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Regular Article |
a Animal Developmental Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Daejeon 305-600, Korea
b National Livestock Research Institute, Chonan 330-800, Korea
c Department of Biotechnology, Taegu University, Kyungsan 712-714, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, implantation, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was performed to address the hypothesis that disproportionate allocations of ICM and TE lineages in embryos result in fetal losses during subsequent postimplantation development. In general, mean ratios of ICM:total cells for bovine blastocysts produced in vitro or in vivo range from 20% to 40% [13–15]. Until now, little information has been available concerning the structural composition of ICM and TE cells in NT bovine embryos, besides previous studies with in vitro fertilization (IVF) and in vivo embryos in the cow [14, 16, 17]. Thus, we examined number and relative proportions of the ICM and TE cells in NT embryos and compared these values with those in normally fertilized counterparts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise mentioned, all chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The bovine oocyte maturation medium consisted of TCM-199 with Eagle salts and L-glutamine supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL, Grand Island, NY), 1 µg/ml estradiol, 1 µg/ml FSH-P (Schering-Plough Animal Health Corp., Kenilworth, NJ), and 25 mM NaHCO3. IVF of bovine oocytes was performed as described by Han et al. [18]. The medium, Fert-TALP [19], consisted of bicarbonate buffered Tyrode solution supplemented with BSA (6 mg/ml), 25 µg/ml heparin, and 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine (PHE). The culture medium for embryo development was CR1aa [20] supplemented with 1 mM glutamine and 1x Eagle essential amino acids solution (Gibco BRL).
In Vitro Maturation and IVF of Bovine Oocytes
Immature bovine oocytes obtained from the ovaries of slaughtered Holstein cows were cultured in 0.5 ml of maturation medium containing 1 µg/ml estradiol and 1 µg/ml FSH-P for 22 h at 38.57°C in 5% CO2 in humidified air. After in vitro maturation, the oocytes were fertilized with frozen-thawed sperm at a concentration of 2 x 106 spermatazoa/ml in 50 µl of fertilization medium. When sperm were added to the fertilization drops, 25 µg/ml heparin and PHE were also added. Sperm and oocytes were coincubated at 38.5°C in 5% CO2 in air. After 20–22 h of insemination, cumulus-enclosed oocytes were stripped by gentle pipetting and then cultured in CR1aa supplemented with 3 mg/ml BSA (fatty acid free). After culture for 3 days, cleaved embryos were further cocultured in each well of a four-well culture plate containing 750 µl CR1aa (with 10% FBS) on a mouse embryonic fibroblast monolayer for 4 days at 38.5°C in 5% CO2 in air [21]. After 7 days of culture, blastocyst formation was observed (7 days from IVF).
Donor Cells
Experiments were conducted according to the Animal Care and Use Committee guidelines of the National Livestock Research Institute of Korea. Bovine fetal fibroblasts were derived from a 45-day fetus and prepared as described previously [22]. The head of the fetus was removed using iris scissors, and soft tissues such as liver and intestine were also discarded by scooping out with two watchmaker's forceps. After twice washing with PBS (Gibco BRL), the carcass was minced with a surgical blade on a 100-mm culture dish. These procedures were performed at room temperature. The minced tissues were incubated in 10 ml of 0.05% (w/v) trypsin/0.53 mM EDTA solution (Gibco BRL) in an incubator at 38.5°C for 30 min. Trypsin was inactivated by adding an equal volume of cell culture medium supplemented with 10% FBS. The cell culture medium was composed of Dulbecco modified Eagle medium (Gibco BRL), 10% FBS, 1000 units of penicillin (Gibco BRL), and 1000 µg/ml of streptomycin (Gibco BRL). After vigorous pipetting, the supernatant was centrifuged at 150 x g for 5 min. The cells were suspended, adjusted to a final concentration of 2 x 106 cells/ml, and then cultured in 10 ml of the culture medium at 38.5°C in 5% CO2 in air in 175-cm2 tissue culture flasks (Nunc, Roskilde, Denmark) until confluent. The fetal fibroblast cells were passaged three or four times before use as donor nuclei.
Somatic Cell NT
Mature oocytes were transferred to 500 µl of TL-Hepes [23] supplemented with 0.1% hyaluronidase and were freed of cumulus cells by mechanical pipetting. The zonae pellucida of oocytes were partially dissected using a fine glass needle [24]. Oocyte manipulations such as enucleation and cell injection were performed by using a micromanipulator equipped with an inverted microscope (Leitz, Ernst Leitz Wetzlar GmbH, Germany). The medium used for manipulation was TL-Hepes containing 7.5 µg/ml cytochalasin B. The first polar body and partial cytoplasm presumptively containing metaphase II chromosomes were removed together by using a micropipette with an inner diameter of 20 µm. Successful enucleation was confirmed by Hoechst 33342 staining and visualization under ultraviolet light. Single cells were individually transferred to the perivitelline space of the recipient cytoplast. The cell-cytoplast complexes were equilibrated in a 50-µl drop of cell fusion medium for 10–20 sec and then transferred to a fusion chamber with two electrodes 1 mm apart overlaid with cell fusion medium. The cell fusion medium consisted of 0.3 M mannitol, 0.5 mM Hepes, 0.01% BSA, 0.1 mM CaCl2, and 0.1 mM MgCl2. The cell-cytoplast complexes were induced to fuse with a single pulse of direct current of 1.6 kV/cm for 20 µsec each by an Electro Cell Manipulator 2001 (BTX, San Diego, CA). These procedures were performed at room temperature. Reconstructed embryos without visible somatic cells were determined as fused eggs 1 h after the fusion pulse. For activation of fused embryos, we used a modified method described by Cibelli et al. [4]. At 4 h after electrofusion, the fused eggs were activated with 5 µM ionomycin for 5 min, followed by treatment with 2.5 mM 6-dimethyl-aminopurine in CR1aa supplemented with 10% FBS for 3.5 h at 38.5°C in 5% CO2 in air. Thereafter, the embryos were cultured as described above.
In Vivo-Derived Embryos
Embryos were collected from superovulated cows (n = 5) on Day 7 after artificial insemination. The superovulation regime consisted of intramuscular injection of 400 mg NIH-FSH-P1 Follotropin-V (Vetrepharm Canada Inc., London, ON, Canada) administered on Day 11 of the estrous cycle followed by 25 mg injection (48 h apart) of prostaglandin F2
. Artificial insemination with frozen-thawed semen was performed twice at 12 h and 24 h after the onset of estrus. Morulae and blastocysts were nonsurgically flushed from the uterus of the cow using a Foley catheter (Agtech, Manhattan, KS).
Differential Staining
Differential staining of ICM and TE cells of blastocysts (Day 7) was performed [25]. The zona pellucida of each blastocyst was removed by incubating in 0.5% pronase solution for 1 min. After rinsing in TL-Hepes medium containing 1 mg/ml polyvinyl alcohol, denuded embryos were exposed to a 1:5 dilution of rabbit anti-pig whole serum for 1 h, rinsed three times for 5 min each in TL-Hepes, and placed into a 1:5 dilution of guinea pig complement containing 10 µg/ml propidium iodide and 10 µg/ml bisbenzimide (Hochest 33342) for 1 h. After briefly rinsing in TL-Hepes, the embryos were mounted on slides under coverslips and examined under ultraviolet light using an epifluorescent microscope (Olympus, Tokyo, Japan). Blue and red colors were designated as ICM and TE cells, respectively.
Statistical Analysis
Four or five replicates for each experiment were conducted. All percentage data were subjected to arcsine transformation. All percentage data and data sets obtained from this study were expressed as mean plusmn; SD. Differences in developmental rate and cell numbers of the embryos between experimental groups were analyzed by a Student t-test and a Duncan multiple range test using the general linear models procedure of the Statistical Analysis System (Cary, NC). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Developmental potential of NT embryos was compared with that of IVF embryos (Table 1). The rates of cleavage and development to the blastocyst stage of NT embryos (80.3% ± 18.6% and 27.3% ± 8.9%, respectively) were similar to those of IVF embryos (81.6% ± 5.6% and 28.2% ± 7.7%, respectively). Thus, no difference in the preimplantation development was detected between IVF and NT embryos.
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Allocations of ICM Versus TE Cells in NT Blastocysts
In the next experiment, blastocysts produced by NT, IVF, and in vivo systems were individually stained by a differential staining method and then evaluated for total cell number. The total cell number of NT embryos (98.0 ± 44.3, n = 55) was equivalent to that of IVF embryos (108.2 ± 33.3, n = 52) but was significantly lower than that of in vivo-derived embryos (122.5 ± 21.6, n = 26; P < 0.05). The NT blastocysts had a higher ICM:total cell ratio (50.1 ± 17.9) as compared with IVF (42.6 ± 14.9) and in vivo-derived (34.9 ± 8.9) embryos (P < 0.05; Table 2). Representative fluorescence micrographs of bovine blastocysts (Day 7) derived from an in vivo system, IVF, and NT after differential staining are shown in Figure 1, D, E, and F, respectively. Various allocation types of ICM and TE cells are shown in NT blastocysts (Fig. 2).
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Distributions of NT Embryos According to Ratio of ICM:Total Cells
The next experiment was conducted to examine how many NT embryos represented an abnormal ratio of ICM:total cells. In vivo-derived blastocysts differentially stained for ICM and TE nuclei were individually classified into four groups (group I: <20%; group II: 20–40%; group III: 40–60% group IV: >60%) according to the ratio of ICM:total cells, and these embryos were compared with IVF-derived and NT embryos. The proportion of blastocysts classified into group II in in vivo-derived, IVF-derived, and NT embryos was 80.8% (21/26), 55.8% (29/52), and 30.9% (17/55), respectively (Fig. 3A). Group II embryos appear to be normal, and most of the in vivo-derived embryos were in this group. Thus, differences were detected in the distribution of presumptive normal embryos among the NT, IVF-derived, and in vivo-derived embryos (P < 0.05). In contrast to the in vivo-derived blastocysts, 67% of the NT blastocysts were allocated to groups III and IV. When the proportion is based on the ratio of TE:total cells, the distribution patterns of the embryos show a reverse tendency of the ICM distribution. The proportin of in vivo-derived, IVF-derived, and NT blastocysts having high TE ratio (group IV) was 80.8% (21/26), 57.7% (30/52), and 34.5% (19/55), respectively (Fig. 3B). Thus, NT blastocysts showed a significant difference in the proportion of TE:total cells as compared with in vivo- and IVF-derived blastocysts (P < 0.05). There was also a difference between in vivo- and IVF-derived blastocysts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because bovine embryos produced by somatic cell NT could also be cultured to the blastocyst stage, at which time they can be transferred to recipients, it is unclear whether the increase in the number of ICM cells or the decrease in the number of TE cells is due to the NT or to in vitro culture. Thus, neither the environmental factors causing the increase in ICM cells or the decrease in TE cells nor the mechanisms by which NT embryos have aberrant proportions of ICM:TE cells are known. In this study, however, our findings suggest that the aberrant ICM:TE ratio may be due to somatic cell NT itself and not in vitro culture (Table 2), although IVF-derived embryos also showed a moderate increase in the ICM:total cell ratio as compared with in vivo-derived embryos.
It has not been possible to determine whether the developmental failures of NT embryos with somatic cell nuclei were due to incomplete nuclear reprogramming or to the cloning procedure itself. In general, it has been accepted that a nucleus from a differentiated somatic cell that is transferred into the cytoplasm of an enucleated oocyte should become reprogrammed to restore normal embryonic development. Regarding nuclear reprogramming, lack of telomere restoration of donor chromatin [27, 28], aberrant expression of specific genes [29], aberrant methylation patterns [8], and different culture systems [30] may be key factors affecting developmental failures of NT embryos. These studies provide indirect evidence that the developmental failures of NT embryos may be due to the inefficient epigenetic reprogramming of donor genomic DNA. In the present cloning system, we proposed that the primary cause of cloned fetus loss is placental abnormality [3–5, 7]. More than 80% of the deaths occurred between Days 30 and 60, with half of these deaths occurring between Days 30 and 40 and half occurring between Days 40 and 60 [10]. These findings suggest that cloned fetuses are variable in the degree of placental development, with more advanced placentas being able to sustain fetal growth for a longer period. Our hypothesis that aberrant ratios of ICM or TE to total cells may be one factor affecting developmental failures is supported by the result of some of our experiments (Table 2 and Fig. 3), although the hypothesis that the decrease of TE cells may be related to early abortion during embryonic development remains to be tested. A correlation between the number of TE cells and the number and/or size of the placentomes has not yet been demonstrated. Nonetheless, we provide additional evidence that developmental failures of bovine NT embryos probably are due to the incomplete blastocyst formation or disproportionate allocations of ICM and/or TE cells.
In general, the viability of in vitro-produced bovine embryos is inferior to that of in vivo-derived counterparts, possibly because of decreased embryo quality resulting from suboptimal in vitro conditions [31]. The reduced viability may result in an increased incidence of fetal mortality following embryo transfer. Abortion rates of in vivo-produced embryos ranged from 9% to 47% during the first or second trimesters of gestation [32–36]. The present cloning system shows relatively high rates of early and late abortion and neonatal and early postnatal deaths [1, 4, 7, 37–39]. Abortion frequency may be related to a specific deficiency or combined deficiencies in either the in vitro culture system or the NT procedure itself, leading to incomplete nuclear reprogramming of the donor cells [3]. In the present study, most NT blastocysts had abnormalities in the ratio of ICM:total cells, or a decrease in TE cells (Fig. 3). These results suggest that high frequencies of abortion and neonatal death in the present cloning system are likely related to aberrant proportions of NT embryos having reduced TE cells at the preimplantation stage.
In a previous study on in vivo-derived embryos, 40% of morula embryos did not display inner cells, and the mean number of inner cells of the morula embryos (approximately 32 cells) ranged from 2.9 to 4.0 [14]. However, inner cells are detected in most NT morula embryos. Moreover, differences in the mean number of inner cells at the morula stage were observed between NT and IVF-derived embryos (data not shown). These findings indicate that disproportionate allocations of ICM cells in bovine NT embryos begin during the morula stage.
In this study, allocations of ICM or TE cells detected in NT bovine embryos were obviously different from those of normal embryos produced in vitro or in vivo. In addition, anomalies of the NT embryo originated from early developmental stages such as the morula or blastocyst stage. These results suggest that the NT embryos having fewer TE cells in blastocyst stage may form abnormal placentas, eventually leading to fetal loss. At this time, our hypothesis that NT embryos having lower percentages of TE cells may fail during gestation cannot be tested because the embryo must be disassembled via experimental procedures. It is also uncertain whether the TE cells are restored during the later developmental stages to support the full-term development of the NT embryos. Alternatively, the ability of NT embryos to develop to term may be restricted to those embryos having a relatively normal ratio of ICM:total cells in the early developmental stages. These anomalies may also lead to inappropriate expression of genes essential for early embryonic development. Expression of a few genes that are exclusively expressed in the TE was heavily affected by the cloning procedure [30], supporting the hypothesis that abnormal placentation may be a major cause of fetal loss after transfer of NT embryos. Thus, expression profiles of known imprinted genes that are important for the embryonic survival should be compared for NT, IVF-derived, and in vivo-derived blastocysts. In addition, profiles of ICM and TE cells in blastocysts grown under different culture conditions remain to be examined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by grants NLM0050111 and HSC0130134 from the Ministry of Science and Technology, Seoul, Korea. 
2 Correspondence: Yong-Mahn Han, Animal Developmental Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Daejeon 305-600, Korea. FAX: 82 42 860 4608; e-mail: ymhan@mail.kribb.re.kr
Accepted: February 14, 2002.
Received: December 31, 2001.
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REFERENCES |
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