The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events.

At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote's cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

Hyaluronic acid and epidermal growth factor improved the bovine embryo quality by regulating the DNA methylation and expression patterns of the focal adhesion pathway.

Focal adhesion pathway is one of the key molecular pathways affected by suboptimal culture conditions during embryonic development. The epidermal growth factor (EGF) and hyaluronic acid (HA) are believed to be involved in the focal adhesion pathway function by regulating the adherence of the molecules to the extracellular matrix. However, regulatory and molecular mechanisms through which the EGF and HA could influence the embryo development is not clear. Therefore, this study aimed to investigate the effect of continued or stage specific supplementation of EGF and/or HA on the developmental competence and quality of bovine preimplantation embryos and the subsequent consequences on the expression and DNA methylation patterns of genes involved in the focal adhesion pathway. The results revealed that, the supplementation of EGF or HA from zygote to the blastocysts stage reduced the level of reactive oxygen species and increased hatching rate after thawing. On the other hand, HA decreased the apoptotic nuclei and increased blastocyst compared to EGF supplemented group. Gene expression and DNA methylation analysis in the resulting blastocysts indicated that, combined supplementation of EGF and HA increased the expression of genes involved in focal adhesion pathway while supplementation of EGF, HA or a combination of EGF and HA during the entire preimplantation period changed the DNA methylation patterns of genes involved in focal adhesion pathway. On the other hand, blastocysts developed in culture media supplemented with EGF + HA until the 16-cell stage exhibited higher expression level of genes involved in focal adhesion pathway compared to those supplemented after the 16-cell stage. Conversely, the DNA methylation level of candidate genes was increased in the blastocysts obtained from embryos cultured in media supplemented with EGF + HA after 16-cell stage. In conclusion, supplementation of bovine embryos with EGF and/or HA during the entire preimplantation period or in a stage specific manner altered the DNA methylation and expression patterns of candidate genes involved in the focal adhesion pathway which was in turn associated with the observed embryonic developmental competence and quality.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation.

BACKGROUND: Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. RESULTS: The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. CONCLUSION: In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

MicroRNA-424/503 cluster members regulate bovine granulosa cell proliferation and cell cycle progression by targeting SMAD7 gene through activin signalling pathway.

BACKGROUND: The granulosa cells are indispensable for follicular development and its function is orchestrated by several genes, which in turn posttranscriptionally regulated by microRNAs (miRNA). In our previous study, the miRRNA-424/503 cluster was found to be highly abundant in bovine granulosa cells (bGCs) of preovulatory dominant follicle compared to subordinate counterpart at day 19 of the bovine estrous cycle. Other study also indicated the involvement of miR-424/503 cluster in tumour cell resistance to apoptosis suggesting this miRNA cluster may involve in cell survival. However, the role of miR-424/503 cluster in granulosa cell function remains elusive Therefore, this study aimed to investigate the role of miRNA-424/503 cluster in bGCs function using microRNA gain- and loss-of-function approaches. RESULTS: The role of miR-424/503 cluster members in granulosa cell function was investigated by overexpressing or inhibiting its activity in vitro cultured granulosa cells using miR-424/503 mimic or inhibitor, respectively. Luciferase reporter assay showed that SMAD7 and ACVR2A are the direct targets of the miRNA-424/503 cluster members. In line with this, overexpression of miRNA-424/503 cluster members using its mimic and inhibition of its activity by its inhibitor reduced and increased, respectively the expression of SMAD7 and ACVR2A. Furthermore, flow cytometric analysis indicated that overexpression of miRNA-424/503 cluster members enhanced bGCs proliferation by promoting G1- to S- phase cell cycle transition. Modulation of miRNA-424/503 cluster members tended to increase phosphorylation of SMAD2/3 in the Activin signalling pathway. Moreover, sequence specific knockdown of SMAD7, the target gene of miRNA-424/503 cluster members, using small interfering RNA also revealed similar phenotypic and molecular alterations observed when miRNA-424/503 cluster members were overexpressed. Similarly, to get more insight about the role of miRNA-424/503 cluster members in activin signalling pathway, granulosa cells were treated with activin A. Activin A treatment increased cell proliferation and downregulation of both miRNA-424/503 members and its target gene, indicated the presence of negative feedback loop between activin A and the expression of miRNA-424/503. CONCLUSION: This study suggests that the miRNA-424/503 cluster members are involved in regulating bovine granulosa cell proliferation and cell cycle progression. Further, miRNA-424/503 cluster members target the SMAD7 and ACVR2A genes which are involved in the activin signalling pathway.

Cellular and exosome mediated molecular defense mechanism in bovine granulosa cells exposed to oxidative stress.

Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 µM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo) or those released by granulosa cells without oxidative stress (NormalExo) were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein), altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells exposed to oxidative stress released exosomes enriched with mRNA of Nrf2 and candidate antioxidants. Subsequent co-incubation of StressExo with cultured granulosa cells could alter the relative abundance of cellular oxidative stress response molecules including Nrf2 and antioxidants CAT, PRDX1 and TXN1. The present study provide evidences that granulosa cells exposed to oxidative stress conditions react to stress by activating cascades of cellular antioxidant molecules which can also be released into extracellular environment through exosomes.

MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation.

BACKGROUND: Oocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach. METHODS: For this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture. RESULTS: The luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial activity, while inhibition of miR-130b during preimplantation embryo development significantly reduced morula and blastocyst formation. CONCLUSION: This study demonstrated that in vitro functional modulation of miR-130b affected granulosa and cumulus cell proliferation and survival, oocyte maturation, morula and blastocyst formation suggesting that miR-130b is involved in bovine oocyte maturation and preimplantation embryo development.

Intrafollicular Oocyte Transfer (IFOT) of Abattoir-Derived and In Vitro-Matured Oocytes Results in Viable Blastocysts and Birth of Healthy Calves.

There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13-14 mm delivered significantly (P < 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9-10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P < 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa + BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa + ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa + BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first time we were able to show that IFOT is a feasible technique to generate bovine blastocysts by transferring in vitro-matured oocytes derived from slaughterhouse ovaries. These IFOT-derived blastocysts closely resemble in vivo-derived blastocysts in terms of lipid content and freeze survival. Thus, the present study laid the groundwork for newly created scientific experiments enabling novel analytical possibilities. Nevertheless, IFOT-derived embryos still reached lower pregnancy rates by trend compared with in vivo-derived embryos, also implicating an important role for the maturational environment in further developmental characteristics.

Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway.

In present study, we sought to examine the ability of preimplantation bovine embryos to activate the NF-E2-related factor 2 (NRF2)-mediated oxidative-stress response under an oxidative stress environment. In vitro 2-, 4-, 8-, 16-cell-, and blastocyst-stage embryos were cultured under low (5%) or high (20%) oxygen levels. The expression of NRF2, KEAP1 (NRF2 inhibitor), antioxidants downstream of NRF2, and genes associated with embryo metabolism were analyzed between the embryo groups using real-time quantitative PCR. NRF2 and KEAP1 protein abundance, mitochondrial activity, and accumulation of reactive oxygen species (ROS) were also investigated in blastocysts of varying competence that were derived from high- or low-oxygen levels. The expression levels of NRF2 and its downstream antioxidant genes were higher in 8-cell, 16-cell, and blastocyst stages under high oxygen tension, whereas KEAP1 expression was down-regulated under the same conditions. Higher expression of NRF2 and lower ROS levels were detected in early (competent) blastocysts compared to their late (noncompetent) counterparts in both oxygen-tension groups. Similarly, higher levels of active nuclear NRF2 protein were detected in competent blastocysts compared to their noncompetent counterparts. Thus, the survival and developmental competence of embryos cultured under oxidative stress are associated with activity of the NRF2-mediated oxidative stress response pathway during bovine pre-implantation embryo development.

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

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

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression.

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.

Transcriptome profile of bovine elongated conceptus obtained from SCNT and IVP pregnancies.

In the present study we analyzed the gene expression changes induced by somatic cell nuclear transfer (SCNT) and in vitro production (IVP) in bovine elongated embryos using Affymetrix bovine genome array. For this, Day-16 bovine embryos from SCNT, IVP, and artificial insemination (AI) were recovered from recipients and used for transcriptome analysis. Despite comparable in vivo development rates, considerable reduction in elongation size was observed in SCNT compared to non-cloned embryos (93.3 mm for SCNT vs. 186.6 mm and 196.3 mm for IVP and AI embryos, respectively). Gene expression analysis revealed that the transcript levels of 477 genes, which are involved in various pathways including arginine and proline or glycerolipid and fatty acid metabolism, were significantly altered in SCNT compared to AI embryos. Similarly, 365 genes were differentially expressed in IVP embryos compared to AI. Thus, several pathways including TNRF-1 signaling and tight junction pathways were affected. To predict whether the altered transcripts were associated with culture condition or errors in transcriptional reprogramming, unique or common differentially expressed genes were analyzed in SCNT and IVP embryos compared to AI or fibroblast donor cells. Accordingly, 71 transcripts were found to be not transcriptionally reprogrammed, as their expression resembled the donor cells more than AI embryos; the remaining transcripts were either partially or incompletely reprogrammed. In conclusion, the present study identified deviations in elongation size, gene expression, and the corresponding molecular pathways in Day-16 SCNT and IVP conceptuses compared to their AI counterparts, which may subsequently be associated with the outcome of fetal development.

Aberrant placenta gene expression pattern in bovine pregnancies established after transfer of cloned or in vitro produced embryos.

In the present study, we used the global transcriptome profile approach to identify dysregulated genes, molecular pathways, and molecular functional alterations in bovine placentas derived from somatic cell nuclear transfer (SCNT) and in vitro embryo production (IVP) pregnancies compared with their artificial insemination (AI) counterparts at day 50 of gestation. For this, day 7 blastocysts derived from AI, IVP, or SCNT were transferred to oestrus-synchronized cows. The pregnant animals were slaughtered at day 50 of gestation, and the placentas were then recovered and used for transcriptome analysis using Affymetrix GeneChip bovine genome array. Results showed the SCNT placenta to be different from its AI counterpart in the expression of 1,196 transcripts. These genes were found to be associated with alterations in key biological processes and molecular pathways in SCNT placenta, and the dysregulation of 9% (n = 110) of these genes was due to transcriptional reprogramming error. IVP placenta also displayed alterations in the expression of 72 genes, of which 58 were common to SCNT placenta. Gene enrichment analysis revealed that the expression of genes involved in organ development, blood vessel development, extracellular matrix organization, and the immune system was affected in both SCNT and IVP placentas. However, 96% of the affected genes in SCNT were not significantly altered in IVP groups. Thus, the higher transcriptome dysregulation in SCNT placenta followed by IVP would reflect the degree of placental abnormality in SCNT and IVP pregnancies at day 50 of the gestation, which may have a profound effect on subsequent fetal development and health of the offspring.

Transcriptome fingerprint of bovine 2-cell stage blastomeres is directly correlated with the individual developmental competence of the corresponding sister blastomere.

To date, gene expression profiles of bovine preimplantation embryos have only been indirectly related to developmental potential due to the invasive nature of such procedures. This study sought to find a direct correlation between transcriptome fingerprint of blastomeres of bovine 2-cell stage embryos with developmental competence of the corresponding sister blastomeres. Isolated blastomeres were classified according to the sister blastomere's development into three groups: two groups displayed developmental incompetency, including those blastomeres whose corresponding sister blastomeres either stopped cleaving after separation (2CB) or were blocked after two additional cleavages before embryonic genome activation (8CB). In the third group were competent blastomeres, which were defined as those whose sister blastomeres developed to the blastocyst stage (BL). As a result, developmental capacity of corresponding sister blastomeres was highly similar. Microarray analysis revealed 77 genes to be commonly differentially regulated among competent and incompetent blastomeres as well as blocked blastomeres. Clustering of differentially expressed genes according to molecular functions and pathways revealed antioxidant activity, NRF2-mediated oxidative stress response, and oxidative phosphorylation to be the main ontologies affected. Expression levels of selected candidate genes were further characterized in an independent model for developmental competence based on the time of first cleavage postfertilization. Moreover, overall results of this study were confirmed by higher developmental rates and more beneficial expression of CAT and PRDX1 when cultured in an antioxidative environment. These results will help us to understand molecular mechanisms defining developmental destination of individual bovine preimplantation embryos.

Molecular mechanisms and pathways involved in bovine embryonic genome activation and their regulation by alternative in vivo and in vitro culture conditions.

Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity.

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.