| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Departments of Veterinary Physiology and Pharmacology,
b Veterinary Anatomy and Public Health,
c Center for Animal Biotechnology and Genomics, and
d Large Animal Medicine and Surgery, Texas A&M University, College Station, Texas 77843
e Ultimate Genetics, Franklin, Texas 77856
ABSTRACT
The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30–90 of gestation. Fetal and placental characteristics were studied from Days 30–90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30–90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35–60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.
IVF/ART, pregnancy
INTRODUCTION
Success has now been achieved with somatic cell cloning in four animal species using both adult and fetal cells [1–11]. The potential applications of cloning in agriculture, animal and human medicine, and the propagation of rare animals clearly have great commercial and conservational benefit [12–14]. However, the efficiency of this process is poor, with less than one animal born per 100 reconstructed nuclear transfer (NT) embryos [10]. Much of this inefficiency results from low initial pregnancy rates and early pregnancy losses, which have resulted in survival rates of cloned bovine blastocysts to term of 10%–80% [3, 7]. First-trimester losses of more than 50% are common for NT pregnancies in cattle, sheep, and goats [1–3, 6, 9, 15], whereas 2%–4% of naturally conceived, Day 30 bovine pregnancies and 11% of in vitro-produced embryos would be expected to be lost by Day 60 [16–18]. To our knowledge, this lack of normal in vivo development has occurred in each species studied so far and is delaying commercial application of the NT technique.
In general, early fetal losses may result from abnormalities of the embryo or its placenta, alterations in maternal uterine environment, or fetomaternal interactions [19]. In naturally conceived pregnancies, fetal abnormalities are a major cause of pregnancy loss [19]. However, in animals derived from cloning or in vitro culture procedures, placental abnormalities occur at a high incidence in both early and late gestation [4, 20–24], and this may be the cause of any observed fetal abnormalities. Accordingly, in this experiment, we used both in vitro- and in vivo-generated embryos as controls for the NT embryos.
The normal bovine placenta progressively attaches to the endometrium almost throughout the first trimester, in contrast to the more rapid and invasive attachment phase in humans and rodents. At Day 30, histological examination revealed the presence of placentomes, microvilli, and tenuous attachment of maternal and fetal epithelia [25]. The initial placental contact with a maternal caruncle induces villous processes to undergo hypertrophy and hyperplasia to form cotyledons that, by Day 42, progress to form larger, more complex placentomes [25, 26]. Binucleate cells in the cow form transient fetomaternal syncitia, which are proposed to be central to villous expansion and subsequent maintenance of placental and endometrial apposition [27]. The binucleate cells of the bovine trophoblast produce pregnancy specific protein b (PSPb), and its detection has been used for pregnancy testing in this and other species [28]. Placentomes with extensive villous formation are thought to be the primary site of transport for easily diffusible, small molecules such as oxygen, carbon dioxide, and also amino acids and glucose, whereas macromolecules are transported in the interplacentomal areas adjacent to uterine gland openings [27, 29].
Lack of placentome development was proposed as the likely cause for early fetal loss in NT fetuses derived from embryonic stem cells [22], and in the current study, we evaluated this hypothesis using somatic cell cloned fetuses. The goal of this study was to document the time of greatest loss of NT fetuses and to investigate the hypothesis that failure of placental development is the major cause of this loss. Fetal development was monitored indirectly by transrectal ultrasonography and by maternal PSPb concentrations. Direct evaluation of fetuses and their placentas was made after slaughter of a randomly selected group of cloned and control pregnancies.
MATERIALS AND METHODS
Donor Cell Lines
The NT embryos used in this experiment derived from adult and fetal fibroblast cell lines. The age of the adults at sample collection ranged from 7–21 yr, and the fetuses ranged between 45–55 days of age. Adult cell lines were derived from three animals: a 21-yr-old Brahman bull, a 8-yr-old Charolais cow, and a 7-yr-old Angus bull. The Days 40–50 fetuses derived from an Angus male, a Holstein female, a Brahman male (same genotype as the adult Brahman), and a Charolais female (same genotype as the adult Charolais cow).
Small pieces of tissue (2–5 mm) were prepared from adult skin biopsy specimens and fetal tissue using a razor blade, then transferred into 25-mm2 flasks containing Dulbecco modified Eagles medium (DMEM-F12; Gibco Laboratories, Grand Island, NY) plus 10% (v:v) fetal bovine serum (FBS; Summit, Fort Collins, CO) plus 1% (v:v) penicillin/streptomycin (Gibco; 10 000 U/ml of penicillin G, 10 000 µg/ml of streptomycin), then cultured at 37°C in air containing 5% C02. When confluency was achieved, cells were trypsinized for 5 min and the recovered cells centrifuged, washed, and then either frozen in DMEM-F12 containing 10% dimethyl sulfoxide before storage at -80°C or transferred into a new 25-mm2 flask containing DMEM-F12 plus 10% FBS. Cells for NT were passaged fewer than six times (<25 days in culture) for each cell line except the Brahman fetal cells, which were used at either passages 2–4 or 18–20.
Nuclear Transfer
The NT technique has been described elsewhere [30]. Briefly, the recipient oocytes were slaughterhouse derived and matured for 17 h in Medium 199 (M 199; Gibco) supplemented with 10% (v:v) fetal calf serum (FCS; Gibco), 0.1 U/ml of FSH (Sioux Biochem, Sioux City, IA), 0.1 U/ml of LH (Sioux Biochem), 1 µg/ml of estradiol (Sigma, St. Louis, MO), 28 µg/ml of pyruvate (Sigma), 0.05 µg/ml of epidermal growth factor (Sigma), and 1% penicillin/streptomycin. Metaphase II oocytes enucleated at 19 h after maturation using a beveled, 25 µm-diameter glass pipette and donor cells of median diameter were combined with enucleated oocytes, then the oocyte-fibroblast couplets were fused with two 20-µsec, 1.6-kV/cm DC fusion pulses delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA). Either serum-fed or serum-starved cells were used. Serum-starved cells were placed in DMEM-F12 containing 0.05% serum for 5 days before NT.
Oocyte activation was performed 3–5 h after fusion by incubation in 5 µM ionomycin (Calbiochem, San Diego, CA) [31] and followed by transfer into 100 µM Butyrolactone-I (Biomol, Plymouth Meeting, PA) in M 199 for 4 h. Embryos were cultured in CR1aa [32] plus 10% FCS with buffalo rat liver coculture for 7 days. Two or three blastocysts were nonsurgically transferred into each recipient at Day 7 after a natural heat. A total of 243 cloned embryos were transferred into 120 recipient cows. Of these 243 embryos, 163 were derived from fetal and 80 from adult somatic cells.
Production of Control Fetuses
Seventeen blastocysts were recovered nonsurgically from three donor cows after routine superovulation and embryo recovery procedures [33], then transferred into 17 recipient cows 7 days after a natural heat. Four in vitro produced (IVP) blastocysts were produced from the in vitro fertilization of slaughterhouse eggs, which was followed by culture in serum-free modified synthetic oviductal medium [34]. These four embryos were then transferred into two recipient cows 7 days after a natural heat.
Fetal Monitoring and Survival Analysis
Diagnosis of pregnancy was made from Day 30 after NT by transrectal ultrasonography with a 5-MHz linear array probe and an Aloka 500 connected to a video printer. A viable pregnancy was defined as the presence of one or more fetuses with a detectable heartbeat. Recipients were examined weekly for the presence of fetal heartbeats, and the crown-rump (CR) length was determined for each live NT, IVF, and ET (embryo transfer of in vivo-produced embryo) fetus by freezing a sagittal-section image of each fetus before measurement with electronic calipers.
PSPb Analysis
Serum samples from cows pregnant with at least one viable fetus were analyzed for PSPb concentrations to determine if these values were predictive for survival of pregnancy to Day 90. Blood samples were taken from subsets of pregnant recipient cows at Day 30 (14 cows), Day 35 (9 cows), and Day 50 (13 cows). Serum from two nonpregnant cows was also analyzed as a control. Blood samples were collected into heparinized vacutainer tubes and centrifuged, and the plasma was then separated and stored at -20°C until transport to BioTracking (Moscow, ID) for analysis. Samples were assayed in triplicate for PSPb immunoreactivity by a double radioimmunoassay using rabbit antiserum to bovine PSPb, with an expected value for pregnant animals of greater than 1.0 ng/ml [28].
Placentas Retrieved at Slaughter
Ten pregnant recipients were randomly allocated for slaughter between Days 35 and 60 of gestation. Of these 10 cows, 6 carried cloned pregnancies (3 derived from adult cells, 3 from fetal cells), and the remaining 4 were control pregnancies (3 ET and 1 IVP pregnancy). The chorioallantois was examined grossly to assess vascularization and placentome development according to the method described by King et al. [25, 29].
The terminated pregnancies were randomly selected, although the examiners were aware of from which group the pregnancies derived. Samples from fetal cotyledons, intercotyledonary areas, maternal caruncles, and intercaruncular areas adjacent to the fetus were preserved for histopathology by fixation in 4% paraformaldehyde for 24 h, which was followed by transfer into 70% ethanol. Paraffin-embedded thin sections of cotyledons and intercotyedonary segments were stained using hematoxylin and eosin and by periodic acid-Schiff to highlight multinucleate giant cells.
Statistics
The conditional probability of a recipient cow carrying a viable pregnancy from Days 30 to 90 was assessed using the Kaplan-Meier survival function [35], and survival to Day 90 was compared using the chi-square test. The day of pregnancy failure was deemed to be midway between the day of no detectable heartbeat and the previous examination. Multiple regression analysis was used to determine the effect of group (NT, control), gestation length, and survivorship at Day 90 on CR length. Estimates were adjusted for the random effect of repeated measurement by cow [36], and ANOVA was used for comparison of PSPb values.
RESULTS
Ultrasonographic Evaluations
Survival analysis Day 30 pregnancy rates were similar for recipients that carried cloned and control pregnancies (45% [54/120] vs. 58% [11/19]; P > 0.1). A direct comparison of the recipient cow pregnancy rate may not be valid, because multiple cloned embryos were transferred into each recipient. No significant difference was found between pregnancy rates for recipients that carried fetuses cloned from fetal (54% [21/39]) or adult (41% [33/81]) somatic cells. Of the 54 cloned pregnancies, 23 derived from adult and 31 from fetal donor cells, whereas in the control group, 9 were in vivo and 2 were in vitro derived. Further analysis of the cloned pregnancies showed that 9 of 54 recipients were diagnosed by ultrasonography to be carrying twins, three sets of which survived to Day 90. A set of IVF twins also survived to Day 90.
The survival rates to Day 90 were 100% (11/11) for control fetuses and only 19% (10/54) for clones (Fig. 1). Fetal deaths were evenly spread throughout Days 30–60, with 35% of Day 30 fetuses dead by Day 40, whereas a further 32% failed from Days 40–60 and 15% died between Days 60–90. Cloned fetuses derived from fetal donor cells were significantly less likely to survive to Day 90 than those derived from adult donor cells (13% vs. 29%; P < 0.001). In those fetuses that survived to Day 90, placentomes were visualized on ultrasonograms from approximately Day 50 of gestation (Fig. 2).
|
|
Pregnancy rates for recipients carrying serum-fed and serum-starved NT embryos were similar (46% [25/54] vs. 44% [29/66]). Of the 10 cloned pregnancies that survived to Day 90, 6 derived from serum-starved and 4 from serum-fed cells, and 6 derived from adult and 4 from fetal cells. Of the these 10 cloned fetuses, 4 were born live, 4 aborted between 4–7 mo of gestation, and 2 were surgically removed by Day 100 of gestation for further study (both with normal placentas). Of the four live births, only one remains alive as of this writing. Two calves died by 5 days of age from cardiopulmonary problems, and one of these calves also had a peracute Clostridium perfringens gut infection. The third calf died at 1 mo of age from a chronic systemic bacterial infection. All control fetuses were born live.
CR lengths Although mean CR lengths were significantly less in NT than in ET fetuses (P < 0.05), CR length was, in fact, only less in those NT fetuses that failed to survive to Day 90 (P < 0.05; Figs. 3 and 4). Thus, no difference was found between the CR lengths of the surviving NT or non-NT fetuses (P > 0.1) when adjusted for the covariates during multiple regression.
|
PSPb Measurements
No significant difference was found between Day 30 and Day 50 PSPb values for pregnancies that failed or that did progress to Day 90 (Table 1). Serum PSPb values taken at Day 30, therefore, were not predictive of fetal survivability to Day 90. At Day 35, PSPb values were actually significantly higher (P < 0.05) in the cloned pregnancies that failed to progress to Day 90 compared with those in the ET pregnancies that would be viable at Day 90. Of the 14 cows sampled at Day 30, 11 carried cloned pregnancies (two sets of twins), and 3 carried ET pregnancies. Of the 13 cows sampled at Day 50, 7 carried cloned pregnancies (two sets of twins), and 6 carried control pregnancies (one IVP set of twins and five ET singletons).
|
Placental Examinations
The placentas from four of six NT pregnancies were abnormal, and these fetuses were small for age (Table 2). The most striking variations from normal were flat, cuboidal chorionic epithelium with a marked decrease in vascularity in two of six NT pregnancies or a reduced number of barely visible cotyledons with normal-appearing chorionic epithelium in two of six NT pregnancies (Figs. 5 and 6). The remaining two NT placentas were grossly normal, except that one of these possessed approximately half the number of grossly visible cotyledons as the age-matched controls.
|
|
Histopathology of the rudimentary cotyledonary areas from Figure 5b revealed apparently normal epithelium with villous formation (Fig. 6b). The fetus from one of these rudimentary placentas possessed a pale liver, with retarded development of the limbs and head (Fig. 7).
|
|
The two "normal" NT pregnancies possessed normal gross development of cotyledons and vasculature, other than some hemorrhagic areas in the cotyledons and chorioallantois from the Day 50 placenta (Fig. 5c). The histopathology of the two normal placentas (Days 50 and 60) closely resembled that of the controls.
DISCUSSION
This study documented that 82% of Day 30 NT fetuses did not survive to Day 90, and that these fetal deaths occurred throughout this 60-day period. These results are in agreement with the first-trimester losses reported in previous cloning experiments [1–3, 6, 9, 10, 15, 24]. More than 80% of the deaths in the current study occurred between Days 30–60, with half of these occurring between Days 30–40 and half between Days 40–60. This suggests that cloned fetuses possess varying degrees of placental development, with more advanced placentas being able to sustain fetal growth for a longer period.
Of the NT placentas examined, two were very underdeveloped, and two lacked normal cotyledon development and number. However, two appeared to be normal, although one of these possessed approximately half the expected number of cotyledons. The most underdeveloped placentas possessed flat, cuboidal chorionic epithelium, with a marked decrease in allantoic blood vessels. These two rudimentary placentas represent a placental phenotype that is inefficient enough to cause fetal death before significant placentome formation. A similar reduction in allantoic epithelial development and vascularization was also reported in preliminary studies [37–39]. A second phenotype was found in two other NT placentas, in which a reduced number of barely visible cotyledons were present whereas the chorionic epithelium appeared to be normal. These placentas appeared to be similar to those reported by Stice et al. [22], who found no placentome formation in the placentas from five of five NT fetuses that died in utero during Days 35–55. The placenta does not appear to be able to produce normal cotyledons, so the decrease in fetomaternal exchange area would cause starvation and death of the fetus around Day 60. The remaining two NT placentas appeared to be normal. However, we hesitate to call the placentas of this third group normal, because placental abnormalities occur frequently in third-trimester and term cloned fetuses [5, 6, 23]. In late term cloned fetuses, the number of placentomes may be reduced to 20% of normal among some late gestation and term cloned calves, which suggests that the completeness of placental development varies widely in cloned animals [23].
We propose that the primary cause of cloned fetus loss is placental abnormality, but fetal abnormalities are a major cause of pregnancy loss in naturally conceived pregnancies [19]. In vitro manipulation of embryos has been linked to abnormal placental development and function, and this may adversely affect an otherwise normal fetus [20, 21, 40, 41]. In our study, an NT fetus on Day 56 was undeveloped for its age and possessed an abnormal liver, which was most likely caused by fatty infiltration. Taken together, this suggests fetuses with deficient placentas that survive beyond Day 50 undergo severe nutrient deficiency that retards further growth and ends in death caused by fetal starvation.
Although we found that significantly more NT fetuses derived from adult than from fetal somatic cells survived to Day 90, this observation needs to be viewed with caution, because many factors may confound this result. One of the fetal cell lines in this experiment was subsequently found to be infected with the bovine viral diarrhea virus, which is recognized as being a cause of early embryonic death [42]. Multiple fetuses also may compromise the development of each fetus because of overcrowding [43]. This may have affected our observations, because two of the three cloned multiple pregnancies, when examined after slaughter, had small-for-age fetuses with abnormal placentas. The third multiple pregnancy showed normal development of the twin fetuses and placentas. The objective of this experiment was to gain an overview regarding the likely causes of early cloned fetal by comparing the results for several months of NT embryo transfers, but variability exists between our methodology and those of other NT studies. Important factors such as age, type and treatment of donor cells, and embryo culture methods may influence survival rates in the first trimester and neonatal viability at term. To our knowledge, the only report with high rates of first-trimester fetal survival is by Kato et al. [7], who found an 80% survival rate after ET. Other recent results in cattle have shown that 50%–60% of cloned pregnancies were lost by Day 100 [3, 15]. Our embryo culture technique may have contributed to the high rate of fetal death seen both before and after Day 90, and future experiments will use synthetic oviduct fluid media and NT techniques that have resulted in improved neonatal viability [3]. In addition, we will investigate the effect of donor cell type and treatment (e.g., age, strain, cell type, culture conditions) on placental development.
The PSPb level and CR length were measured to develop an early test for the prediction of fetal survival. Because PSPb is produced by the binucleate cells of the trophoblast [44], we investigated it as a marker for the presence of these cells. We had anticipated that small-for-age fetuses would have inadequate placentas, and that low PSPb values in maternal serum would result. However, maternal PSPb values were of no benefit in determining which pregnancies were destined to fail before Day 90. The observation that CR lengths were less only in those NT fetuses destined to fail before Day 90 is valuable, because small-for-age NT fetuses can be monitored closely to determine if they should be recovered for further experiments or if the recipient cow can be aborted and prepared for future embryo transfers.
Histopathology of the NT placentas showed a range of trophoblastic epithelial development, from low cuboidal to tall columnar. In the cloned placentas with rudimentary placentomes, villous formation was present in these areas, but the reduced number and size of these areas would cause a corresponding decrease in the placental surface area. The lack of placentome development could be the result of diminished or aberrant production of key regulatory hormones, such as insulin-like growth factors (IGFs) or abnormalities in maternal-placental cell communication [45]. Abnormalities in placentation have been observed in IGF-II knockout mice, which possess very small placentas, and in mice that overexpress IGF-II, which have increased body size and placental edema [46], all features that have been observed in cloned placentas [5, 6, 23].
In summary, we observed a range of placental phenotypes, from chorioallantoic hypoplasia through partial placental development, with a reduced number of barely visible cotyledons, and, finally, normal placental development in a minority of NT fetuses. Based on the survival plots and recovered placentas, we propose that three placental phenotypes determine the survival of cloned fetuses from Days 30–90. Within each group is variability in placental architecture that corresponds to varying degrees of placental insufficiency. Those fetuses that die before Day 45 possess placentas with grossly deficient chorionic epithelium and blood vessel formations that cannot support further fetal development. Fetuses that progress beyond this early stage have better developed trophoblastic epithelium that can maintain the nutrient supply to the rapidly growing fetus until functional placentomes are required to increase the transport of soluble nutrients. at approximately Day 50, those fetuses with subnormal placentome formation will slowly starve to death, whereas the third group that develop enough placentomes to support further growth will progress to the second trimester. These experiments provide preliminary data on which to build more intensive research efforts to identify the factors causing failure of normal placental development in cloned fetuses.
|
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance with embryo production provided by Ms. Jane Prior and the skilled embryo transfer management performed by Mr. Don Wideman and Dr. Marcelo Costa.
FOOTNOTES
First decision: 14 March 2000.
1 Supported by grants from the Department of Large Animal Medicine and Surgery (J.R.H.); the Texas Coordinating Board of Higher Education, Advanced Technology Program (M.E.W.); and grant P30-ESO09106 from the National Institute of Environmental Health Science to the Center for Environmental and Rural Health at Texas A&M University (R.C.B.). 
2 Correspondence: Jonathan Hill, Box 34, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. FAX: 607 2533531; jrh35{at}cornell.edu
Accepted: July 28, 2000.
Received: February 16, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  N. Mansouri-Attia, O. Sandra, J. Aubert, S. Degrelle, R. E. Everts, C. Giraud-Delville, Y. Heyman, L. Galio, I. Hue, X. Yang, et al. Endometrium as an early sensor of in vitro embryo manipulation technologies PNAS, April 7, 2009; 106(14): 5687 - 5692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bauersachs, S. E. Ulbrich, V. Zakhartchenko, M. Minten, M. Reichenbach, H.-D. Reichenbach, H. Blum, T. E. Spencer, and E. Wolf The endometrium responds differently to cloned versus fertilized embryos PNAS, April 7, 2009; 106(14): 5681 - 5686. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Palmieri, P. Loi, G. Ptak, and L.D. Salda REVIEW PAPER: A Review of the Pathology of Abnormal Placentae of Somatic Cell Nuclear Transfer Clone Pregnancies in Cattle, Sheep, and Mice Vet. Pathol., November 1, 2008; 45(6): 865 - 880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Everts, P. Chavatte-Palmer, A. Razzak, I. Hue, C. A. Green, R. Oliveira, X. Vignon, S. L. Rodriguez-Zas, X. C. Tian, X. Yang, et al. Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer Physiol Genomics, October 8, 2008; 33(1): 65 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. I Alexopoulos, P. Maddox-Hyttel, P. Tveden-Nyborg, N. T D'Cruz, T. R Tecirlioglu, M. A Cooney, K. Schauser, M. K Holland, and A. J French Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss Reproduction, October 1, 2008; 136(4): 433 - 445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S L Rodriguez-Zas, K Schellander, and H A Lewin Biological interpretations of transcriptomic profiles in mammalian oocytes and embryos Reproduction, February 1, 2008; 135(2): 129 - 139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wuensch, F. A. Habermann, S. Kurosaka, R. Klose, V. Zakhartchenko, H.-D. Reichenbach, F. Sinowatz, K. J. McLaughlin, and E. Wolf Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle Biol Reprod, June 1, 2007; 76(6): 983 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C J Fletcher, C T Roberts, K M Hartwich, S K Walker, and I C McMillen Somatic cell nuclear transfer in the sheep induces placental defects that likely precede fetal demise Reproduction, January 1, 2007; 133(1): 243 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lucifero, J. Suzuki, V. Bordignon, J. Martel, C. Vigneault, J. Therrien, F. Filion, L. C. Smith, and J. M. Trasler Bovine SNRPN Methylation Imprint in Oocytes and Day 17 In Vitro-Produced and Somatic Cell Nuclear Transfer Embryos Biol Reprod, October 1, 2006; 75(4): 531 - 538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-I. Chae, S.-K. Cho, J.-W. Seo, T.-S. Yoon, K.-S. Lee, J.-H. Kim, K.-K. Lee, Y.-M. Han, and K. Yu Proteomic Analysis of the Extraembryonic Tissue from Cloned Porcine Embryos Mol. Cell. Proteomics, September 1, 2006; 5(9): 1559 - 1566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R Arnold, V. Bordignon, R. Lefebvre, B. D Murphy, and L. C Smith Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos. Reproduction, August 1, 2006; 132(2): 279 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ono and T. Kono Irreversible Barrier to the Reprogramming of Donor Cells in Cloning with Mouse Embryos and Embryonic Stem Cells Biol Reprod, August 1, 2006; 75(2): 210 - 216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Constant, M. Guillomot, Y. Heyman, X. Vignon, P. Laigre, J.L. Servely, J.P. Renard, and P. Chavatte-Palmer Large Offspring or Large Placenta Syndrome? Morphometric Analysis of Late Gestation Bovine Placentomes from Somatic Nuclear Transfer Pregnancies Complicated by Hydrallantois Biol Reprod, July 1, 2006; 75(1): 122 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Somers, C. Smith, M. Donnison, D. N Wells, H. Henderson, L. McLeay, and P L Pfeffer Gene expression profiling of individual bovine nuclear transfer blastocysts. Reproduction, June 1, 2006; 131(6): 1073 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sagirkaya, M. Misirlioglu, A. Kaya, N. L First, J. J Parrish, and E. Memili Developmental and molecular correlates of bovine preimplantation embryos. Reproduction, May 1, 2006; 131(5): 895 - 904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Jouneau, Q. Zhou, A. Camus, V. Brochard, L. Maulny, J. Collignon, and J.-P. Renard Developmental abnormalities of NT mouse embryos appear early after implantation. Development, April 1, 2006; 133(8): 1597 - 1607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Armstrong, M. Lako, W. Dean, and M. Stojkovic Epigenetic Modification Is Central to Genome Reprogramming in Somatic Cell Nuclear Transfer Stem Cells, April 1, 2006; 24(4): 805 - 814. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bertolini, C. R Wallace, and G. B Anderson Expression profile and protein levels of placental products as indirect measures of placental function in in vitro-derived bovine pregnancies Reproduction, January 1, 2006; 131(1): 163 - 173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P Tveden-Nyborg, T T Peura, K M Hartwich, S K Walker, and P Maddox-Hyttel Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos Reproduction, November 1, 2005; 130(5): 681 - 694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Miles, C. E. Farin, K. F. Rodriguez, J. E. Alexander, and P. W. Farin Effects of Embryo Culture on Angiogenesis and Morphometry of Bovine Placentas During Early Gestation Biol Reprod, October 1, 2005; 73(4): 663 - 671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Dawson, T. J. King, I. Wilmut, L. M. Harkness, B. G. Kelly, and S. M. Rhind Immunohistochemical Characterization of Cloned Lamb Nephropathy J. Histochem. Cytochem., December 1, 2004; 52(12): 1657 - 1664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M Bertolini, A L Moyer, J B Mason, C A Batchelder, K A Hoffert, L R Bertolini, G F Carneiro, S L Cargill, T R Famula, C C Calvert, et al. Evidence of increased substrate availability to in vitro-derived bovine foetuses and association with accelerated conceptus growth Reproduction, September 1, 2004; 128(3): 341 - 354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. V. Dindot, P. W. Farin, C. E. Farin, J. Romano, S. Walker, C. Long, and J. A. Piedrahita Epigenetic and Genomic Imprinting Analysis in Nuclear Transfer Derived Bos gaurus/Bos taurus Hybrid Fetuses Biol Reprod, August 1, 2004; 71(2): 470 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Piedrahita, B. Mir, S. Dindot, and S. Walker Somatic Cell Cloning: The Ultimate Form of Nuclear Reprogramming? J. Am. Soc. Nephrol., May 1, 2004; 15(5): 1140 - 1144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
S. Hiendleder, K. Prelle, K. Bruggerhoff, H.-D. Reichenbach, H. Wenigerkind, D. Bebbere, M. Stojkovic, S. Muller, G. Brem, V. Zakhartchenko, et al. Nuclear-Cytoplasmic Interactions Affect In Utero Developmental Capacity, Phenotype, and Cellular Metabolism of Bovine Nuclear Transfer Fetuses Biol Reprod, April 1, 2004; 70(4): 1196 - 1205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Ohgane, T. Wakayama, S. Senda, Y. Yamazaki, K. Inoue, A. Ogura, J. Marh, S. Tanaka, R. Yanagimachi, and K. Shiota The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice Genes Cells, March 1, 2004; 9(3): 253 - 260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 K. Hochedlinger and R. Jaenisch Nuclear Transplantation, Embryonic Stem Cells, and the Potential for Cell Therapy N. Engl. J. Med., July 17, 2003; 349(3): 275 - 286. [Full Text] [PDF]
|
|
|
|
|
|
J. B. Gurdon and J. A. Byrne The first half-century of nuclear transplantation PNAS, July 8, 2003; 100(14): 8048 - 8052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wrenzycki, D. Herrmann, and H. Niemann Timing of Blastocyst Expansion Affects Spatial Messenger RNA Expression Patterns of Genes in Bovine Blastocysts Produced In Vitro Biol Reprod, June 1, 2003; 68(6): 2073 - 2080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
D. Humpherys, K. Eggan, H. Akutsu, A. Friedman, K. Hochedlinger, R. Yanagimachi, E. S. Lander, T. R. Golub, and R. Jaenisch Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei PNAS, October 1, 2002; 99(20): 12889 - 12894. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. J. Yin, T. Tani, I. Yonemura, M. Kawakami, K. Miyamoto, R. Hasegawa, Y. Kato, and Y. Tsunoda Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes Biol Reprod, August 1, 2002; 67(2): 442 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-B. Koo, Y.-K. Kang, Y.-H. Choi, J. S. Park, H.-N. Kim, K. B. Oh, D.-S. Son, H. Park, K.-K. Lee, and Y.-M. Han Aberrant Allocations of Inner Cell Mass and Trophectoderm Cells in Bovine Nuclear Transfer Blastocysts Biol Reprod, August 1, 2002; 67(2): 487 - 492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
M. M. Pace, M. L. Augenstein, J. M. Betthauser, L. A. Childs, K. J. Eilertsen, J. M. Enos, E. J. Forsberg, P. J. Golueke, D. F. Graber, J. C. Kemper, et al. Ontogeny of Cloned Cattle to Lactation Biol Reprod, July 1, 2002; 67(1): 334 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
J. G. Knott, K. Poothapillai, H. Wu, C. L. He, R. A. Fissore, and J. M. Robl Porcine Sperm Factor Supports Activation and Development of Bovine Nuclear Transfer Embryos Biol Reprod, April 1, 2002; 66(4): 1095 - 1103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.P. Enright, M. Taneja, D. Schreiber, J. Riesen, X.C. Tian, J.E. Fortune, and X. Yang Reproductive Characteristics of Cloned Heifers Derived from Adult Somatic Cells Biol Reprod, February 1, 2002; 66(2): 291 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Heyman, P. Chavatte-Palmer, D. LeBourhis, S. Camous, X. Vignon, and J.P. Renard Frequency and Occurrence of Late-Gestation Losses from Cattle Cloned Embryos Biol Reprod, January 1, 2002; 66(1): 6 - 13. [Abstract] [Full Text]
|
|
|
|
|
|
S. Tanaka, M. Oda, Y. Toyoshima, T. Wakayama, M. Tanaka, N. Yoshida, N. Hattori, J. Ohgane, R. Yanagimachi, and K. Shiota Placentomegaly in Cloned Mouse Concepti Caused by Expansion of the Spongiotrophoblast Layer Biol Reprod, December 1, 2001; 65(6): 1813 - 1821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Cross Factors affecting the developmental potential of cloned mammalian embryos PNAS, May 22, 2001; 98(11): 5949 - 5951. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
