Magnesium is a critical element for competent development of bovine embryos.

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2-2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P < 0.05). Supplementation of 1.4 mM MgCl2 into the medium increased the blastocyst rate in the ICPbio BSA group (29.4%) but not in the PVA group (5.4%; P < 0.05) to a level comparable to that of the FBS group (33.7%; P > 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P < 0.05), further maintained at 44.9% (2.1 mM) and 43.4% (2.8 mM) (P > 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P < 0.05). Comparable blastocyst development was achieved in both ICPbio BSA (30.0-33.1%) and Sigma BSA (37.4-38.7%) groups when 1.4 mM Mg2+ was supplemented regardless of its source (MgCl2 vs. MgSO4; P > 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P < 0.05). These results demonstrate that Mg2+ is a key ion that promotes competent blastocyst and term development. Therefore, a simple and efficient defined medium (CR1-Mg2+-BSA) can successfully replace complex serum and somatic cell co-culture.

Oct4 promoter activity in stem cells obtained through somatic reprogramming.

Multiple methods exist that can reprogram differentiated cells to a pluripotent state similar to that of embryonic stem cells (ESCs). These include somatic cell nuclear transfer (SCNT), fusion-mediated reprogramming (FMR) of somatic cells with ESCs, and the production of induced pluripotent stem cells (iPSCs). All of these methods yield cells in which the endogenous Oct4 gene is reactivated. We were interested in comparing the activity of the Oct4 promoter in three different classes of pluripotent cells, including normal ESCs, FMR cells (FMRCs), and iPSCs. We prepared cells of all three types that harbor a transgene composed of the mouse Oct4 promoter driving green fluorescent protein (Oct4-GFP). All cell derivations started with a characterized transgenic Oct4-GFP mouse, and from this we derived ESCs, FMRCs, and iPSCs with the Oct4-GFP transgene present in an identical genomic integration site in all three cell types. Using flow cytometry we assessed Oct4 promoter expression, cell cycle behavior, and differentiation kinetics. We found similar levels of GFP expression in all three cell types and no significant alterations in pluripotency or differentiation. Our results suggest that the pluripotent condition is a potent "local attractor" state, because it can be achieved through three vastly different avenues.

Follicular oocytes better support development in rabbit cloning than oviductal oocytes.

This study was conducted to determine the effect of rabbit oocytes collected from ovaries or oviducts on the developmental potential of nuclear transplant embryos. Donor nuclei were obtained from adult skin fibroblasts, cumulus cells, and embryonic blastomeres. Rabbit oocytes were flushed from the oviducts (oviductal oocytes) or aspirated from the ovaries (follicular oocytes) of superovulated does at 10, 11, or 12 h post-hCG injection. The majority of collected oocytes were still attached to the sites of ovulation on the ovaries. We found that follicular oocytes had a significantly higher rate of fusion with nuclear donor cells than oviductal oocytes. There was no difference in the cleavage rate between follicular and oviductal groups, but morula and blastocyst development was significantly higher in the follicular group than in the oviductal group. Two live clones were produced in follicular group using blastomere and cumulus nuclear donors, whereas one live clone was produced in the oviductal group using a cumulus nuclear donor. These results demonstrate that cloned rabbit embryos derived from follicular oocytes have better developmental competence than those derived from oviductal oocytes.

Indistinguishable transcriptional profiles between in vitro- and in vivo-produced bovine fetuses.

During the past several decades, in vitro fertilization (IVF) has been increasingly used both in animal production and human infertility treatment. Animals derived from in vitro manipulation are occasionally associated with abnormal offspring syndrome (AOS) and other developmental abnormalities. By studying gene expression of in vitro-produced (IVP) embryos/animals, we gain an indicator of how well this procedure mimics the in vivo environment. Most previous studies of this nature have focused on only a few genes at a time or have been limited to studying the pre-implantation stage; a global view of how gene transcription may be influenced by in vitro procedures during fetal development has yet to be ascertained. To this end, we collected liver and placental tissue samples from IVP and in vivo control bovine fetuses at days 90 and 180 of gestation. We used a bovine 13K oligonucleotide microarray to investigate the transcriptional profiles in both tissues from IVP fetuses, and compared them with those of their age-matched in vivo counterparts. Surprisingly, in both liver and placental tissues, the transcriptional profiles between IVP and control fetuses, at either 90 or 180 days of gestation, were indistinguishable. A total of 879 genes were found to be significantly regulated during liver development from 90 to 180 days of gestation, but there were no gene expression changes in the placental tissue during this developmental period. Quantitative real-time RT-PCR on 11 selected genes confirmed these results. Our results have certain implications for IVF technologies, both in agriculture and in human medicine.

Simulated microgravity compromises mouse oocyte maturation by disrupting meiotic spindle organization and inducing cytoplasmic blebbing.

In the present study, we discovered that mouse oocyte maturation was inhibited by simulated microgravity via disturbing spindle organization. We cultured mouse oocytes under microgravity condition simulated by NASA's rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation. While the rate of germinal vesicle breakdown did not differ between 1 g gravity and simulated microgravity, rate of oocyte maturation decreased significantly in simulated microgravity. The rate of maturation was 8.94% in simulated microgravity and was 73.0% in 1 g gravity. The results show that the maturation of mouse oocytes in vitro was inhibited by the simulated microgravity. The spindle morphology observation shows that the microtubules and chromosomes can not form a complete spindle during oocyte meiotic maturation under simulated microgravity. And the disorder of γ-tubulin may partially result in disorganization of microtubules under simulated microgravity. These observations suggest that the meiotic spindle organization is gravity dependent. Although the spindle organization was disrupted by simulated microgravity, the function and organization of microfilaments were not pronouncedly affected by simulated microgravity. And we found that simulated microgravity induced oocytes cytoplasmic blebbing via an unknown mechanism. Transmission electron microscope detection showed that the components of the blebs were identified with the cytoplasm. Collectively, these results indicated that the simulated microgravity inhibits mouse oocyte maturation via disturbing spindle organization and inducing cytoplasmic blebbing.

Altered gene expression profiles in the brain, kidney, and lung of one-month-old cloned pigs.

Although numerous mammalian species have been successfully cloned by somatic cell nuclear transfer (SCNT), little is known about gene expression of cloned pigs by SCNT. In the present study, expression profiles of 1-month-old cloned pigs generated from fetal fibroblasts (n = 5) were compared to those of age-matched controls (n = 5) using a 13K oligonucleotide microarray. The brain, kidney, and lung were chosen for microarray analysis to represent tissues from endoderm, mesoderm, and ectoderm in origin. In clones, 179 and 154 genes were differentially expressed in the kidney and the lung, respectively (fold change >2, p < 0.05, false discovery rate = 0.05), whereas only seven genes were differentially expressed in the brain of clones. Functional analysis of the differentially expressed genes revealed that they were enriched in diabetic nephropathy in the kidney, delayed alveologenesis as well as downregulated MAPK signaling pathways in the lung, which was accompanied with collapsed alveoli in the histological examination of the lung. To evaluate whether the gene expression anomalies are associated with changes in DNA methylation, global concentration of the methylated cytosine was measured in lung DNA by HPLC. Clones were significantly hypermethylated (5.72%) compared to the controls (4.13%). Bisulfite-pyrosequencing analyses of the promoter regions of differentially expressed genes, MYC and Period 1 (PER1), however, did not show any differences in the degree of DNA methylation between controls and clones. Together, these findings demonstrate that cloned pigs have altered gene expression that may potentially cause organ dysfunction.

Reprogramming of mouse fibroblasts to an intermediate state of differentiation by chemical induction.

Induced pluripotent stem cells (iPSCs) generated by forced expression of four transcription factors offer promises for regenerative and therapeutic uses in human diseases. However, it is necessary to overcome the risk of tumorigenicity caused by the use of potent oncogenes and the use of randomly integrating vectors before the iPSC technology can be applied to human medicine. Stem cells and cancer cells share many features in common, implying that there are similar underlying mechanisms in their development. Small molecules have been used to induce cell reprogramming for lineage trans-differentiation and for maintaining pluripotency of stem cells. In this study, we investigated the possibility of replacing all reprogramming viral factors with small molecules. To this end, we evaluated the effects of carcinogens at nongenotoxic levels on somatic cells. We identified 16 candidate chemicals through biology-oriented in silico high-throughput screening with commercially available inventories from Sigma-Aldrich for cancer research, and established a reprogramming protocol of 16-day treatment followed by 5 days of recovery. This protocol was applied to B6/129 mouse embryonic fibroblasts (MEFs) at passage 3. From recovery day 2, colonies appeared at an efficiency of 0.02%. These colonies were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1) at a comparable level to those of mouse embryonic stem cells (ESCs). Global gene expression analysis with a 38K gene MEEBO microarray revealed that the colonies had 564 (1.5%) differentially expressed genes compared to MEFs at day 0 of treatment, and these genes were enriched in "neuromuscular differentiation." Moreover, 122 differentially expressed genes in the colonies were ESC-enriched, including downregulated somatic markers and upregulated stem cell markers. In conclusion, combined chemical treatment of MEFs herein might have caused these cells to transverse to an intermediate state within the mesodermal lineages.

Functionality and transduction condition evaluation of recombinant Klf4 for improved reprogramming of iPS cells.

The induced pluripotent stem cell (iPSC) technology holds great potential in regenerative therapy. iPSCs could be induced by proteins (piPSC) linked with poly-arginine cell-penetrating peptides (CPPs) without the risk of genomic alteration, athough with extremely low efficiency and delayed reprogramming. We aimed to evaluate the reprogramming potency of purified mouse Klf4 proteins linked with the CPP of HIV transactivator of transcription (TAT) or Drosophila Penetratin protein at the N- or C-terminus. Eukaryotically expressed recombinant Klf4 targeted cell nucleus while the purified proteins localized in the cytoplasmic and peri-nuclear region. However, using a combined transduction of Klf4 protein and retroviruses expressing Oct4, Sox2, and c-Myc (OSM), we found both TAT- and penetratin-linked Klf4 proteins significantly induced mouse iPSC formation at the nanomolar level in 2 to 4 weeks. Klf4 protein with TAT at the N-terminus showed no reprogramming activity while the fusion protein, with Discosoma red fluorescent protein (DsRed) between TAT and Klf4, exhibited significant iPSC induction function. The iPSCs induced by Klf4 protein and retroviral OSM combinations were pluripotent. Using the protein/retroviral OSM reprogramming assay, we further evaluated Klf4 protein transduction conditions and showed that four continued transductions by purified Klf4 proteins are sufficient for effective iPSC induction. Our results demonstrated for the first time that TAT- and Penetratin-linked Klf4 proteins can effectively replace viral Klf4 in reprogramming fibroblasts, and provided a valuable strategy to evaluate recombinant proteins and transduction conditions for the improvement of piPSC induction efficiency.

Impact of phase transition on the mouse oocyte spindle during vitrification.

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.

A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.

Oocyte source and hormonal stimulation for in vitro fertilization using sexed spermatozoa in cattle.

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, P = .001). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, P = .001). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

Efficient derivation of embryonic stem cells from nuclear transfer and parthenogenetic embryos derived from cryopreserved oocytes.

Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.

Developmental stage-specific imprinting of IPL in domestic pigs (Sus scrofa).

Imprinted in placenta and liver (IPL) gene has been identified as an imprinted gene in the mouse and human. Its sequence and imprinting status, however, have not been determined in the domestic pigs. In the present study, a 259 base pair-specific sequence for IPL gene of the domestic pig was obtained and a novel SNP, a T/C transition, was identified in IPL exon 1. The C allele of this polymorphism was found to be the predominant allele in Landrace,Yorkshire, and Duroc. The frequency of CC genotype and C allele are different in Duroc as compared with Yorkshire (P = .038 and P = .005, resp.). Variable imprinting status of this gene was observed in different developmental stages. For example, it is imprinted in 1-day old newborns (expressed from the maternal allele), but imprinting was lost in 180-day-old adult (expressed from both parental alleles). Real-time PCR analysis showed the porcine IPL gene is expressed in all tested eight organ/tissues. The expression level was significantly higher in spleen, duodenum, lung, and bladder of 180-day-old Lantang adult compared to that in 1-day-old newborns Lantang pigs (P < .05). In conclusion, the imprinting of the porcine IPL gene is developmental stage and tissue specific.

Acceptance of embryonic stem cells by a wide developmental range of mouse tetraploid embryos.

Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P > 0.05) and blastocyst (16.7%, P < 0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.

Rapid elimination of the histone variant MacroH2A from somatic cell heterochromatin after nuclear transfer.

Oocytes contain a maternal store of the histone variant MacroH2A, which is eliminated from zygotes shortly after fertilization. Preimplantation embryos then execute three cell divisions without MacroH2A before the onset of embryonic MacroH2A expression at the 16-cell stage. During subsequent development, MacroH2A is expressed in most cells, where it is assembled into facultative heterochromatin. Because differentiated cells contain heterochromatin rich in MacroH2A, we investigated the fate of MacroH2A during somatic cell nuclear transfer (SCNT). The results show that MacroH2A is rapidly eliminated from the chromosomes of transplanted somatic cell nuclei by a process in which MacroH2A is first stripped from chromosomes, and then degraded. Furthermore, MacroH2A is eliminated from transplanted nuclei by a mechanism requiring intact microtubules and nuclear envelope break down. Preimplantation SCNT embryos express endogenous MacroH2A once they reach the morula stage, similar to the timing observed in embryos produced by natural fertilization. We also show that the ability to reprogram somatic cell heterochromatin by SCNT is tied to the developmental stage of recipient cell cytoplasm because enucleated zygotes fail to support depletion of MacroH2A from transplanted somatic nuclei. Together, the results indicate that nuclear reprogramming by SCNT utilizes the same chromatin remodeling mechanisms that act upon the genome immediately after fertilization.

The oocyte spindle is preserved by 1,2-propanediol during slow freezing.

OBJECTIVE: To investigate the specific changes in oocyte spindle subjected to severe challenges of low temperature, as well as to examine the effect of cryoprotectants in preserving oocyte spindle during cryopreservation. DESIGN: In vitro experimental study. SETTING: Academic research laboratory. ANIMAL(S): B6D2F1 (C57BL/6 X DBA/2) mice. INTERVENTION(S): Mouse oocytes were cryopreserved using a slow freezing method in a sodium-depleted medium with 1.5 mol/l 1,2-propanediol (PROH) and 0.3 M sucrose. To examine the spindle, oocytes were fixed before, during, and after cryopreservation, and oocytes were analyzed by immunocytochemistry and confocal microscopy. RESULT(S): The MII spindle was preserved during the slow freezing, because the cryoprotectant PROH was found to support the organization of MII spindle in resisting the subzero temperature. In contrast, the MII spindle was disassembled gradually during the thawing process with or without PROH. Most of the oocytes were able to recover the MII spindle after thawing, but a portion of thawed oocytes could not sustain the meiotic spindle because of parthenogenetic activation. CONCLUSION(S): 1,2-Propanediol can support the organization of MII spindle to defy the subphysiologic temperature; however, the PROH cannot sustain oocyte spindle structure after the subsequent thawing process.

Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation.

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate = 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate = 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-tau (IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.

Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle.

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression. The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O'Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.

Endometrium as an early sensor of in vitro embryo manipulation technologies.

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy.