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We recently developed and validated a methodology based on early morphokinetics to predict 6 categories of bovine embryos, including 4 categories of blastocysts: early hatching blastocysts (EHB), hatching blastocysts (HB), low hatching blastocysts (LHB), and arrhythmic blastocysts (AB) (Reis et al. 2018 Anim. Reprod. 15, 601). We hypothesised that different early morphokinetic pathways could (1) be accompanied by different biological patterns (EGA, etc.) and (2) influence interaction with environmental constraints (cryopreservation, etc.). The objective of this study was (1) to investigate whether transcriptomic differences between the predicted EHB, HB, LHB, or AB could be observed immediately after the fourth embryonic cycle (just after EGA); (2) to assess cryotolerance of these morphokinetic categories. Time lapses were produced during 4 days post invitro insemination (dpi) (Reis et al. 2018). Study 1: 128 embryos having finished the fourth embryonic cycle were individually dry frozen at 4.7 dpi, classified as EHB, HB, LHB, or AB, and pooled into four batches of eight embryos/category (total 16 samples). Total RNA was extracted, amplified using the SMARTseq V4 ultralow input kit (Clontech), libraries were prepared using the Nextera XT Illumina library preparation kit, and sequenced (paired-end 50-34bp) on an Illumina NextSEqn 500 instrument. Identification of differentially expressed genes was achieved using Limma package (R). The P-values were adjusted using the Benjamini and Hochberg false discovery rate (Saenz-de-Juano et al. 2014). Fold change >2 or 135µm and −2µm at zona pellucida) were slow frozen at 6.2 or 7 dpi, classified as EHB (n=8), HB (n=29), LHB (n=32), or AB (n=21), and further thawed and cultured invitro for 72h. Cryotolerance was evaluated at 24 and 72h post-thawing. Statistical analysis was performed using chi-square or Fisher test. Study 1: the AB category presented 258, 699, and 899 upregulated genes and 40, 62, and 60 downregulated in comparison to LHB, HB, and EHB, respectively. The functional comparison of the two extremes (EHB vs. AB), at 4.7 dpi, showed that AB embryos present intense transcription regulation activity. The monocarboxylic acid binding pathway was upregulated in EHB. Study 2: the post-thawing invitro survival was similar between EHB, HB, LHB, and AB (50%, 34.5%, 31.3%, and 42.9%, respectively). The hatching/alive ratio in EHB category tended to be lower than AB (1/4 vs. 8/9; P=0.052). Frozen/thawed EHB and HB presented reduced hatching rates, whereas LHB and AB significantly increased the hatching potential after thawing compared with their not-frozen counterparts of the learning database (25% vs. 100%; 50% vs. 100%; 70% vs. 11%; 88.9% vs. 18%, respectively). Morphokinetic prediction highlighted a new classification of bovine embryos with different biological features. This could contribute to improve practice on IVF embryo transfer taking into account these differences.

Remerciements à l'Unité UCEA de Bressonvilliers, à V. Gelin et A. Neveu pour leur aide.; Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2. Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2. Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.

In a previous work, we proposed an algorithm to select a subset of discriminant morphokinetical parameters within a larger predefined set (116 parameters) and to predict 6 major bovine embryo profiles of in vitro development. The algorithm relies on flexible combinations of the discriminant parameters selected within the four first cleavage cycles. The retained profiles were arrested embryos (embryos without mitotic activity, showing signs of life); dead embryos (embryos with all cells dead); anarchic embryos (embryos with abnormal morphological and/or kinetical development: some of these embryos can result in a blastocyst); not hatched blastocysts (blastocysts not hatching by 8 dpi); hatching blastocysts (blastocysts hatching in vitro from 7.3 to 8 dpi); and early hatching blastocysts (blastocysts hatching from 6 to 7.2 dpi) (Reis et al. 2018 Anim. Reprod.). The aim of the present work was to develop a ready-to-use software (EasyPickAndPredict) allowing the extension of this methodology to other embryologists. The software of high portability was built with the JAVA language and embedded with the classifier (R language, R Foundation for Statistical Computing, Vienna, Austria) and a user’s help for annotation (Adobe Premiere Pro CS5, San Jose, CA, USA). The predictive software is easy to handle, fast to load, and has high portability. The “manual annotation” function is based on click actions to annotate the discriminant parameters. The “prediction” function calls the embedded classifier. The “report” function creates customised reports including the embryo classification, the summary of the measures, and the accuracy of the prediction (vote system). The “help” function calls an audiovisual guideline with annotation specifications for all the morphokinetic parameters currently described (including the discriminant subset of parameters). This document includes help to produce a good time-lapse video (16min 51s); annotation specifications for the cleavage cycles 1 to 4 (36 min 26s); and a summary with examples of the 6 major embryonic morphokinetical profiles (16min 47s). The predictive graphical interface is easy to manipulate and automatically extracts the value of each discriminant parameter on the time-lapse picture as validated by the user click. The functions “manual annotation”, “automatic prediction”, “help”, and “report” supply embryologists with a standard approach to predict and analyse morphokinetical profiles of their embryos. This standardised approach is necessary to improve the capacity of comparison of morphokinetical works in different laboratories and enhance knowledge about in vitro-produced bovine embryos.

The somatic cell nuclear transfer (SCNT) procedure requires nuclear remodeling to return differentiated somatic nuclei to the totipotent undifferentiated stage. We hypothesize that mechanical constraints might occur upon SCNT and thereby affect nuclear remodeling. Therefore, we analyzed the nuclear structures upon SCNT using as donors either wild-type fibroblasts with a dense vimentin network or vimentin-deprived cells [embryonic stem cells (ESCs) and fibroblasts invalidated for vimetin]. We demonstrated that following nuclear transfer of wild-type fibroblasts, vimentin intermediate filaments (IFs) persisted around the transplanted nuclei and 88% of them presented severe distortions. We also showed that the presence of vimentin filaments in the reconstructed embryos was correlated with DNA damage, as evidenced by γH2A.X foci. On the other hand, when ESCs or vimentin-null (Vim(-/-)) fibroblasts devoid of IFs were used as nuclear donors, no nuclear distortion and less DNA damage were observed. Altogether we believe that the introduction of vimentin into recipient oocytes during SCNT induces a mechanical constraint on the transplanted nucleus that is responsible for nuclear distortions and DNA damage. This could lead to incomplete reprogramming that would be detrimental to further embryonic development.

The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (?60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage.

Notice à reprendre pour la pagination officielle; In in vitro-produced (IVP) bovine embryos, a burst in transcriptional activation of the embryonic genome (EGA) occurs at the 8-16-cell stage. To examine transcriptional regulation prior to EGA, notably in response to heat stress, we asked (1) whether the spontaneous expression of a luciferase transgene that is driven by the minimal mouse heat-shock protein 1b (hspa1b) gene promoter paralleled that of HSPA1A during EGA in IVP bovine embryo and (2) whether expression of the endogenous heat-inducible iHSPA group member HSPA1A gene and the hspa1b/luciferase transgene were induced by heat stress (HS) prior to EGA. Using two culture systems, we showed that luciferase activity levels rose during the 40-h long EGA-associated cell cycle. In contrast, iHSPA proteins were abundant in matured oocytes and in blastomeres from the two-cell to the 16-cell stages. However, normalised results detected a rise in the level of HSPA1A and luciferase mRNA during EGA, when transcription was required for their protein expression. Prior to EGA, HS-induced premature luciferase activity and transgene expression were clearly inhibited. We could not, however, establish whether this was also true for HSPA1A expression because of the decay of the abundant maternal transcripts prior to EGA. In bovine embryos, heat-induced expression of hspa1b/luciferase, and most likely of HSPA1A, was therefore strictly dependent on EGA. The level of the heat-shock transcription factor 1 molecules that were found in cell nuclei during embryonic development correlated better with the embryo's capacity for heat-shock response than with EGA-associated gene expression.

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International audience; Pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. In bovine, however, little information is available about dynamics of pluripotency genes during these processes. In this study, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, having similar in vitro but different full term development rates. Our findings affirm: firstly, the core triad of pluripotency genes is probably not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Secondly, an earlier ICM specification of transcripts and proteins of SOX2 and NANOG makes them pertinent candidates of bovine pluripotent lineage specification than OCT4. Thirdly, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development.

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.

The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.

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 (