
Fibroblasts that had been cultured without serum for at least 3 days [16] were used as donor cells. Somatic cells were introduced into young bovine MII cytoplasts immediately after removal of bovine nuclear DNA. Manipulation was done in 150-µl drops of TALP-Hepes supplemented with 7.5 µg/ml cytochalasin B. A single fibroblast was placed into the perivitelline space of an enucleated oocyte. NT was completed by 30 min after enucleation. The couplets were placed into TALP-Hepes supplemented with 3 mg/ml fatty acid-free BSA and kept on a warm plate prior to fusion. Recipient cytoplasts and donor nuclei were fused by a double electric pulse within 30 min of NT. Prior to fusion, NT units were placed into fusion medium (0.25 M sorbitol, 100 µM calcium-acetate, and 100 µM magnesium-acetate) for 10 min. They were then transferred into a fusion chamber consisting of two wires, 0.5 mm apart, and overlaid with the fusion medium. Two electric pulses (1.8–2.0 KV/cm, 30 µsec each) were applied. A 15-min recovery in TALP-Hepes containing 20% FCS followed fusion to allow membranes to return to their normal appearance. NT units were activated between 24 and 28 h after the start of maturation (6–10 h post-NT) using a procedure described by Susko-Parrish et al. [28]. The NT units were washed through Ca2+- and Mg2+-free TALP-Hepes and then exposed to 5 µM ionomycin for 4 min, washed in 30 mg/ml BSA in TALP-Hepes, and incubated in 1.9 mM 6-dimethylaminopurine-6-DMAP [28] in CR1aa [29] for 4 h at 39°C and 5% CO2 in air.
Embryo Culture, Transfer, and Pregnancy Monitoring
After activation, the NT units were washed again and placed into CR1aa for embryo culture. CR1aa was used for culturing of embryos produced from all five species [29]. The embryos were stained with Hoechst 33342 (5 µg/ml) to examine fusion efficiency. Some of the zygotes containing DNA were fixed with acid-ethanol (acetic acid:ethanol 1:3) and stained with orcein (1% orcein in 45% acetic acid) to assess chromatin morphology by phase-contrast microscopy. Samples of NT embryos were examined 1 h after fusion and 1, 7, 24, and 36 h after activation. The remaining NT embryos were transferred into 50-µl drops of CR1aa medium containing 10% FCS after 3 days. Embryos were examined 24 h after activation for initial cleavage and monitored every 24 h for progression of development through Day 9. At various times throughout embryo culture, a few embryos were selected and stained with Hoechst 33342 to confirm the presence of embryonic nuclei in individual blastomeres. Cow-cow NT blastocysts (a single blastocyst per animal) were transferred nonsurgically into recipients on Day 7 postestrus. NT embryos produced from pig and sheep donor fibroblasts were transferred at the 8- to 16-cell stage into the uterine horns by laparoscopy. Two embryos were transferred into each uterine horn of surrogate sheep on Day 4 after estrus, and 5–6 embryos into each uterine horn of surrogate sows on Day 3 after estrus. Embryos produced from rat fibroblasts were transferred at the 2- to 4-cell stage into rat oviducts. Pregnancies were determined by ultrasound on Day 35 in cows and Day 25 in ewes and sows. Surrogate rats were transabdominally palpated on Day 14 after embryo transfer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryos created from all donor fibroblast species were examined during the first 36 h after fusion. Twenty-three NT embryos were fixed in acetic acid:ethanol (1:3) and stained with aceto-orcein. Successfully fused NT units contained the somatic cell nucleus at the periphery of the oocyte cytoplasm (Fig. 1C), and the nucleus did not change in size during exposure to MII cytoplasm. The nuclear envelope remained intact, and no premature chromosome condensation was observed. Enlargement of the nucleus was apparent by the time the activation protocol was complete (Fig. 1D) and persisted throughout zygotic interphase (12 h postactivation) in intra- as well as in interspecies NT units. Decondensed chromatin was contained within a nuclear envelope and no chromatin fragmentation was observed at any time point examined (Fig. 1E). First cleavage occurred between 16 and 24 h after activation regardless of the donor nucleus species (Fig. 1F), and the second mitosis could be observed 36 h after activation (Fig. 1G).
Timing of Embryonic Cleavage Divisions and Developmental Potential of Interspecies Embryos
The biggest challenge in producing NT units using fibroblasts proved to be fusion by electroporation, the current method of choice in NT. The small size of fibroblasts used as nuclear donors (12–20 µm) provided less extensive contact area between the oocyte cytoplasm and the donor plasma membrane (in comparison to embryonic blastomeres), making fusion rates low. The removal of a portion of the oocyte cytoplasm during enucleation increased the size of the perivitelline space—size that could not be compensated for by a small somatic cell. Even though the donor cell was wedged between the zona and the oocyte plasma membrane after insertion, it was often displaced by the time the units were being fused. Absence of a close contact between the membranes made fusion unsuccessful in numerous attempts to create NT units (Table 1). However, from fused units, proportions of those that completed the first cell cycle were 55.8%, 66%, 52.2%, 85.7%, and 90.2% for cow, sheep, pig, monkey, and rat NT embryos, respectively. Timing of the first two cleavage divisions corresponded more closely to the timing of cleavage observed in bovine in vitro-produced embryos regardless of the donor nucleus species. However, with every progressive cleavage division the embryos started approaching the donor species-specific timing of development (Fig. 2). Embryonic nuclei were visualized by Hoechst 33342 fluorochrome staining under UV illumination [30, 31] to exclude the possibility of cytoplasm fragmentation in the absence of DNA (Fig. 1, H and I). We further observed that a number of embryos failed to undergo compaction. In these noncompacting embryos, numerous well-defined small blastomeres occupied the center of the intrazonal space, leaving a substantial gap between the embryo and the zona pellucida (Fig. 1J). No flattening of the outer cells was observed, and cells remained distributed evenly throughout the embryo. The embryos failed to undergo blastocoele formation after prolonged culture even though they remained viable and the cell number was increasing. The embryos that underwent compaction, however, started forming a blastocoele cavity (Fig. 1K). Proportions of interspecies embryos that developed to the blastocyst stage were not significantly different from those for intraspecies NT embryos (Table 1). With progression of embryo culture, expansion of the blastocoele cavity, as well as flattening of outer cells on one pole and concentration of inner cells at the other pole, was observed in these embryos (Fig. 1K). The time of the onset of blastocoele cavity formation corresponded more closely with the time of this event in the species of the donor nucleus. Sheep and pig blastocysts were observed as early as Day 5, and bovine and monkey blastocysts not before Day 6 and 8 after NT, respectively (Fig. 2). All of the early embryos (2- to 4-cell stage) produced from rat fibroblasts were transferred into uterine horns of surrogate rats, and therefore further developmental results were not available.
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Embryo Transfer and Detection of Pregnancy
Pregnancy results are presented in Table 2. None of the embryos transferred to surrogates progressed to a developmental stage when heartbeats should have been detected by transabdominal (Day 25 after transfer in sheep and pigs) or transvaginal (Day 35 after transfer in cows) ultrasonography. No fetuses were detected by palpation on Day 14 after transfer in rats. Ten surrogate cows displayed extended estrous cycles (35–48 days after embryo transfer). Two surrogate sheep displayed sacks of fluid within uterine horns and were hysterectomized shortly after ultrasonography. Both uterine horns in both animals had distinctly developed caruncles; however, no fetal membranes or fetal remnants were found.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fusion rates were lower than those observed after intraspecies fusion, due at least in part to the smaller size of fibroblasts of various species. Efficient fusion depends on not only healthy cell membranes but also on the extent of the contact between the donor cell plasma membrane and the oocyte plasma membrane. In addition, cell membrane properties and their compatibility with bovine oocyte plasma membrane may be determining the optimal time and field strength of electrical pulsing. Nevertheless, fused NT units initiated cleavage divisions and progressed to the 16-cell stage at high rates. The earliest stages of embryogenesis in normal embryos are regulated by maternally inherited gene products stored within the oocyte cytoplasm. Progression of development becomes dependent on embryonic gene activation at a species-specific developmental stage (reviewed in [40]). This occurs at the 2- to 4-cell stage in rats [41], late 4-cell stage in cattle [42] and pig [43], and 8-cell stage in sheep [44]. To our knowledge the timing of the maternal-to-embryonic transition in nonhuman primate embryos has not been reported; in human embryos it occurs at the 4- to 8-cell stage [45]. One of the morphological features that characterize the time of the transition is a developmental block in nonpermissive in vitro culture conditions [44]. In our study, high proportions of NT units progressed beyond the transcription-requiring, species-specific developmental stage. We can propose at least two scenarios explaining our results. Continuation of development could be a consequence of efficient reprogramming of the donor nucleus, regardless of the species, followed by now embryonic gene expression. Alternatively, it could indicate incompatibilities between the new components synthesized by the donor nucleus and the components left over from the recipient cytoplasm. In this case, the introduced fibroblast nucleus would be directing cell proliferation, and the resulting multicellular structure would have few or no embryonic characteristics. Our observations of embryo-like structures containing a high number of cells that would not undergo compaction support the latter scenario. Expression of specific genes [46, 47] has been shown to be required for compaction and cavitation in developing embryos [48]. Absence of compaction in some embryos in this study would suggest lack of or impairment in transcription of genes required for these differentiation events to occur [48].
The embryos that underwent compaction started forming a blastocoele cavity. Since bovine oocyte DNA had been removed and its removal confirmed by UV illumination, it is reasonable to assume that development in interspecies embryos was directed by the introduced fibroblast nucleus. As expected, disparity in the number of chromosomes between the species used for NT (60 in cattle, 54 in sheep, 38 in pigs, 42 in monkeys, 42 in rats) does not seem to be limiting for this developmental success. Even though embryonic development seemed "normal," possible ploidy problems have to be considered [25, 36]. High proportions of cleaving embryos, regardless of the donor nucleus species, developed to advanced stages in CR1aa medium, the medium designed for bovine embryo culture [29]. It is well established that embryos from different mammalian species require species-specific embryo culture conditions (reviewed in [49]). The ability of CR1aa to support embryonic development of interspecies units may be attributable to the fact that this development is driven by bovine cytoplasm alone. Since bovine oocyte cytoplasm supports the first three embryonic cell cycles in the absence of embryonic transcription, this seems like a reasonable possibility. Alternatively, CR1aa may be supporting embryo development of embryos of other species as successfully as it supports bovine embryos.
Timing of the first two cleavage divisions was not different in NT embryos produced regardless of the donor nucleus species. These results are not surprising, since the first cleavages occur in several mammalian species embryos at very similar times (reviewed in [50]). As development progresses, the length of embryonic cell cycles begins to vary between species. There is a considerable species variation in the length of preimplantation period as well as time of blastocyst formation [50], but the overall structural and morphological characteristics of the developing embryos and ultimately blastocysts are similar in most mammals. Cow-cow NT units that successfully fused and activated started cleaving and developed to specific embryonic stages in accordance with the timing of these events observed in in vitro-fertilized bovine embryos. Timing of cleavage divisions in the sheep-cow embryos was accelerated as compared to that in bovine-bovine counterparts. Sheep-cow embryos started forming blastocyst-like structures on Day 5 after activation, similar to in vitro fertilization-produced sheep embryos [51]. Similarly, timing for NT embryos produced from nuclei of other species resembled timing of development in vitro similar to that for in vitro fertilization-produced embryos: units produced from pig fibroblasts started forming blastocoele cavity 5 days after activation [52], and those produced from monkey fibroblasts 8 days after activation [53]. This would argue for an active role of the donor nucleus during development that was donor species specific.
At present we do not have sufficient data to attribute our developmental observations to the nuclear reprogramming of the fibroblast nucleus. Despite a number of studies that have addressed reprogramming after NT [5, 6, 20, 23, 54–57], molecular descriptors of successful reprogramming have yet to be described. The only criterion that when satisfied proves dedifferentiation, is a pregnancy carried to term. Our results show that the cytoplasm of a mature cow oocyte has the ability to support several mitotic cell cycles directed by newly introduced nuclear DNA. Whether this introduced differentiated DNA is reprogrammed, is modified, or simply remains unchanged is currently under investigation.
Before the usefulness of interspecies NT can be judged, the extent and faithfulness of nuclear reprogramming, as well as compatibilities between the somatic cell's and the recipient's cytoplasmic components, such as mitochondrial DNA, have to be examined. Nevertheless, the genome of a differentiated somatic cell from a variety of mammalian species can sustain development and form a blastocyst-like structure with distinct blastocyst morphology (inner cell mass, trophectoderm, and blastocoele cavity) when placed into bovine oocyte cytoplasm. With respect to this ability, bovine oocyte competency does not discriminate among the species investigated, chromosome number, or the age of the animals donating somatic nuclei. If safety and efficiency are proven, a common mammalian oocyte cytoplasm may be a suitable host for dedifferentiation of somatic nuclei of various mammals, including humans. Much more work is required to evaluate long- and short-term effects of mixing of nuclear and cytoplasmic components of various species. With increasing success of embryonic stem cell technology [58, 59], the embryonic cell lines grown from these interspecies embryos could be used to address some of the concerns.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Tanja Dominko, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97007. FAX: 503 614 3725; dominkot{at}ohsu.edu
Accepted: January 26, 1999.
Received: November 23, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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