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Regular Article |
a Division of Gene Expression and Development, Roslin Institute, Roslin EH25 9PS, United Kingdom
b South Adelaide Research and Development Institute, Turretfield Research Center, Rosedale SA 5350, Australia
ABSTRACT
Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.
conceptus, developmental biology, implantation/early development, placenta
INTRODUCTION
The cloning of viable animals by nuclear transfer (NT) is a highly inefficient procedure that suffers from high rates of abortion throughout gestation and postnatal death, even for those species in which it is effective. Using somatic cells as nuclear donors, experiments in sheep and cattle suggest that as few as 1%–2% of reconstructed eggs or as much as 5% of NT blastocysts can develop to term [1–4]. Similar success rates have been reported in the mouse using either somatic or embryonic stem cells as nuclear donors [5–8]. The reasons for cloned embryo/fetal failure are poorly understood. At least half of all reconstructed embryos fail to complete the early developmental events necessary to reach the blastocyst stage. Such failure may be related to DNA damage resulting from cell-cycle incompatibilities between donated nuclei and recipient egg cytoplasts [9]. Because development to the blastocyst stage and beyond is also absolutely dependent on regulated gene expression, early and late clone failure may also reflect deficiencies in nuclear reprogramming. Global changes in mRNA expression reflecting nuclear reprogramming are apparent by the blastocyst stage following somatic cell NT in cattle [10]. At this stage, aberrant expression of both known and unknown genes is suspected [10, 11]. After implantation, a common feature of cloned fetuses that fail during gestation is retarded development [12]. Cloned fetal mortality at birth is also associated with cardiopulmonary deficiencies. This is exacerbated by a lack of spontaneous parturition [12, 13].
In sheep, embryonic attachment to the wall of the uterus, begins around Day 14 of gestation (D14) and is completed by D28–35 [14]. During this period, the embryo transforms itself from an elongated trophoblast completing gastrulation to a highly defined fetus. The extraembryonic membranes of the fetus also transform, beginning with an avascular chorion, to a vascular chorioallantoic structure, which itself becomes highly specialized. Vascularization of the chorioallantois originates from the mesodermal component of the allantois that grows from a diverticulum of the hindgut of the D15–16 ovine embryo [15]. As in all ruminants, attachment of fetal membranes to the uterine endometrial mucosa and their specialization occurs at discrete sites called caruncles. Contact with caruncles causes these membranes to become highly vascularized villous structures, known as cotyledons, that undergo hyperplasia and hypertrophy. Together, a fetal cotyledon and a maternal caruncle comprise a placentome, the functional unit of the placenta for maternal/fetal exchange of nutrients and metabolic wastes. Although it is unknown exactly when an absolute requirement for a functional placenta occurs in the sheep, it is presumably required after attachment is completed by D35.
In the present study, our objective was to evaluate the normalcy of cloned fetal development at D35. This was done by comparing fetuses cloned from somatic cells with age-matched fetuses conceived in vivo by mating or produced by in vitro maturation (IVM), fertilization (IVF), and culture (IVC) of oocytes and embryos. Our results reveal a high incidence of developmental retardation associated with deficiencies in chorioallantoic vascularization in cloned fetuses. These findings are consistent with the hypothesis that the failure of cloned embryos to develop beyond the blastocyst stage to term is related, at least in part, to deficiencies in the establishment of adequate placentation.
MATERIALS AND METHODS
This study was conducted following approval by the Roslin Institute Animal Ethics Committee and within a project license issued under the Animal (Scientific Procedures) Act of 1986. Data were collected in two experimental sessions during the autumn/winter of 1998–1999. In the first experiment, fetuses were created by mating of Black Welsh or Blackface ewes with a Black Welsh ram or by cloning from D26 Black Welsh fetal fibroblast cells (BLW1) that previously yielded live offspring [1]. In the second experiment, fetuses were derived from the IVF and IVC of oocytes from slaughterhouse ovaries or cloned from Black Welsh Fetal fibroblast cells (BLW6) isolated from a D35 fetus produced by natural mating in the first experiment.
Mating and Superovulation
Groups of cycling adult sheep to be mated or superovulated were first synchronized with an intravaginal progestagen sponge (Veramix; Pharmacia and Upjohn, Corby, Northamptonshire, UK) for 14 days. Ewes were mated by introduction of Black Welsh rams 24 h after sponge removal. Superovulation of ewes for recovery of oocytes for NT was achieved by one of two methods. In the first, ewes received eight 1-unit s.c. injections of ovine FSH (oFSH; Ovagen; Immuno-Chemical Products Ltd., Aukland, New Zealand) at 12-h intervals over 4 days, commencing at 0700 h on Day 11 after sponge insertion. A single i.m. injection of 400 IU of eCG (PMSG-Intervet; Intervet UK, Milton Keyes, Buckinghamshire, UK) was given with the second oFSH injection. Sponges were removed at the seventh oFSH injection at 0700 h and followed 24 h later by an i.m. injection of 2 ml of gonadorelin-releasing hormone (GnRH; Receptal; Intervet UK). This method was used for all ewes in experiment 1 and a number of ewes in experiment 2. A second method used in experiment 2 involved the administration of a single bolus of oFSH (1 unit) and 400 IU of eCG 36 h before sponge removal. The GnRH was administered 26 h after sponge removal. For both methods, oocytes were surgically retrieved from oviducts 26–29 h after GnRH injection.
Nuclear Transfer
Unless otherwise noted, all chemical reagents used during egg micromanipulation and culture were purchased from Sigma (Poole, Dorset, UK). Somatic cell NT was based on the method described by Wilmut et al. [1]. Oocytes for NT were collected from superovulated Blackface ewes in PBS plus 1% sheep or fetal calf (SeraQ; Thorpeworks, Tunbridge Wells, Kent, UK) serum and transferred immediately to calcium-free, Hepes-buffered ovine synthetic oviductal fluid (oSOF) [16] for removal of cumulus cells and enucleation. If necessary, cumulus was removed by pipetting in 600 IU/ml of hyaluronidase. Oocytes were enucleated manually in the presence of 7.5 µg/ml of cytochalasin B following treatment for 15 min in 5 µg/ml of Hoechst 33342 and 7.5 µg/ml of cytochalasin B. Enucleated oocytes were incubated in Hepes-buffered oSOF beginning 1 h before reconstruction and activation. Sheep fetal fibroblasts were cultured for 4–6 days in serum-reduced medium (0.5% fetal calf serum) before use as karyoplast donors. The NT fusion and activation was by three 80-µsec pulses of 1.25 kV/cm2 in 0.3 M mannitol, 0.1 mM MgCl2, and 0.05 mM CaCl2. Reconstructed embryos were incubated overnight in an atmosphere of 5% O2, 5% CO2, and 90% N2 (5:5:90) at 38°C. One-day postactivation embryos were embedded in 1–2% agar chips in Hepes-buffered oSOF and transferred to the ligated oviduct of an estrus-synchronized recipient for a further 6 days. Morula- and blastocyst-stage embryos recovered 7 days postactivation were transferred to the uteri of estrus-synchronized ewes (1–2 per recipient).
In Vitro Embryo Production
In vitro production (IVP) of embryos by IVM/IVF/IVC was based on the method described by Walker et al. [16]. Ovaries from Scottish Blackface and Scottish Blackface-cross ewes were collected at the abattoir and transported to the laboratory in PBS at 30–32°C. Cumulus-oocyte complexes (COCs) were aspirated from 2- to 8-mm follicles and washed three times in Hepes-buffered Tissue Culture Medium (TCM)-199 (Gibco BRL Life Technologies, Paisley, UK) containing 2% sheep serum and twice in bicarbonate-buffered TCM-199 containing 20% sheep serum before maturation. The COCs were matured in groups of 40–50 per 800 µl of bicarbonate-buffered TCM-199 containing 20% sheep serum, 5 µg/ml of FSH (Foltrophin; Vetrepharm, Kingston, ON, Canada), 5 µg/ml of LH (Lutrophin; Vetrepharm), and 1 µg/ml of estradiol in 5% CO2 in air at 38°C. Matured COCs were incubated with spermatozoa approximately 24 h after the commencement of maturation, after the removal of excess cumulus by a 10- to 15-sec treatment with TCM-199 containing 2% sheep serum and 300 IU/ml of hyaluronidase. Approximately 1 h before insemination, frozen semen was thawed. Motile sperm were harvested by a "swim-up" procedure in which 0.3 ml of thawed semen were layered below 1 ml of IVF medium (oSOF + 2% sheep serum) and, after a 1-h incubation period, the top fraction was collected. The COCs were inseminated in groups of 40–50 in 450 µl of IVF medium under oil in 5% CO2 in air at 38°C. Approximately 24 h after insemination, putative embryos were cleaned of remnant cumulus cells by pipetting in a fine-bored mouth pipette (first wash) using embryo culture medium consisting of oSOF containing 1x essential and nonessential amino acids and 4 mg/ml of BSA (oSOFaaBSA). Embryos were subsequently washed twice in oSOFaaBSA and incubated in 800 µl of the same at 38°C in a 5:5:90 atmosphere as groups of 20–40. Embryo cleavage was assessed on Day 2 of development, with Day 0 being the day of insemination. On Day 6 of development, embryos were reassessed, with morula and blastocysts being transferred to final recipients (
2 per ewe).
Fetal Recovery and Cytogenetic Analysis
At D35, pregnant ewes were killed with an i.v. injection of 20% sodium pentobarbitone (Euthatal; Merial Animal Health Ltd., Harlow, Essex, UK) at a dosage of 1 ml per 1.4 kg body weight. Following hysterectomy of killed ewes, fetuses were recovered and evaluated before being decapitated and eviscerated in sterile PBS with 1% gentamicin. Fetal carcasses were then minced and incubated for 2–5 min at 37°C in 2 ml of trypsin-EGTA (TEG) solution (2.5 g/L of trypsin, 0.1 M NaCl, 0.7 mM Na2HPO4, 1.5 mM KH2PO4, 4 mM KCl, 5 mM D-glucose, 22 mM Tris, 1 mM EGTA, 0.01% PVA [cat. p8136, Sigma]; 30–70 kDa). The TEG/tissue preparations were transferred to a conical tube to which 13 ml of Glasgow Minimum Essential Medium (GMEM; Sigma), modified by the addition of 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 1x nonessential amino acids (all components in MEM, except serum, from Gibco BRL Life Technologies; serum from SeraQ), were added. After allowing 5 min for larger pieces of tissue to settle, cellular material still in suspension was removed and centrifuged at 1000 rpm for 5 min, followed by one wash with modified GMEM. After a second centrifugation, pelleted cells were resuspended in 7 ml of modified GMEM and placed in a 25-cm tissue-culture flask. Cells were incubated at 37°C in 5% CO2, and medium was replaced with fresh modified GMEM after 24 h. By 48 h, flasks were normally confluent. Mitotic spreads were prepared from fetal fibroblasts as described previously [17].
Statistical Analysis
Development of NT embryos to the blastocyst stage was tested using a Fisher exact test. Analysis of fetal measurements was by a one-way ANOVA on log-transformed data followed by paired Tukey-Kramer comparisons.
RESULTS
Preimplantation Development of NT Embryos
In vitro produced (IVM/IVF/IVC) embryos were generated during three trials in February 1999. A total of 185 COCs were inseminated, of which 56% cleaved, and 39% of cleaved embryos formed blastocysts by the time of transfer (6 days postinsemination). The NT embryos were produced using two different serum-deprived Black Welsh fetal fibroblast cell lines in two separate sessions, with each session consisting of three to four trials. Using BLW1 as karyoplasts, 288 ovulated oocytes were recovered, of which 61% fused, and 9% of fused embryos formed blastocysts. Using BLW6 as karyoplasts, 160 ovulated oocytes were recovered, of which 66% fused, and 21% of fused embryos formed blastocysts. No difference was found in the production of blastocysts in separate sessions by NT from different fetal fibroblast lines (P = 0.3203).
Recovery and Evaluation of Fetuses at D35
Sheep pregnancies were assessed by ultrasonography at D26 and D35 and confirmed surgically on D35. All pregnancies detected during the first scan remained positive at D35 (Table 1). All mated ewes were surgically confirmed as pregnant and yielded four purebred and five crossbred fetuses. Transfer of 71 IVP morula and blastocysts into 30 ewes resulted in 22 (73%) pregnant ewes. Ten pregnant ewes that had received 21 IVP embryos were killed to recover 17 fetuses (81%). Ultrasound examination of ewes receiving NT embryos yielded an approximately 50% pregnancy rate. Surgical recovery of fetuses from ultrasound-positive ewes revealed that for each line of NT embryos (BLW1 or -6), approximately 30% of transferred embryos were recovered as fetuses.
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Fetal appearance at D35 was evaluated according to the method described by Green and Winters [15] and arbitrarily categorized (Table 2) according to gross morphology of the head, limb, and body as being equivalent to fetuses at D16–17, D19–21 (see Fig. 2b, left side), D25–28, D30–33 (Fig. 1a, left side), or D34–35 (Fig. 1b). In most cases, both mated and IVP fetuses were similar and conserved in their development (Table 3). All fetuses recovered from pure- and crossbreeding had developed fully to D35 equivalence. Two of the 17 IVP fetus (12%) were retarded in their development (D30–33 and D25–28), with the latter showing signs of morphological degradation. Although one D34–35 IVP fetus possessed normal gross morphology, it suffered from dermal hemorrhaging. This anomaly was a more prevalent feature of NT fetuses (see below). Of the seven NT fetuses cloned from the BLW1 cell line, only three (43%) had developed to D35 equivalence. The remaining four (57%) were developmentally retarded at various developmental stages (one at D25–28, two at D19–21, and one at D16–17). For the eight NT-BLW6 fetuses, only two (25%) were morphologically equivalent to D35. Of the six remaining, three showed comparable ranges of developmental retardation as observed in fetuses cloned from the NT-BLW1 cell line (one at D25–28, one at D19–21, and one at D16–17), with the remaining three being only slightly retarded (D30–33).
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Four major anomalies were observed in NT fetuses (Tables 3 and 4). The least common, which was observed in one NT-BLW1 and one NT-BLW6 D34–35 fetus, consisted of an accumulation of fluid in the fourth ventricle of the brain (Figs. 1a and 2b). This was not obviously correlated with any of the other anomalies observed. A more common anomaly, occurring in five of eight (63%) NT-BLW6 fetuses and in one NT-BLW1, was an enlarged liver that was either bloodless or paler than expected (Fig. 1 and Tables 3 and 4). This feature was observed in both developmentally arrested (D16–17 and D25–28) and more advanced (D30–35) fetuses. When occurring in the latter, it was also accompanied by a third anomaly, consisting of moderate to severe dermal hemorrhaging throughout the trunk and head (Fig. 1). Interestingly, the most severe hemorrhaging observed was in a D34–35 fetus, in which the liver was threefold smaller than normal.
The fourth anomaly consisted of a lack of placental vascular development reflected by reduced or absent numbers of cotyledons (Table 4). This defect was independent of the abundance of chorionic membrane present (Fig. 2d) or amnion formation (data not shown), and it was most striking in NT fetuses arrested at D28 or earlier. In D35-equivalent NT fetuses, the range of cotyledon counts (8–22) (Table 4) was also either notably below or only approaching the average for mated purebred, crossbred, or IVP fetuses (mean ± SEM, n: purebred, 32 ± 3, 4; crossbred, 25 ± 4, 5; IVP 25 ± 2, 15). The variable incidence of twinning among all fetal groups and the low number of developmentally advanced NT fetuses precluded a meaningful statistical comparison of fetal size between groups. However, the range of fetal crown-rump lengths for D35-equivalent NT fetuses (20–24 mm) also fell below the average for mated purebred, crossbred, or IVP fetuses, which were identical to each other (mean length [mm ± SEM], 25 ± 0.5). Similarly, the range of D35-equivalent NT fetal weights (1.1–1.8 g) was mostly below the averages for control fetuses (mean wt [g ± SEM], n: purebred, 1.7 ± 0.1, 4; crossbred, 1.9 ± 0.1, 5; IVP, 1.9 ± 0.1, 15). Enlarged livers and dermal hemorrhaging in NT fetuses equivalent to D30 or older were correlated with placenta possessing 10 or fewer cotyledons (Table 4). This was approximately half the number observed in the most normally developed NT fetal placenta (20–22) and a third of the average count for mated purebred fetuses (see above).
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Development to Term
One NT-BLW6 pregnancy that was allowed to go to term yielded a dead lamb. The lamb was of normal size and weight for its breed (Purebred Black Welsh). Although it was not known if the fetus was born dead, a postmortem examination revealed that its lungs had never expanded. Other defects unlikely to have been terminal included a 2-mm perforation in the ventral septa of its heart and the absence of a short section of its rectum (atresia ani) that led to an overextended large bowel. No evidence of physical abnormalities was found in 17 lambs born from 12 IVP pregnancies diagnosed at D35 and permitted to go to term, at which no evidence of oversize was found in these lambs (i.e., all birth weights were within population limits).
Cytogenetic Analysis of Fibroblasts from D35 Fetuses
To investigate the cellular normalcy of the fetuses isolated, nonclonal fibroblast cultures were derived from both NT and mated-purebred progeny (Table 5). The karyotype of these cells was then examined along with freshly resuscitated aliquots of frozen fibroblast cell lines that had served as nuclear donors. Fibroblasts were only obtained from fetuses that had developed to D30–35 equivalence. The NT fetuses used were either morphologically normal or suffering from the deficient placental vascularization and liver/hemorrhaging anomalies described. Attempts to establish fibroblast cultures from D25–28 NT fetuses were unsuccessful, possibly because of the previous degeneration of this tissue. Evaluation of early passage cells (p3) revealed no appreciable differences in the frequency of a normal complement of chromosomes (n = 54) either between or within fetal groups.
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DISCUSSION
Through the purposeful evaluation of fetal development early during gestation, our study has revealed the extent and nature of cloned fetal failure in sheep. Using current methods, only 30% of transferred NT morulae and blastocysts were recovered as fetuses within the first quarter of gestation, with more than 50% being developmentally retarded and possibly dead or dying. Although categorization of developmental age is problematic because of variations between and within fetuses in the timing of differentiative events [18], cloned fetal retardation in our study was made striking by the morphological uniformity observed among IVP and mated fetuses of various genetic backgrounds. Comparable rates of pregnancy failure soon after the establishment of pregnancy have been previously reported for NT pregnancies in cattle [11, 19]. The high incidence of developmental delay observed in our study suggests that fetal loss is determined long before it is detected externally or physiologically.
Retarded development of cloned fetuses was strikingly associated with avascular or hypovascular placentas, which were equally prevalent in fetuses from both fibroblast donor cell lines. Absence of cotyledonary tissue and a concurrent, hemorrhagic response at the surface of uterine caruncles have been previously described in bovine embryonic cell-derived NT conceptuses presumed to have been aborted at D38 (term, D276) [19]. A similar placental pathology to that which we observed has also been recently described within the first trimester of bovine somatic cell NT conceptuses [20]. Recent attempts to clone mice from primordial germ cells have also encountered associated midgestational failure with deficiencies in embryo-derived placental vasculature, specifically in the formation of labyrinthine trophoblast [21]. The condition of avascular ovine placentas observed in the present study were remarkably similar to that of allantoic aplasia, previously reported in as much as 20% of pregnancies derived from IVP cattle embryos [22]. However, the characterization of gross placental morphology in our study was insufficient to resolve whether the lack of vascularization we observed stemmed from a lack of allantoic outgrowth or an absence of vasculogenesis. Interestingly, little evidence of vascular deficiencies was found in our own fetuses from IVP embryos.
A complete absence of placental vascularization would have clearly precluded fetal growth and differentiation beyond that which could be supported by yolk sac-derived nutrition. It is also likely that the severity of vascular deficiencies in cloned fetal membranes early during gestation would be directly related to limitations in subsequent development and resulting pathologies. This idea is supported by studies regarding intrauterine growth restriction in sheep induced experimentally by prepregnancy removal of endometrial caruncles. Restriction of the maternal/fetal circulatory interface in this way predictably alters the supply of O2 and nutrients for growth and the clearance of fetal metabolites such as CO2, lactate, and pyruvate. This results in an overall restriction of fetal growth and birth weight, in addition to an alteration in the proportionate weight of individual organs such as the brain, kidney, and adrenals with respect to body weight [23]. Changes in the proportionate weight of these organs are accompanied by alterations in their differentiation and function, creating further physiological imbalances [24–28]. Intrauterine growth restriction induced experimentally in sheep and studied epidemiologically in human infants also results in aberrant lung development, with adverse affects on respiratory function demonstrated at birth [29].
In near-term sheep, the artificial elevation of fetal plasma lactate osmotically draws fluid from the maternal to the fetal circulation, leading to hydramnios (excessive amniotic fluid accumulation) or hydrops fetalis (fetal edema) [30]. The latter of these, edematous placental pathologies such as enlarged umbilical vessels and membranes, and hydrallantois (excessive allantoic fluid accumulation) have all been previously reported in NT sheep and cattle during late gestation or at term [4, 12, 13, 31]. Somatic NT mice that develop to term also have placental weights that are twofold higher than those of noncloned neonates, but it is not clear from that study whether such increases could be attributed to hyperplasia or edema [7]. Although occurring substantially earlier, the placental deficiencies observed in our study could, therefore, have contributed to the fetal anomalies observed, namely the enlargement of fetal liver (an active site of metabolism), dermal hemorrhaging, and possibly, swelling of brain structures. This concept is supported by the chronological sequence of the developmental stages in which these fetal defects were first detected and their correlation with low cotyledonary counts.
In IVP cattle, allantoic aplasia is also associated with anomalies in the development of the hind region [22]. Such defects are also observed in NT cattle and sheep at term and include aberrant development of the rectum and urogenital tract [12] (unpublished results). Other defects that are confined to NT conceptuses include aberrant septation of the heart [12, 31] (unpublished results) and postnatal death by anemia resulting from lymphoid hypoplasia [32, 33]. A common origin for all of these pathologies and the avascular placentation observed in our study is not impossible. Progenitors of hematopoietic stem cells derive from intraembryonic, para-aortic mesoderm, which forms from the same caudal mesodermal region from which the allantois emerges [34].
The molecular mechanisms responsible for failure and deficiencies of NT fetuses remain largely unknown. Placental defects observed in mice cloned from early germ cells, which are devoid of epigenetic imprints, are reminiscent of those seen in homozygous null mutant mice for Mash-2, a well-known, epigenetically imprinted gene [21, 35]. Combined expression and methylation analysis of genes normally subject to epigenetic regulation in such cloned fetuses revealed aberrations supporting the hypothesis that gametic imprints are essential for normal development [21]. Our sheep fibroblasts would not be expected to be devoid of epigenetic imprints, but aberrant imprinting of one or several genes induced by NT or intrinsic to the donor cells may be responsible for some (or all) of the defects observed [36]. An alternative, and not mutually exclusive, possibility is that aberrant clone development stems from chromosomal instability resulting from in vitro manipulations. Both IVC and parthenogenetic activation in themselves can lead to aneuploidies in bovine embryos [37]. In humans, aneuploidies consisting of triploidy, monosomy X, or trisomies of chromosomes 8, 13, or 21 account for 50% of all early pregnancy failures, with such failures being associated with reduced placental vascularization, fetal dermal hemorrhaging, and ultimately, fetal hydrops [38]. Our study failed to reveal gross karyotypic anomalies in the fibroblasts derived from cloned fetuses. However, more subtle aberrations, such as insertions or translocations of chromosome segments, cannot be ruled out. Interestingly, attempts to rescue NT cattle and mice conceptuses by chimeric aggregation have been found only to extend gestation, not to enable development to term [19, 21]. Such studies, therefore, suggest that placental deficiencies in NT pregnancies may be noncell autonomous, that is, the result of aberrant interaction between one or more cell types.
In summary, the results of our study demonstrate that cloned fetal development is characterized by a high incidence of developmental retardation that is associated with deficiencies in the establishment of placental vasculature. Neither placental nor fetal anomalies detected in cloned fetuses could be correlated with chromosome instability reflected by changes in chromosome number. Our results suggest, however, that the developmental transition from yolk sac to allantoic-derived nutrition represents an ideal focal point at which to base future studies regarding cloned fetal failure.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical contribution of dedicated embryological and surgical staff at the Roslin Institute, whose efforts made this study possible. Specifically, Mr. Bill Ritchie and Ms. Ailsa Travers for embryo micromanipulation; Ms. Tricia Ferrier and Ms. Judy Fletcher for cell culture; Mr. John Bracken, Mr. Whim Bosma, Ms. June Bowen, Mr. Mike Malcolm-Smith, and Mrs. Marjorie Ritchie for surgery; and Mr. Roddy Field for photography. The authors would also like to thank Drs. Andras Dinnyes and Jane McCracken for helpful discussions. Sheep semen was kindly provided by Edinburgh Genetics, Midlothian, UK.
FOOTNOTES
First decision: 17 November 2000.
1 Funded by Geron Bio-med, Roslin, Midlothian, UK. 
2 Correspondence. FAX: 44 0131 4400434; paul.desousa{at}bbsrc.ac.uk
Accepted: February 8, 2001.
Received: September 27, 2000.
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J. R. Miles, C. E. Farin, K. F. Rodriguez, J. E. Alexander, and P. W. Farin Angiogenesis and Morphometry of Bovine Placentas in Late Gestation from Embryos Produced In Vivo or In Vitro Biol Reprod, December 1, 2004; 71(6): 1919 - 1926. [Abstract] [Full Text] [PDF]
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S. R. Ravelich, A. N. Shelling, A. Ramachandran, S. Reddy, J. A. Keelan, D. N. Wells, A. J. Peterson, R. S.F. Lee, and B. H. Breier Altered Placental Lactogen and Leptin Expression in Placentomes from Bovine Nuclear Transfer Pregnancies Biol Reprod, December 1, 2004; 71(6): 1862 - 1869. [Abstract] [Full Text] [PDF]
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D. O. Brandao, P. Maddox-Hyttel, P. Lovendahl, R. Rumpf, D. Stringfellow, and H. Callesen Post Hatching Development: a Novel System for Extended in Vitro Culture of Bovine Embryos Biol Reprod, December 1, 2004; 71(6): 2048 - 2055. [Abstract] [Full Text] [PDF]
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N. Beaujean, J. Taylor, J. Gardner, I. Wilmut, R. Meehan, and L. Young Effect of Limited DNA Methylation Reprogramming in the Normal Sheep Embryo on Somatic Cell Nuclear Transfer Biol Reprod, July 1, 2004; 71(1): 185 - 193. [Abstract] [Full Text] [PDF]
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M. Guillomot, A. Turbe, I. Hue, and J.-P. Renard Staging of ovine embryos and expression of the T-box genes Brachyury and Eomesodermin around gastrulation Reproduction, April 1, 2004; 127(4): 491 - 501. [Abstract] [Full Text] [PDF]
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O. V. Patel, O. Yamada, K. Kizaki, T. Takahashi, K. Imai, S. Takahashi, Y. Izaike, L. A. Schuler, T. Takezawa, and K. Hashizume Expression of Trophoblast Cell-Specific Pregnancy-Related Genes in SomaticCell-Cloned Bovine Pregnancies Biol Reprod, April 1, 2004; 70(4): 1114 - 1120. [Abstract] [Full Text] [PDF]
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W. V Holt, A. R Pickard, and R. S Prather Wildlife conservation and reproductive cloning Reproduction, March 1, 2004; 127(3): 317 - 324. [Abstract] [Full Text] [PDF]
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S. R. Ravelich, B. H. Breier, S. Reddy, J. A. Keelan, D. N. Wells, A. J. Peterson, and R. S.F. Lee Insulin-Like Growth Factor-I and Binding Proteins 1, 2, and 3 in Bovine Nuclear Transfer Pregnancies Biol Reprod, February 1, 2004; 70(2): 430 - 438. [Abstract] [Full Text] [PDF]
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R. S.F. Lee, A. J. Peterson, M. J. Donnison, S. Ravelich, A. M. Ledgard, N. Li, J. E. Oliver, A. L. Miller, F. C. Tucker, B. Breier, et al. Cloned Cattle Fetuses with the Same Nuclear Genetics Are More Variable Than Contemporary Half-Siblings Resulting from Artificial Insemination and Exhibit Fetal and Placental Growth Deregulation Even in the First Trimester Biol Reprod, January 1, 2004; 70(1): 1 - 11. [Abstract] [Full Text] [PDF]
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 S. Schutt, A. R. Florl, W. Shi, M. Hemberger, A. Orth, S. Otto, W. A. Schulz, and R. H. Fundele DNA Methylation in Placentas of Interspecies Mouse Hybrids Genetics, September 1, 2003; 165(1): 223 - 228. [Abstract] [Full Text] [PDF]
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 C. R. Cogle, S. M. Guthrie, R. C. Sanders, W. L. Allen, E. W. Scott, and B. E. Petersen An Overview of Stem Cell Research and Regulatory Issues Mayo Clin. Proc., August 1, 2003; 78(8): 993 - 1003. [Abstract] [PDF]
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G. G. Cezar, M. S. Bartolomei, E. J. Forsberg, N. L. First, M. D. Bishop, and K. J. Eilertsen Genome-Wide Epigenetic Alterations in Cloned Bovine Fetuses Biol Reprod, March 1, 2003; 68(3): 1009 - 1014. [Abstract] [Full Text] [PDF]
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T. T. Peura, D. O. Kleemann, S. R. Rudiger, G. S. Nattrass, C. J. McLaughlan, and S. K. Walker Effect of Nutrition of Oocyte Donor on the Outcomes of Somatic Cell Nuclear Transfer in the Sheep Biol Reprod, January 1, 2003; 68(1): 45 - 50. [Abstract] [Full Text] [PDF]
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S. Hiendleder, V. Zakhartchenko, H. Wenigerkind, H.-D. Reichenbach, K. Bruggerhoff, K. Prelle, G. Brem, M. Stojkovic, and E. Wolf Heteroplasmy in Bovine Fetuses Produced by Intra- and Inter-Subspecific Somatic Cell Nuclear Transfer: Neutral Segregation of Nuclear Donor Mitochondrial DNA in Various Tissues and Evidence for Recipient Cow Mitochondria in Fetal Blood Biol Reprod, January 1, 2003; 68(1): 159 - 166. [Abstract] [Full Text] [PDF]
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G. Ptak, M. Clinton, M. Tischner, B. Barboni, M. Mattioli, and P. Loi Improving Delivery and Offspring Viability of In Vitro-Produced and Cloned Sheep Embryos Biol Reprod, December 1, 2002; 67(6): 1719 - 1725. [Abstract] [Full Text] [PDF]
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 J. G. Thompson, K. L. Kind, C. T. Roberts, S. A. Robertson, and J. S. Robinson Epigenetic risks related to assisted reproductive technologies: Short- and long-term consequences for the health of children conceived through assisted reproduction technology: more reason for caution? Hum. Reprod., November 1, 2002; 17(11): 2783 - 2786. [Abstract] [Full Text] [PDF]
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J. R. Hill, D. H. Schlafer, P. J. Fisher, and C. J. Davies Abnormal Expression of Trophoblast Major Histocompatibility Complex Class I Antigens in Cloned Bovine Pregnancies Is Associated with a Pronounced Endometrial Lymphocytic Response Biol Reprod, July 1, 2002; 67(1): 55 - 63. [Abstract] [Full Text] [PDF]
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P. Loi, M. Clinton, B. Barboni, J. Fulka Jr., P. Cappai, R. Feil, R. M. Moor, and G. Ptak Nuclei of Nonviable Ovine Somatic Cells Develop into Lambs after Nuclear Transplantation Biol Reprod, July 1, 2002; 67(1): 126 - 132. [Abstract] [Full Text] [PDF]
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P. Chavatte-Palmer, Y. Heyman, C. Richard, P. Monget, D. LeBourhis, G. Kann, Y. Chilliard, X. Vignon, and J.P. Renard Clinical, Hormonal, and Hematologic Characteristics of Bovine Calves Derived from Nuclei from Somatic Cells Biol Reprod, June 1, 2002; 66(6): 1596 - 1603. [Abstract] [Full Text] [PDF]
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 W. M. Rideout III, K. Eggan, and R. Jaenisch Nuclear Cloning and Epigenetic Reprogramming of the Genome Science, August 10, 2001; 293(5532): 1093 - 1098. [Abstract] [Full Text] [PDF]
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