Preimplantation Stage Embryos Research Articles

Presence of Nitric Oxide Synthase Immunoreactivity and mRNA in Buffalo (Bubalus bubalis) Oocytes and Embryos

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two-, four- and eight-cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM-199 + 10% FBS + 5 μg/ml pFSH + 1 μg/ml estradiol-17β + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 μg/ml gentamycin sulphate for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2-4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross-reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi-quantitative expression of NOS genes by RT-PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre-implantation stage embryos.

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.

Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage

BackgroundLipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known.ResultsHere we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically.ConclusionsThese results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin.

Ontological aspects of pluripotency and stemness gene expression pattern in the rhesus monkey

Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative “stemness” and “pluripotency” genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.

Production of Transgenic Bovine Cloned Embryos Using Piggybac Transposition

Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

330 REDUCED HYPERACUTE REJECTION BY TRIPLE TRANSGENIC EXPRESSION OF HUMAN COMPLEMENT REGULATORY FACTORS (hDAF and hCD59) AND H-TRANSFERASE

The present study was conducted to establish a porcine transgenic cell line with human CRPs and HT genes, focused on hyperacute rejection (HAR) considering clinical xenotransplantation as alternative sources of human organs. As a first step towards establishing the stable cell line, the cDNA for 3 genes encoding human DAF, CD59, and H-transferase were cloned and sequenced. A tricistronic expression vector was constructed with the aid of 2 IRES elements (pCMV-hDAF_IRES-hHT_IRES-hCD59). The CMV-based expression vector was then introduced into miniature pig ear fibroblast cells by electroporation. Reverse transcription PCR analysis revealed that cell lines stably expressing human transgene-specific transcripts were established. The inhibitory effect of immune response in the established transgenic cell lines was measured by human serum-mediated cytolysis assay, as measured by ELISA. Under the assay conditions (based on human serum from 10 to 50%), the transgenic cell group showed significantly greater survival rate under various serum concentrations than did the nontransgenic cell control group. Moreover, the transgenic cell lines used as nuclear donors for a subsequent NT experiment were confirmed to be expressing their transgene transcripts in vitro developed preimplantation stage embryos. These results indicated that the established cell lines with human transgenes might have an inhibitory effect against lysis by human complement. It is possible that these transgenic cells could serve as nuclear donors to produce transgenic cloned pigs for xenotransplantation.

193 BOVINE EMBRYO GENOTYPING USING A 50K SINGLE NUCLEOTIDE POLYMORPHISM CHIP

Genomic tools are now available for most livestock species and are used routinely for marker-assisted selection (MAS) and genomic selection (GS) in cattle. Recently, multiple-marker detection has been achieved from biopsies of preimplantation stage embryos, thus allowing embryos to be selected before transfer (Le Bourhis et al. 2009 Reprod. Fertil. Dev. 21, 192 abst). This strategy provides the opportunity to estimate some traits of particular interest, the presence of genetic abnormalities, or both. The present work aimed to assess the efficiency of MAS/GS evaluation from biopsied bovine embryos by using the bovine 50K single nucleotide polymorphism (SNP) Illumina chip. A biopsy of 5 to 10 cells was obtained under laboratory conditions, using a microblade under a stereomicroscope, from 29 in vitro-cultured morulae and blastocysts. Biopsies were transferred individually as dry samples in tubes and sent frozen (n = 13) or at room temperature (n = 16) to the genotyping laboratory. The genomic DNA of each biopsy was amplified using a whole-genome amplification (WGA) kit according to the manufacturer’s instructions (Qiagen REPLI-g® Mini Kit, Qiagen, Valencia, CA). Following WGA, DNA concentration was determined by using PicoGreen. For subsequent genotyping, a custom CRV 50K Illumina chip was used. Call rates were calculated from 50 905 SNP. Percentage of allele drop-out (%ADO), which was estimated from the number of heterozygous markers [%ADO = (calculated hetero – observed hetero)/calculated hetero]. Parentage error was estimated from 12 embryos by using the genotypes of the parents of the embryos. Both groups of transport conditions were compared using Student’s t-test. Results are presented as mean ± SEM. A greater quantity of DNA was obtained after amplification of biopsies that were sent frozen to the laboratory when compared with those at room temperature (P < 0.05). However, the SNP call rate, %ADO, and parentage error did not differ between groups. These results indicate that genotyping from embryo biopsies following WGA can be achieved with good efficiency when using high-density marker chips. To validate the use of MAS/GS from early embryos in breeding schemes, a larger number of in vivo embryos are currently genotyped under field conditions. This will allow the reliability of this method to be assessed and the correlation between embryo and calf genetic evaluation to be quantified with the current WGA efficiency. Table 1.Amount of DNA after WGA and genotyping results

Association between the extent of DNA damage in the spermatozoa, fertilization and developmental competence in preimplantation stage embryos

To examine the fertilizing ability and DNA damage response of preimplantation stage embryos derived from the γ-irradiated mouse sperm carrying varying amounts of DNA strand-breaks. The DNA damage in the sperm was induced by exposing the testicular area to different doses of γ-radiation. After mating with healthy female mice, sperm zona binding, fertilizing ability of DNA damaged sperm and developmental competence of embryos derived from the DNA damaged sperm were assessed. The in vivo zona binding ability and fertilizing ability of DNA damaged sperm was significantly affected in the 5.0 and 10.0 Gy sperm irradiation groups. Although the development of the embryos derived from the DNA damaged sperm was not significantly affected until day 2.5 post-coitus, further development was significantly altered, as evidenced by the total cell number in the embryos. The sperm carrying DNA strand breaks still has the ability to fertilize the oocyte normally. However, the events like zona-binding and successful fertilization depend on the extent of sperm DNA fragmentation. The study has also showed a great heterogeneity in embryonic development at peri-implantation period with respect to the degree of sperm DNA damage.

Adiponectin and adiponectin receptors in the mouse preimplantation embryo and uterus

Adiponectin (Adipoq), a protein secreted by adipocytes in inverse proportion to the adipose mass present, modulates energy homeostasis and increases insulin sensitivity. Tissue Adipoq signaling decreases in settings of maternal diabetes, polycystic ovary syndrome (PCOS) and endometriosis, conditions which are associated with reproductive difficulty. Our objective was to define the expression and hormonal regulation of Adipoq and its receptors in the mouse preimplantation embryo and uterus. By real-time quantitative PCR, mRNA transcripts for Adipoq, AdipoR1, AdipoR2, Ppara, Ppard, FATP1 (SLC27A1) and acyl CoA oxidase (Acox1) were identified in mouse 2-cell and 8-cell embryos, while blastocyst stage embryos and trophoblast stem (TS) cells expressed mRNA for all genes except Adipoq. Protein expression of Adipoq, AdipoR1, AdipoR2, the insulin sensitive transporters GLUT8 (Slc2A8), GLUT12 (Slc2A12) and p-PRKAA1 was identified by immunofluorescence staining in all stages of preimplantation embryos including the blastocyst. In situ hybridization demonstrated the presence of Adipoq, AdipoR1 and AdipoR2 mRNA in the mouse decidual cells of the implantation site and in artificially decidualized cells, and the expression of these proteins was confirmed by western blotting. Flow cytometry confirmed cell surface expression of AdipoR1 and AdipoR2 in TS cells and decidual cells. These results suggest for the first time that Adipoq signaling may play an important role in preimplantation embryo development and uterine receptivity by autocrine and paracrine methods in the mouse. Implantation failures and pregnancy loss, specifically those experienced in women with maternal metabolic conditions such as diabetes, obesity and PCOS, may be the result of aberrant Adipoq and AdipoR1 and AdipoR2 expression and suboptimal decidualization in the uterus.

Immunolocalization of Methylcytosine Indicates Dynamic Global Reprogramming of this Modification During the First Two Cell-Cycles of Mouse Embryo Development.

The purpose of this study was to re-examine the pattern of cytosine methylation (5MeC) in mouse preimplantation stage embryos. It is currently considered that progressive demethylation of the genome occurs throughout the preimplantation stage and new methylation occurs at or soon after implantation. Given the essential requirement for DNA methylation for genomic stability this prolonged relative demethylation creates a biological paradox. We have therefore undertaken a reassessment of this phenomenon. The immunofluorescence staining method was used to test the 5MeC in different stages of preimplantation stage embryos. We found that global demethylation of both the paternal and maternal genome progressively occurs throughout the zygote stage and is complete by syngamy. This demethylation can be perturbed after IVF or prolonged culture. By the 2-cell stage, embryos collected direct from the oviduct showed some remethylation high levels of cytosine methylation, and interphase nuclei 4-cell and 8-cell embryos stained extensively for 5meC. The remethylation during the early 2-cell stage was inhibited by the selective DNMT inhibitor, RG108 (5 µM) - late stage zygotes where incubated for 16h with or without RG108 and the resulting 2-cell embryos stained for 5meC. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (p<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. These results indicate that the current paradigms of epigenetic reprogramming in the early embryo require reassessment. (poster)

Obésité et fertilité de la femme

Weight, fat mass and obesity have been shown to play a major role in female reproduction. Obese women have a greater risk than nonobese women of infertility and they fail to become pregnant in both natural and assisted conception cycles. This cannot be explained only by their lack of ovulation. There are several potential mechanisms. On one hand, the endometrium seems to be partially responsible for this low fecundity in obese women. On the other hand, the oocyte seems to be implied. In a model of obese mouse, maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and an increased generation of reactive oxygen species (ROS). Furthermore, compared with controls, obese mice have significantly more decreased embryonic IGF-IR staining, smaller fetuses and smaller pups. In this model, all weaned pups have been fed with a regular diet. At 13 weeks, pups delivered from obese mice were significantly larger, and these pups demonstrated early development of a metabolic-type syndrome. These findings suggest that maternal obesity has adverse effects as early as the oocyte and preimplantation embryo stages and that these effects may contribute to lasting morbidity in offspring, underscoring the importance of optimal maternal weight and nutrition before conception.

Temporal expression pattern of insulin-like growth factors (IGF-1 and IGF-2) ligands and their receptors (IGF-1R and IGF-2R) in buffalo ( Bubalus bubalis) embryos produced in vitro

The objective of the study was to assess the temporal expression of genes for IGF-1, IGF-2 and their receptors in different pre-implantation stage buffalo embryos (from oocytes to blastocyst) and to evaluate the effect of IGF-1 on culture media during in vitro buffalo embryo development. Buffalo oocytes were retrieved from slaughterhouse derived ovaries. In vitro matured oocytes were fertilized with frozen buffalo bull semen. Presumptive zygotes were cultured in modified synthetic oviductal fluid (mSOF supplemented with β-mercaptoethanol (100 μM) and without (control) or with IGF-1 (100 ng/ml) till blastocyst stage. For each treatment five replicates were taken. Supplementation of IGF-1 to embryo culture media resulted in significantly higher blastocyst formation rate (29.7% versus 25.6%; p < 0.05) than the control group. In vitro produced embryos of different stages were stored in small amount of PBS at − 70 °C till used for RNA isolation. Total RNA isolated from oocytes and pre-implantation embryos was reverse transcribed to cDNA in total volume of 25 μl reaction mixture using Moloney-Murine Leukemia Virus Reverse Transcriptase (MMLV-RT). Polymerase chain reaction (PCR) was performed using cDNA from different pools of oocytes and embryos. RT-PCR revealed that IGF-1 mRNA was neither expressed in oocytes (immature, matured and matured vitrified oocytes) nor at any pre-implantation stage embryos while mRNA for IGF-1R, IGF-2 and IGF-2R were detected in all oocytes as well as in all pre-implantation stage embryos. It can be concluded that supplementation of IGF-1 is useful for in vitro oocyte maturation and embryo development in buffalo.

Porcine Induced Pluripotent Stem Cells Produce Chimeric Offspring

Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Review: Role of tubal environment in preimplantation embryogenesis: application to co-culture assays

The culture of early preimplantation stage embryo is still delicate and the metabolic pathways of embryos are not completely understood. Embryo needs are evolutionary during the preimplantation development, consequently it is difficult to meet embryo needs in vitro. Culture conditions have to respect several physical and chemical equilibria: such as redox potential, pH, osmotic pressure, metabolic flux of energetic compounds, endogenous pools of amino acids and transcripts, etc. Embryo culture media are generally supplemented with amino acids, glucose, other energetic metabolites and antioxidant compounds, vitamin, and growth factors etc. Furthermore autocrine and paracrine regulation of embryo development probably exist. In fact embryo culture conditions have to be as non-toxic as possible. Various types of co-culture systems have been devised to overcome these problems. Complex interrelations exist between embryos and co-cultured cells. The beneficial effects of co-cultured cells may be due to continuous modifications of the culture medium, i.e. the elimination of toxic compounds and/or the supply of embryotrophic factors.

Diet-induced obesity model: abnormal oocytes and persistent growth abnormalities in the offspring.

Associations between maternal obesity and adverse fetal outcomes are well documented, but the mechanisms involved are largely unknown. Most previous work has focused on postconceptional events, however, our laboratory has shown pre- and periconceptional aberrations in maternal glucose metabolism have adverse effects on oocytes and embryos that carry on to the fetus. To demonstrate effects of maternal obesity in the pre- and periconceptional periods, we compared reproductive tissues from diet-induced obese female mice to those of control mice. Ovaries were either stained for follicular apoptosis or dissected and evaluated for oocyte size and meiotic maturation. Mice were also mated and followed for reproductive outcomes including preimplantation embryonic IGF-I receptor (IGF-IR) immunostaining, midgestation fetal growth, and midgestational placental IGF receptor 2 (Igf2r) mRNA. Delivered pups were followed for growth and development of markers of metabolic syndrome. Compared with controls, obese mice had significantly more apoptotic ovarian follicles, smaller and fewer mature oocytes, decreased embryonic IGF-IR staining, smaller fetuses, increased placental Igf2r mRNA, and smaller pups. All weaned pups were fed a regular diet. At 13 wk pups delivered from obese mice were significantly larger, and these pups demonstrated glucose intolerance and increased cholesterol and body fat suggesting early development of a metabolic-type syndrome. Together, our findings suggest maternal obesity has adverse effects as early as the oocyte and preimplantation embryo stage and that these effects may contribute to lasting morbidity in offspring, underscoring the importance of optimal maternal weight and nutrition before conception.

The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20 degrees C/min to -70 degrees C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; -7.2 degrees C) vs. 2) those undergoing IIF within 1 degrees C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80-90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from -37 degrees C to -43 degrees C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, -23.1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

A-type lamin dynamics in bovine somatic cell nuclear transfer embryos

The persistence of A-type nuclear lamin in somatic cell nuclear transfer (SCNT) embryos has been proposed as a marker for incomplete nuclear reprogramming. Using monoclonal antibodies to A/C- (A/C-346 and A/C-131C3) and B-type lamin, we compared distribution during early development of bovine IVF, parthenogenetic and SCNT embryos. A/C-346 staining was observed in the pronuclei of IVF embryos and in nuclei at the two-cell stage, but was not detected in subsequent cleavage stages up to and including hatched blastocysts. In contrast, A/C-131C3 and anti-lamin B2 stained all preimplantation stage embryos. Parthenogenetic and SCNT embryos had similar staining patterns to IVF embryos for all three antibodies, demonstrating correct nuclear architecture reprogramming. Inhibiting protein synthesis with cycloheximide (CHX) in parthenogenetic and SCNT embryos did not affect lamin A/C localisation, suggesting that lamin A/C is maternal in origin. However, activation with CHX delayed lamin A/C incorporation compared with 6-dimethylaminopurine activation. In SCNT embryos, staining for both A/C- and B-type lamin was delayed compared with parthenotes, although lamin B2 incorporation preceded lamin A/C in both. In conclusion, the lamin A/C distribution in SCNT bovine embryos paralleled that of IVF and parthenogenetic controls and therefore is not a marker of incomplete reprogramming.

Embryo culture: can we perform better than nature?

Culture of preimplantation-stage embryos has always been a key element of laboratory embryology and has contributed substantially to the success of many assisted reproduction procedures. During the past decade, its importance has increased as extended in-vitro embryo culture and single blastocyst transfer have become indispensable parts of the approach to decreasing the chance of multiple pregnancy while preserving the overall efficiency of the treatment. However, in spite of the scientific and commercial challenge stimulating research worldwide to optimize embryo culture conditions, a consensus is missing even in the basic principles, including composition and exchange of media, the required physical and biological environment and even the temperature of incubation. This review attempts to summarize the controversies, demonstrate the fragility of some widely accepted dogmas and generate an open-minded debate towards rapid and efficient optimization. New approaches expanding the traditional frames of mammalian embryo culture are also discussed. Although some researchers suppose that the efficiency of the presently applied in-vitro culture systems have already approached the biological limits, authors are confident that substantial improvement may be achieved that may expand considerably the possibilities of future assisted reproduction in humans.

160 ATTEMPTS TO CULTURE BIOPSIED CELLS FROM IN VITRO BOVINE BLASTOCYSTS FOR GENOTYPING

Genomic tools have now become available for most livestock species and are being used routinely for marker-assisted selection in cattle. One major challenge in bovine selection is the possibility to detect multiple markers from biopsies of pre-implantation stage embryos which allows to transfer only selected embryos following genotyping. Preliminary studies have shown that 2 ng of DNA collected from 200 embryonic cells (hatched blastocyst) may be sufficient for genotyping based on few markers (&lt;100). However, the present genotyping techniques are much more demanding in terms of DNA. The aim of this work was to test different in vitro culture conditions of biopsied cells issued from bovine blastocysts to produce a large number of cells for genotyping. Bovine embryos were produced in vitro according to a standard protocol (Menck M et al. 1997 Reprod. Nutr. Dev. 37, 141-150). Only grade 1 embryos were biopsied using a microblade under a stereomicroscope. Biopsies had from 5 to 10 cells. Biopsied embryos were in vitro cultured in B2 + 2.5% FCS seeded with VERO cells for 48 h to assess the survival rate. Individual biopsies were cultured in vitro in 4-well culture dishes (Nunc) coated with collagen type 1 at 39°C in a humidified air atmosphere and 5% CO2 under 3 medium conditions. Intact hatched Days 8 to 10 blastocysts were cultured under the same conditions as controls. In condition 1, 43 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 10% FCS and 0.25% ITS (insulin, rransferrin, selenium). In condition 2, 30 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 20% FCS supplemented with 1 mM sodium pyruvate, 1 μg mL-1 of heparin, and 1 μg mL-1 of FGF4. In condition 3, 30 biopsies and 43 control blastocysts were cultured in a complex medium composed of 30% of [DMEM/F12 + 20% FCS] and 70% [DMEM/F12 + 20% FCS conditioned medium using mitomycined VERO cells] supplemented with 1 mM sodium pyruvate, 1.5 μg mL-1 of heparin, and 1.5 μg mL-1 of FGF4 (adapted from Oda et al. 2006 Methods Enzymol. 419, 387-400). Medium was replaced every 3 days. Outgrowths were physically detached and isolated cells were cultured using condition 3. For further passages, monolayers were trypsinized (0.025%) and cells were analyzed by immunofluorescence using anti-cytokeratin 1-8 antibodies. After biopsy and 48 h of in vitro culture, 97.1% (100/103) of embryos survived. For all culture conditions, none of the biopsied cells attached to the coated dishes and no colony were observed after culture. Control intact blastocysts adhered and formed significantly lower rate of outgrowths for condition 1 v. 2 and 3: 77.1% v. 85.7% and 93%, respectively (P &lt; 0.05). After several passages, 3 cell lines were produced and we observed a network of cytokeratin filaments by immunofluorescence suggesting an epithelial cell type for this network. These results show that production of a large number of cells from biopsies was not efficient enough for genotyping. However, the 3 tested culture conditions are favorable for the production and multiplication of cells from intact bovine blastocysts and condition 3 seems to be a suitable medium condition for embryonic cell culture.