Nuclear Transfer Pregnancies Research Articles

Proteomics of bovine endometrium, oocytes and early embryos

Summary. Embryo-maternal communication, as well as recognition and establishment of pregnancy, is predominantly controlled by secretion of hormones and proteins. Furthermore, uterine fluid serves as a histotroph and contains factors essential for embryo development and elongation, making it a meaningful source for proteomic approaches of early embryonic development. The studies included in this review address the analysis of uterine fluid and endometrium during the oestrous cycle, early pregnancy, maternal effects on embryo development and embryo-induced alterations to the maternal environment. Clear differences between the proteome of uterine fluid and blood plasma unravelled the active contribution of the endometrium to the composition of uterine luminal fluid. Proteins were identified which may contribute either to growth enhancement of blastocysts or influence secretion of "Interferon tau". Several studies suggested important roles of intrauterine factors including "Tissue inhibitor of metalloproteinases 2" for pregnancy establishment and "Acyl-CoA-binding protein" for the embryo prior to implantation. A meta-analysis of quantitative proteome studies revealed functional clusters including the terms "degradation of extracellular matrix", "platelet degranulation" and "MHC class II antigen presentation". Proteomic studies targeting oocytes and early embryos provided evidence for the function of "Cyclin B" in mitotic resumption, for "14-3-3 epsilon" to activate "maturation promotion factor" for promotion of mitosis during in vitro maturation, or for the inhibition of proteasomes during the late phase of maturation. Analysis of blastocyst derived trophectoderm cell lines suggested effects of annexin down-regulation on the failure rate of nuclear transfer pregnancies. Finally, absolute quantification of "Y-box-binding protein 2" and "Insulin-like growth factor 2 mRNA-binding protein 3" during early embryogenesis by "selected reaction monitoring" demonstrated the dynamism of proteome alterations during the first embryonic cell divisions.

Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants.

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

Massive deregulation of miRNAs from nuclear reprogramming errors during trophoblast differentiation for placentogenesis in cloned pregnancy

BackgroundLow efficiency of Somatic Cell Nuclear Transfer (NT) has been widely addressed with high incidence of placental abnormalities due to genetic and epigenetic modifications. MiRNAs are shown to be major regulators of such modifications. The present study has been carried out to identify the expression patterns of 377 miRNAs, their functional associations and mechanism of regulation in bovine placentas derived from artificial insemination (AI), in vitro production (IVP) and NT pregnancies.ResultsThis study reveals a massive deregulation of miRNAs as chromosomal cluster or miRNA families without sex-linkage in NT and in-vitro derived IVP placentas. Cell specific localization miRNAs in blastocysts and expression profiling of embryos and placentas at different developmental stages identified that the major deregulation of miRNAs exhibited in placentas at day 50 of pregnancies is found to be less dependent on global DNA methylation, rather than on aberrant miRNA biogenesis molecules. Among them, aberrant AGO2 expression due to hypermethylation of its promoter was evident. Along with other factors, aberrant AGO2 expression was observed to be associated with multiple defects in trophoblast differentiation through deregulation of miRNAs mediated mechanisms.ConclusionThese aberrant miRNA activities might be associated with genetic and epigenetic modifications in abnormal placentogenesis due to maldifferentiation of early trophoblast cell lineage in NT and IVP pregnancies. This study provides the first insight into genome wide miRNA expression, their role in regulation of trophoblast differentiation as well as abnormal placental development in Somatic Cell Nuclear Transfer pregnancies to pave the way to improve the efficiency of cloning by nuclear transfer.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

BackgroundTo investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere.Methodology/Principal FindingsSamples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/SignificanceData suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse.

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG67kDa), and two heterologous RIA with PAG67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.

Proteomic analysis of the major cellular proteins of bovine trophectoderm cell lines derived from IVP, parthenogenetic and nuclear transfer embryos: Reduced expression of annexins I and II in nuclear transfer-derived cell lines

Trophectoderm cell lines were established from 8-day in vitro-cultured embryos of cattle derived from fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 5 V-, 16 NT-, 12 P-, and 16 IVF-derived cell lines were compared by 2D-gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. Common protein spots ( n = 118) were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominent cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, anti-oxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexins I and II. In comparative analysis of the 2D-gels, the NT-derived trophectoderm had less annexins I and II in comparison to the IVF- and P-derived trophectoderm. Because annexins I and II are abundant in the placenta and have functions important to the maintenance of placentation, the down-regulation of the annexin genes in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.

Large Offspring or Large Placenta Syndrome? Morphometric Analysis of Late Gestation Bovine Placentomes from Somatic Nuclear Transfer Pregnancies Complicated by Hydrallantois

Somatic nuclear transfer (NT) in cattle is often complicated by fetal oversize (i.e., large offspring syndrome), hydrallantois, and placentomegaly in late gestation. The aims of this work were to obtain data on the placentome structure in NT-recipient cows with hydrallantois (NTH) and to relate these with fetal and placental weights to better understand the abnormalities observed in NTH pregnancies during the third trimester. Pregnant cows were slaughtered between Gestation Days 180 and 280. The fetuses were weighed, and the placentomes were numbered and weighed. Placentomes were examined by histologic and stereological techniques. Macroscopic data showed that placental overgrowth preceded fetal overgrowth, and the ratio of the fetal to the total placentome weight in the NTH group was lower than that in controls after Gestation Day 220. This suggests that placental overgrowth is due to placental default rather than due to fetal overgrowth, as shown also by stereological analysis showing primary deregulation of the growth of cotyledonary tissues. Observed alterations, such as thinning of the maternal epithelium within placentomes and increased trophoblastic surface, could be secondary adaptations. Thus, placental growth deregulations would be due to modifications of the expression of placental factors. Various examples of placental deficiency were observed, suggesting that some fetal abnormalities observed in NTH calves, such as enlarged heart, enlarged umbilical cord, and abdominal ascites, are consequences of placental dysfunction. Therefore, the condition described by the term "large offspring syndrome" might better be described by "large placenta syndrome," because this syndrome affects an average of 50% of late-gestation NT pregnancies. No conclusion can be drawn from this work on apparently normal pregnancies.

73 PROTEOMIC ANALYSIS OF THE MAJOR CELLULAR PROTEINS OF BOVINE TROPHECTODERM CELL LINES DERIVED FROM IVP, PARTHENOGENETIC, AND NUCLEAR TRANSFER EMBRYOS: REDUCED EXPRESSION OF ANNEXIN I IN NUCLEAR TRANSFER-DERIVED CELL LINES

Trophectoderm cell lines were established from 8-day in vitro cultured bovine embryos that were derived from the fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 2 V-, 16 NT-, 12 P-, and 13 IVF-derived cell lines were compared by 2-D gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. One-hundred and eighteen in common protein spots were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominently expressed cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, antioxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexin I and II. In comparative analysis of the 2-D gels, the NT-derived trophectoderm showed diminished annexin I expression in comparison to the IVF-derived trophectoderm. Since annexin I is abundantly expressed in the placenta and has functions important to the maintenance of placentation, the down-regulation of annexin I in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.

Expression of TGF-β1, TGF-β2, TGF-β3 and the Receptors TGF-βRI and TGF-βRII in Placentomes of Artificially Inseminated and Nuclear Transfer Derived Bovine Pregnancies

Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

Perturbations in the Biochemical Composition of Fetal Fluids Are Apparent in Surviving Bovine Somatic Cell Nuclear Transfer Pregnancies in the First Half of Gestation1

Amniotic and allantoic fluid volumes and composition change dynamically throughout gestation. Cattle that are pregnant with somatic cell nuclear transfer (NT) fetuses show a high incidence of abnormal fluid accumulation (particularly hydrallantois) and fetal mortality from approximately midgestation. To investigate fetal fluid homeostasis in these pregnancies, Na, K, Cl, urea, creatinine, Ca, Mg, total PO(4), glucose, fructose, lactate, total protein, and osmolalities were measured in amniotic and allantoic fluids collected at Days 50, 100, and 150 of gestation from NT pregnancies and those generated by the transfer of in vitro-produced embryos or by artificial insemination. Deviations in fetal fluid composition between NT and control pregnancies were apparent after placental and fetal organ development, even when no gross morphological abnormalities were observed. Individual NT fetuses were affected to varying degrees. Elevated allantoic Na was associated with lower K and increased allantoic fluid volume or edema of the fetal membranes. Total PO(4) levels in NT allantoic and amniotic fluid were elevated at Days 100 and 150. This was not accompanied by hypophosphatemia at Day 150, suggesting that PO(4) acquisition by NT fetuses was adequate but that its readsorption by the kidneys may be impaired. Excessive NT placental weight was associated with low allantoic glucose and fructose as well as high lactate levels. However, the fructogenic ability of the NT placenta appeared to be normal. The osmolality of the fetal fluids was maintained within a narrow range, suggesting that the regulation of fluid composition, but not osmolality, was impaired in NT pregnancies.

70 RECONSTRUCTED BOVINE BLASTOCYSTS COMPRISING NUCLEAR TRANSFER-DERIVED INNER CELL MASS AND TROPHECTODERM FROM IVF EMBRYOS DO NOT IMPROVE IN VIVO DEVELOPMENT OF CLONES

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome) and fetal overgrowth (Lee RSF et al. 2004 Biol. Reprod. 70, 1–11). Early embryonic loss in bovine NT pregnancies may also be due to immunological rejection (Hill JR et al. 2002 Biol. Reprod. 67, 55–63). As a means of overcoming placental abnormalities and improving pregnancy outcome in bovine NT, reconstructed blastocysts were produced by combining immunosurgically isolated inner cell masses (ICM) from Day 7 NT embryos with the trophectoderm (TE) of Day 7 IVF embryos. Oocytes for the production of NT and IVF embryos were obtained from abattoir-collected ovaries of dairy cows. The semen used for IVF was from the bull from which the cell line for NT was derived. The NT blastocysts were produced as described previously (Oback B et al. 2003 Cloning Stem Cells 5, 3–12) except that two one-cell embryos were aggregated together after NT (2NT). Blastocyst reconstruction was achieved using a modified procedure (Rorie RW et al. 1994 Vet. Record 135, 186–187). Embryos from four experimental groups were transferred individually to synchronized recipient heifers on Day 8 of culture: (1) ICM from 2NT embryos reconstructed with IVF TE (R-2NT, n = 15); (2) ICM from IVF embryos reconstructed with IVF TE (R-IVF, n = 15); (3) control 2NT (n = 10); and (4) control IVF (n = 10). Pregnancy rates were recorded and treatments compared using Fisher's exact test. After slaughter between Days 149 and 161 of gestation, morphometric measurements were determined for the fetuses, fetal organ weights, fluid volumes, and placentomes. Data were rank transformed; treatments were compared using Student's t-test with standard errors calculated from the pooled variation. Pregnancy rates on Day 35 were R-2NT (60%), R-IVF (47%), 2NT (90%), and IVF (10%). Pregnancy rates on Day 150 were R-2NT (40%), R-IVF (40%), 2NT (70%), and IVF (10%). The reason for the low IVF pregnancy rate was unknown. Previously, pregnancy rates using the same sire and cell line (but using Day 7 embryo transfer) on Day 35 were 63% (n = 40) and 69% (n = 42) for IVF and single, non-aggregated NT, respectively, and 50% and 33% for IVF and NT on Day 150. The single NT pregnancy rate was not significantly different from that for the 2NT embryos. There was no significant difference in pregnancy rates on Day 35 and Day 150 between R-2NT v. 2NT, R-2NT v. R-IVF, or 2NT v. R-IVF. The blastocyst reconstruction procedure did not have any impact on fetal development or influence pregnancy rates. All fetuses recovered were male. No significant differences were found between R-2NT and 2NT fetuses in terms of fetal weight, fluid volume, total placentome weight, and placentome numbers or in the relative and absolute weights of the brain, heart, liver, and kidneys. Thus, replacement of the TE in NT embryos with TE from IVF embryos did not overcome placental abnormalities or decrease fetal overgrowth prevalence.

Altered placental lactogen and leptin expression in placentomes from bovine nuclear transfer pregnancies.

Appropriate growth, development, and function of the placenta is central to the success of nutrient partitioning between the mother, placenta, and fetus. Hormones such as placental lactogen (PL) and leptin are produced in the bovine placenta and play an important role in nutrient partitioning and regulation of placental and fetal growth. Nuclear transfer pregnancies are associated with a number of fetal and placental abnormalities, including increased placental growth and macrosomia, and hence represent a unique situation to gain insight into fetoplacental growth regulation. We have examined the expression of bovine PL (bPL) and leptin in placentomes of artificially inseminated (AI), in vitro produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation in the cow. Immunolocalization studies showed that spatial and temporal patterns of expression of bPL and leptin were markedly altered in the placentomes of NT pregnancies compared with AI or IVP controls. Concentrations of bPL in allantoic fluid, as determined by radioimmunoassay (RIA), were significantly higher (P < or = 0.001) in NT pregnancies (17.9 +/- 3.2 ng/ml; mean +/- SD) compared with AI (2.03 +/- 1.5 ng/ml), but not IVP (23.4 +/- 12.8 ng/ ml) pregnancies on Day 150 of gestation. In contrast, amniotic fluid levels of bPL were significantly decreased in NT pregnancies at Day 150 gestation. Leptin mRNA expression, as determined by real-time reverse transcription-PCR, was increased 2.4- to 3.0-fold in NT placentomes compared with AI controls at all gestational ages examined. We speculate that the observed dysregulation of expression of bPL and leptin in NT placentomes could contribute to aberrations in cell migration and invasion and subsequently to alterations in placental metabolism and transfer of nutrients to the fetus, thus leading to increased placental and fetal macrosomia in NT pregnancies.

Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses.

Somatic cell nuclear transfer (NT) in cattle is an inefficient process, whereby the production of calves is hindered by low pregnancy rates as well as fetal and placental abnormalities. Interspecies models have been previously used to facilitate the identification of single nucleotide polymorphisms (SNPs) within coding regions of genes to discriminate between parental alleles in the offspring. Here we report the use of a bovine interspecies model (Bos gaurus x Bos taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in Day 40-old female NT-derived fetuses and placenta. Analysis of NT and control pregnancies indicated disruption of genomic imprinting at the X inactivation-specific transcript (XIST) locus in the chorion, but not the fetus of clones, whereas proper allelic expression of the insulin-like growth factor II (IGF2) and gene trap locus 2 (GTL2) loci was maintained in both the fetus and placenta. Analysis of the XIST differentially methylated region (DMR) in clones indicated normal patterns of methylation; however, bisulfite sequencing of the satellite I repeat element and epidermal cytokeratin promoter indicated hypermethylation in the chorion of clones when compared with controls. No differences were detected in methylation levels in the fetus proper. These results indicate that the nuclear transfer process affects gene expression patterns in the trophectoderm- and inner cell mass-derived tissues to different extents.

40PREDICTORS OF CLONED CALF VIABILITY

The high rate of gestational loss in nuclear transfer (NT) pregnancies is a major economic and animal cost, preventing the widespread use of the technique. It is currently not possible to predict which early gestation fetuses will remain viable to term. As placental abnormalties are commonly associated with nonviable fetuses, placental proteins such as pregnancy specific protein (PSP 60, Heyman et al., 2002 Biol. Reprod. 63, 1787–1794; or PSPb Hill et al., 2000 Biol. Reprod. 66, 6–13) have been investigated as possible markers of viable pregnancy. To build upon these studies, we explored the predictive value of Day-35 maternal serum PSPb value to final cloned-calf viability; PSPb profiles throughout pregnancy; and the relationship of maternal plasma estradiol levels to calf viability. Maternal serum was sampled for PSPb at the time of the first ultrasound pregnancy examination (Day 35) to determine fetal number and viability (heartbeat). PSPb values were determined using an RIA by Dr. Garth Sasser (BioTracking, Moscow, ID). Median PSPb values were compared using the Kruskal-Wallis one-way ANOVA test on ranks. PSPb levels from failed single and twin pregnancies were significantly different from those of AI bred controls (P&amp;lt;0.05). PSPb levels from failed singles were not significantly different from those of viable single pregnancies although only nonviable cloned pregnancies had PSPb values &amp;gt;7.5. As PSPb levels at Day 35 were not highly predictive of viable outcome, we detailed the gestational PSPb profile for a different group of cows (n=40) that carried cloned pregnancies beyond Day 90. PSPb was determined at Days 35, 50, 65, and 90, then monthly to Day 240. Although there was no clear statistical significance, PSPb levels from nonviable cloned pregnancies were consistently higher at each time point than for viable pregnancies (14 cows gave birth to live calves) and significantly higher than those of controls. At term, plasma estradiol concentrations were assessed in a group of 5 NT pregnancies. Estradiol was investigated as cloned pregnancies often show a lack of readiness for parturition (e.g. lack of udder development, prolonged gestation, dysmature calves). These 5 pregnancies produced 3 highly viable NT calves: 1 with lowered viability that survived only with a high level of intensive care, and 2 nonviable despite a high level of assistance. In this small group, estradiol levels on the day of birth rose in parallel with viability (viable, 662pg; lowered viability, 170pg; nonviable, 76pg). This is a clinical observation rather than a statistical trend that suggests further investigation may be warranted. In summary, our results suggest maternal PSPb levels have value in identifying the outliers in a population of cloned pregnancies and that term-cloned pregnancies may show abnormally low maternal estradiol concentrations at term. Table 1

52GREATER PHENOTYPIC VARIABILITY IN CLONED CATTLE FETUSES FROM ONE CELL LINE THAN CONTEMPORARY HALF-SIBS GENERATED BY ARTIFICIAL INSEMINATION OR IN VITRO EMBRYO PRODUCTION

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with considerable variation in developmental abnormalities. Some of this variation may be due to the cell types/lines used and the specific production and culture methods for NT embryos. Fetal and placental development were studied in 24 pregnancies generated by NT (Wells et al., 2003 Theriogenology 59, 45–59) from a granulosa cell line. The controls consisted of 11 and 14 pregnancies resulting from in vitro-produced embryos (IVP) and artificial insemination (AI), respectively. All fetuses shared the same Friesian sire; oocytes for the derivation of NT or IVP embryos were obtained from abattoir-derived ovaries of Friesian cows. Morphometric measurements were made on the fetuses, fetal membranes, fluid volumes and placentomes at Days 50, 100 and 150 of gestation after slaughter. Pairwise comparisons of within group variances between the treatment groups were made using the F-test. The pregnancy loss from AI or embryo transfer to Day 150 was ∼40% for the AI and IVP groups. However, 60% of NT embryos transferred had been lost by Day 150. NT fetal weights at Day 100 were more variable than for the AI (P&amp;lt;0.001) and IVP (P&amp;lt;0.05) groups. The NT crown-rump lengths were also more variable compared with the IVP (P&amp;lt;0.05) but not the AI group. At Day 100, NT heart (P&amp;lt;0.01), kidney (P&amp;lt;0.01) and liver (P&amp;lt;0.05) weights were more variable compared with the AI group; both liver (P&amp;lt;0.05) and kidney (P&amp;lt;0.01) weights were similarly more variable in the Day 150 NT group. Part of this variability may be due to disproportionate organ growth in NT fetuses. Increased variability was most evident in the utero-placental tissues. At Day 50, the total fetal membrane weights were more variable in the NT compared with the AI (P&amp;lt;0.001) but not the IVP group. The Day 50 IVP membrane weights were also more variable (P&amp;lt;0.05) than for the AI group. The greater variability of the Day 150 NT membrane weights, when compared with AI (P&amp;lt;0.05) or IVP (P&amp;lt;0.01), could be due partly to greater variation in the allantoic and amniotic fluid volumes. Placentome numbers were significantly lower in the surviving Day 100 NT pregnancies but the weights of the total maternal caruncular tissue of the placentomes were significantly higher and more variable in the NT group (P&amp;lt;0.05 for both AI and IVP). Placentome numbers in surviving NT pregnancies at Day 150 were similar to the controls. However, the total caruncle weights in both the NT and IVP groups were more variable when compared with the AI group (P&amp;lt;0.05 for both). Thus, there is greater variability in fetal membrane and placental development in NT and, to a certain extent, IVP fetuses, when compared with the AI group. The increased variability within this NT group suggests that epigenetic effects arising from incomplete reprogramming of the donor genome and embryo culture can override genetic traits to a certain extent. Supported by FRST C10X0018.

42ANGIOGENESIS IN CLONED AND IVF-DERIVED BOVINE PREGNANCIES AT DAY 30 OF GESTATION

New and expanded microvascular networks in maternal and embryonic tissue are vital to development of a functional placenta. Typically a large proportion of bovine pregnancies derived from somatic cell nuclear transfer (NT) are lost between Days 25 and 45 of gestation. Several laboratories have noted aberrant placental development of NT embryos and fetuses; one factor that may contribute to these defects is faulty maternal or embryonic angiogenesis during placentation. The aim of this study was to determine if angiogenesis in maternal caruncular tissues differs in bovine NT- and IVF-derived pregnancies at the time of placentation. Cloned embryos were produced using cultured ear skin fibroblasts from a 15-year-old Hereford cow; in other experiments, these donor cells resulted in a term live calf and 67% of viable Day-30 embryos dying by Day 51 of gestation. For IVF-derived embryos, IVM oocytes were fertilized with semen from Hereford or Angus bulls. All embryos were cultured in vivo in ligated sheep oviducts, and at Day 8 blastocysts were transferred to synchronized recipient heifers. Whenever possible, one embryo was transferred to each uterine horn. A total of 41 NT and 41 IVF grade 1, 2 and 3 embryos were transferred to 48 recipients. Viable NT (n=9; 22.0% of transferred) and IVF (n=9; 22.0%) embryos were recovered at Day 30, and caruncular tissue adjacent to each embryo was sampled. Expression of genes implicated in angiogenesis was measured by real-time quantitative RT-PCR and normalized to histone 2A expression. In addition, paraformaldehyde-fixed sections were stained by the Periodic Acid-Schiffs method to identify blood vessels. Microvascular density was determined as percentage of total tissue area composed of blood vessels in the luminal caruncular stroma as measured by two observers. Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Institute, Cary, NC, USA). Quantification of gene expression revealed no differences between NT and IVF pregnancies for angiogenesis-promoting growth factors and their receptors: vascular endothelial growth factor-A (VEGF-A), VEGF-C, placental growth factor, VEGF receptor-1 (VEGFR-1/flt-1), VEGFR-2 (flk-1/KDR), VEGFR-3 (flt-4), angiopoietin-1 (Ang-1), and Ang receptor-1 (tie-1). Likewise, NT and IVF pregnancies demonstrated similar mean microvascular densities in the caruncles; however, density differed between individual pregnancies across both groups (P&amp;lt;0.05). For these experiments, the timing of NT and IVF, embryo transfer, and tissue collection on Day 30 was as consistent as possible. Despite these efforts, embryo morphology and developmental stage were highly variable even within the IVF group. It follows that the progress of placentation, and hence the signaling between each embryo and the maternal tissues, may have differed as well, accounting for the variation observed. Our results suggest that failure of maternal tissue to increase angiogenesis during placentation is not a primary cause of aberrant placental development in cloned cattle.

31EFFECTS OF CYCLOHEXIMIDE ON CAPRINE SOMATIC CELL NUCLEAR TRANSFER EMBRYO AND FETAL DEVELOPMENT

The use of protein synthesis inhibitors to down regulate the levels of Maturation Promoting Factor (MPF) following fusion and activation are widely used in the field of Nuclear Transfer (NT). Cycloheximide is a protein synthesis inhibitor that blocks the levels of cyclin B, a component of MPF which is required to maintain MII stage arrest in oocytes. However, it is unclear what, if any, effects these broadbased inhibitors may have on nonspecific protein expression in the oocytes or karyoplasts. The purpose of this study was to examine the effects of treatment with and without cycloheximide on embryo and fetal development to term. Ovulated in vivo MII oocytes from superovulated does during the traditional breeding season were surgically collected and then enucleated and reconstructed with either transfected fetal or adult skin cell karyoplasts or transgenic primary somatic skin cells. Couplets were simultaneously fused and activated with a single electrical pulse between 2.6 and 3.0kVcm−1 for 20μs. An additional electrical pulse was given to fused couplets and non-fused couplets were re-fused. Reconstructed embryos were either treated with 5μgmL−1 cyclohexamide (Sigma, St. Louis, MO, USA) for a minimum of 3h or cultured directly post-fusion and activation. The embryos were cultured in SOF +BSA at 38°C in a humidified modular incubation chamber (Billrups-Rothenberg, USA) containing 6% O2, 5% CO2, and 89% N2 for 24–48h. Cleavage was assessed, and nuclear transfer embryos, with age appropriate development (up to 2 cells at 24h or 2 to 8 cells by 48h), were surgically transferred to the oviduct of surrogate recipient does. There were 18 and 11 confirmed pregnancies by Day 50 post-fusion and activation from the cycloheximide and non cycloheximide groups, respectively. A total of 12 recipients that received cycloheximide treated embryos produced term pregnancies and yielded 15 offspring. Alternatively, 9 recipients that received untreated embryos produced term pregnancies which yielded 12 offspring. A total of 27 NT offspring were produced and one offspring from a set of quadruplets died several days post-natally. While significantly more cycloheximide treated NT embryos cleaved, there was no significant difference in pregnancy rate or offspring born between treatment groups. These results suggest that the use of cycloheximide for embryo development in somatic cell nuclear transfer may not be necessary for establishing and maintaining caprine NT pregnancies. Table 1

Insulin-like growth factor-I and binding proteins 1, 2, and 3 in bovine nuclear transfer pregnancies.

In cloned pregnancies, placental deficiencies, including increased placentome size, reduced placentome number, and increased accumulation of allantoic fluid, have been associated with low cloning efficiency. To assess differences in paracrine and endocrine growth regulation in cloned versus normal bovine placentomes and pregnancies, we have examined the expression of insulin-like growth factor (IGF)-I and -II and their binding proteins (IGFBP)-1 through -3 in placentomes of artificially inseminated (AI), in vitro-produced (IVP), and nuclear transfer (NT) pregnancies at Days 50, 100, and 150 of gestation. Fetal, maternal, and binucleate cell counts in representative placentomes were performed on Days 50-150 of gestation in all three groups. Increased numbers of fetal, maternal, and binucleate cells were present in NT placentomes at all stages of gestation examined. Immunolocalization studies showed that spatial and temporal patterns of expression of IGFBP-2 and -3 were markedly altered in the placentomes of NT pregnancies compared to AI/IVP controls. Concentrations of IGF-I in fetal plasma, as determined by RIA, were significantly higher (P = 0.001) in NT pregnancies (mean +/- SEM, 30.3 +/- 2.3 ng/ml) compared with AI (19.1 +/- 5.5 ng/ml) or IVP (24.2 +/- 2.5 ng/ml) pregnancies on Day 150 of gestation. Allantoic fluid levels of IGFBP-1 were also increased in NT pregnancies. These findings suggest that endocrine and paracrine perturbations of the IGF axis may modulate placental dysfunction in NT pregnancies. Furthermore, increased cell numbers in NT placentomes likely have significant implications for fetomaternal communication and may contribute to the placental overgrowth observed in the NT placentomes.