Differential gene expression in bovine elongated (Day 17) embryos produced by somatic cell nucleus transfer and in vitro fertilization

Somatic cloning in cattle is associated with impaired embryo development, caused by inappropriate epigenetic reprogramming during embryogenesis; however, there is a paucity of data regarding gene expression at the critical elongation and peri-implantation stages. The objective of the present study was to identify genes differentially expressed in bovine cloned embryos at Day 17 of development (Day 0 = day of nucleus transfer or IVF). Day 7 blastocysts (Hand Made Cloned or IVP) were transferred to recipient cattle and collected at Day 17. The efficiency of recovery of elongated embryos was similar, however cloned embryos elongated less than IVP embryos (91.8 ± 45.8 vs. 174 ± 50 mm) and fewer had embryonic discs (63 vs. 83%). Qualitative and quantitative PCR detected expression of OCT4, NANOG, IFNtau, EOMES, FGF4, SOX2, and CDX2 in all IVP embryos. In most cloned embryos, NANOG and FGF4 were absent (verified by qPCR); NANOG, EOMES, and FGF4 were underexpressed, whereas IFNtau was overexpressed in cloned embryos. Based on qPCRs, other genes, i.e., SPARC, SNRB1, and CBPP22, were down-regulated in cloned embryos, whereas HSP70 and TDKP1 were overexpressed. In bovine microarrays, 47 genes (3.6%) were deregulated in cloned embryos, including several involved in trophoblast growth and differentiation. In conclusion, we inferred that these data were indicative of incomplete epigenetic reprogramming after cloning; this could lead to aberrant gene expression and subsequently early pregnancy loss. There was an apparent association between incomplete morphological elongation and aberrant reprogramming of a subset of genes critical for early embryonic development.     

Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.     

Production of a cloned calf using zona-free serial nuclear transfer

The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.     

Identification of genes aberrantly expressed in mouse embryonic stem cell-cloned blastocysts

During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.     

Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P<0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones.     

Gelatin induces trophectoderm differentiation of mouse embryonic stem cells

In this study, we selected gelatin as ECM (extracellular matrix) to support differentiation of mES (mouse embryonic stem) cells into TE (trophectoderm), as gelatin was less expensive and widely used. We found that 0.2% and 1.5% gelatin were the suitable concentrations to induce TE differentiation by means of detecting Cdx2 expression using real-time PCR. Moreover, about 15% cells were positive for Cdx2 staining after 6 days differentiation. We discovered that the expressions of specific markers for TE, such as Cdx2, Eomes, Hand1 and Esx1 were prominently increased after gelatin induction. Meanwhile, the expression of Oct4 was significantly decreased. We also found that inhibition of the BMP (bone morphogenetic protein) signalling by Noggin could promote mES cells differentiation into TE, whereas inhibition of the Wnt signalling by Dkk1 had the contrary effect. This could be used as a tool to study the differentiation and function of early trophoblasts as well as further elucidating the molecular mechanism during abnormal placental development.     

Gene profiling of cattle blastocysts derived from nuclear transfer, in vitro fertilization and in vivo development based on cDNA library

Gene expression analysis of cloned embryos would enable us to better understand the early biological events during preimplantation after NT (nuclear transfer). Routine RT-PCR and Northern-blot were limited because it could not analyze tens of thousands of genes at one time and were impeded by minimum material. Based on the developed RT-PCR methodology, we previously constructed cDNA libraries with equivalent to single embryo from the pooled AI-blastocysts (artificial insemination and in vivo developed blastocysts) of cattle. To identify gene expression profiles in NT- and IVF (in vitro fertilized)-blastocysts, and search for new candidate genes involved during this period, here we created cDNA sources from three types of blastocysts (AI-, IVF- and NT-blastocysts). The expressions of 60 genes previously identified from cDNA library were compared in three types of blastocyst. Results showed that the gene expression profile of NT-blastocysts was more similar to that of AI-blastocysts than that of created from IVF-blastocysts. Several important genes, such as Oct-4 and IFN-ι, only detected in the early embryonic development, were highly expressed in three types of blastocysts and showed no significant difference, it indicated that the donor nuclear undergone efficient reprogramming by the blastocyst stage and gained totipotential after nuclear transfer. The gene expression profiles in three types of blastocysts suggested that nuclear transfer and in vitro culture environments impaired the viability of embryos in different ways.     

Trophectoderm lineage determination in cattle

The trophectoderm (TE) and inner cell mass (ICM) are committed and marked by reciprocal expression of Cdx2 and Oct4 in mouse late blastocysts. We find that the TE is not committed at equivalent stages in cattle, and that bovine Cdx2 is required later, for TE maintenance, but does not repress Oct4 expression. A mouse Oct4 (mOct4) reporter, repressed in mouse TE, remained active in the cattle TE; bovine Oct4 constructs were not repressed in the mouse TE. mOct4 has acquired Tcfap2 binding sites mediating Cdx2-independent repression-cattle, humans, and rabbits do not contain these sites and maintain high Oct4 levels in the TE. Our data suggest that the regulatory circuitry determining ICM/TE identity has been rewired in mice, to allow rapid TE differentiation and early blastocyst implantation. These findings thus emphasize ways in which mice may not be representative of the earliest stages of mammalian development and stem cell biology.     

Cloning of the bovine antiapoptotic regulator, BCL2-related protein A1, and its expression in trophoblastic binucleate cells of bovine placenta

This report studied the identification and sequence of a full-length cDNA for the bovine BCL2 antiapoptotic family member, BCL2-related protein A1 (BCL2A1), and its localized and quantitative expression in the placenta to clarify the regulatory mechanism of trophoblast cell proliferation and differentiation during implantation and placental development. We cloned a full-length bovine BCL2A1 cDNA with 725 nucleotides and an open-reading frame corresponding to a protein of 175 amino acids. The predicted amino acid sequence shared 78% homology with human BCL2A1. All BCL2 homology domains (BH1, BH2, BH3, and BH4) in bovine BCL2A1 were conserved as well as in other mammalian BCL2A1. In the placentomes, in situ hybridization demonstrated that the BCL2A1 was limited in binucleate cells expressing various pregnancy-specific molecules like placental lactogen. BCL2-associated X protein (BAX) was also expressed in binucleate cells. Quantitative real-time RT-PCR detection exhibited a high-level expression of BCL2A1 in the conceptus at Day 21 of gestation, and it was expressed and increased in the extraembryonic membrane, cotyledon, and intercotyledon from implantation to term. BAX expression intensity increased with progression of gestation and remained elevated in postpartum. Caspase-3 protein (CASP3) and mRNA (CASP3) were detected from late gestation to postpartum in placenta as well as in the results of TUNEL detection. We believe that the apoptosis of binucleate cells may be regulated by the balance of the BCL2A1 and BAX. BCL2A1 genes produced a BCL2A1 protein in the mammalian cell-expression system. This molecule is a new candidate for antiapoptotic maintenance of the binucleate cells that support placental functions throughout gestation in bovine.     

Connexin 31 (GJB3) deficiency in mouse trophoblast stem cells alters giant cell differentiation and leads to loss of oxygen sensing

The nonphysiological placental oxidative environment has been implicated in many complications during human pregnancy. Oxygen tension can influence a broad spectrum of molecular changes leading to alterations in trophoblast cell lineage development. In this study, we report that mouse wild-type trophoblast stem cells (TSCs) react to low oxygen (3%) with an enhanced differentiation into the giant cell pathway, indicated by a downregulation of the early stem cell markers Eomes and Cdx2 as well as by a significant upregulation of Tfap2c and the differentiation markers Tpbpa and Prl3d1. Here we demonstrated that connexin 31/GJB3-deficient TSCs failed to stabilize HIF-1A under low oxygen, resulting in nonresponsiveness of different marker genes, such as Cdx2 and Eomes and Tfap2c and Tpbpa. Moreover, connexin 31-deficient TSCs revealed a shift in giant cell differentiation from Prl3d1 expressing parietal giant cells to Ctsq, Prl3b1, and Prl2c2-positive giant cells, probably sinusoidal and canal lining trophoblast giant cells. Thus, loss of connexin 31 led to different giant cell subtypes which bypass the progenitor regulators Tfap2c and Tpbpa under low oxygen conditions.